Antibody that binds to human tpor

Abstract

The present invention pertains to: an antibody or an antibody fragment thereof that binds to human TPOR; a nucleic acid that has a base sequence encoding the antibody or the antibody fragment thereof; a vector containing the nucleic acid; a transformed cell containing the vector; a method for producing the antibody or the antibody fragment thereof; a therapeutic agent containing the antibody or the antibody fragment thereof; and a reagent for detection or a reagent for measurement, containing the antibody or the antibody fragment thereof.

Description

Antibodies that bind to human TPOR

[0001] The present invention relates to an antibody or antibody fragment that binds to human TPOR, a nucleic acid having a base sequence encoding the antibody or antibody fragment, a vector containing the nucleic acid, transformed cells containing the vector, a method for producing the antibody or antibody fragment, a therapeutic agent containing the antibody or antibody fragment, and a detection reagent or measurement reagent containing the antibody or antibody fragment.

[0002] Thrombopoietin (TPO) is a hematopoietic factor that promotes the proliferation of megakaryocytes and platelets in the body. Human TPO is a glycoprotein consisting of 332 amino acid residues, and its N-terminal sequence is known to be important for its activity. It exerts its function by binding to the TPO receptor (TPOR) on the cell membrane.

[0003] Mpl (also known as c-Mpl) is a TPO receptor. Human c-Mpl is a single-pass transmembrane glycoprotein consisting of 635 amino acids including the signal peptide and 610 amino acids in its mature form, and belongs to the type I cytokine receptor family. The messenger RNA and protein sequences of human c-Mpl have already been reported (see Genbank: NM_005373, NP_005364). It is thought that TPO binds to CRM1 and transmits a signal by dimerizing c-Mpl, but the detailed modes of binding and activation have not been elucidated. When c-Mpl dimerizes, the signal transduction kinase bound to the intracellular domain is activated, and a phosphorylation signal is transmitted into the cell. It is known that the Jak-STAT, PI3K-Akt, and Ras-MAPK pathways are activated by the TPO-Mpl signaling pathway. Mice lacking TPO or c-Mpl have been reported to have platelet counts reduced to about 10-20% of those of wild-type mice, indicating that the TPO-Mpl system is a major system for regulating platelet count. c-Mpl expression is found not only in megakaryocytes but also in undifferentiated hematopoietic progenitor cells and hematopoietic stem cells. c-Mpl-positive cell fractions in the bone marrow have been shown to have higher bone marrow reconstitution ability compared to c-Mpl-negative fractions, and it has been found that not only megakaryocytes and platelets but also hematopoietic stem cells are reduced in c-Mpl-deficient mice (Non-patent Literature 1, 2). These findings suggest that the TPO-Mpl system is involved in the hematopoietic system at the stem cell level.

[0004] It is known that romiplostim, an Fc fusion protein of the TPO mimetic peptide, exerts a therapeutic effect against aplastic anemia, which is caused by a decrease in hematopoietic stem progenitor cells, by acting on hematopoietic stem progenitor cells and increasing their number (Non-Patent Literature 3). As for TPOR agonist antibodies, monoclonal antibodies described in Patent Literature 1 and Patent Literature 2 have been reported.

[0005] U.S. Patent No. 8048421, International Publication No. 2009041734

[0006] Miyazaki, Hiroshi, "Future Prospects of Thrombopoietin," Japanese Journal of Transfusion Medicine, 2000, 46(3), 311-316. Murone M et al. Stem Cell, 1998, 16:1-6, Review. Lee JW et al., The Lancet Haematology 2019, 6(11), e562-e572.

[0007] The present invention aims to provide an antibody or antibody fragment that binds to human TPOR and has excellent agonist activity and / or excellent physical properties, a nucleic acid having a nucleotide sequence encoding the antibody or antibody fragment, a vector containing the nucleic acid, transformed cells containing the vector, a method for producing the antibody or antibody fragment, a therapeutic agent containing the antibody or antibody fragment, and a detection reagent or measurement reagent containing the antibody or antibody fragment.

[0008] The inventors of this invention, after diligent research, have found an antibody that solves the above-mentioned problems.

[0009] In other words, the present invention relates to the following: [1] An antibody or antibody fragment comprising a heavy chain variable region (hereinafter referred to as VH) containing an amino acid sequence in which at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is modified, and a light chain variable region (hereinafter referred to as VL) containing an amino acid sequence in which 0 or 1 or more amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 are modified, and which binds to a human thrombopoietin receptor (hereinafter referred to as human TPOR). [2] The antibody or antibody fragment according to [1] above, wherein the position of the modified amino acid residue is one selected from (a) to (d) below. (a) the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH, (b) the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH and the 49th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL, (c) the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH and the 70th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VH, or (d) the 100th and 70th amino acid residues of the amino acid sequence represented by SEQ ID NO: 1 in VH and the 49th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL. [3] The antibody or antibody fragment according to [1] or [2] above, wherein the modification of the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is a substitution with an alanine residue or a glycine residue. [4] The antibody or antibody fragment according to any one of [1] to [3] above, wherein the modification of the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is a substitution with an alanine residue. [5] The antibody or antibody fragment according to any one of [1] to [4] above, wherein the modification of the 70th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is a substitution with a valine residue. [6] The antibody or antibody fragment according to any one of [1] to [5] above, wherein the modification of the 49th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL is a substitution with an alanine residue, a methionine residue, a serine residue, or a threonine residue.[7] The antibody or antibody fragment according to any one of [1] to [6] above, wherein the modification of the 49th amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in the VL is a substitution with an alanine residue or a threonine residue. [8] The antibody or antibody fragment according to any one of [1] to [7] above, wherein the modification of the amino acid residue is one selected from (A) to (D) below. (A) Two modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with an alanine residue; (B) Two modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with a threonine residue; (C) Three modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue and substitution of the 70th amino acid residue with valine, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with an alanine residue; or (D) Three modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue and substitution of the 70th amino acid residue with valine, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with a threonine residue. [9] The antibody or antibody fragment according to any one of [1] to [7] above, wherein the modification of the amino acid residues is three modifications: substitution of the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue and substitution of the 70th amino acid residue with valine, and substitution of the 49th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL with an alanine residue.

[10] The antibody or antibody fragment according to any one of [1] to [7] above, which is one selected from (1) to (4) below.(1) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 3, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO: 4; (2) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 7, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO: 8; (3) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 5, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO: 6; or (4) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 9, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO: 10.

[11] The antibody or antibody fragment according to any one of claims 1 to 10, wherein the subclass of the heavy chain constant region of the antibody is IgG4, and the amino acid sequence of the heavy chain constant region of the antibody includes the amino acid residue substitutions S228P, L235E, and R409K represented by the EU index.

[12] The antibody or antibody fragment according to any one of [1] to

[11] , wherein the heavy chain constant region of the antibody is a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14.

[13] The antibody fragment is Fab, Fab', F(ab'). ２

[14] An antibody fragment according to any one of [1] to

[12] above, which is one selected from a peptide comprising a single-chain antibody (scFv), a dimerized V region (diabody), a disulfide-stabilized V region (dsFv), and a CDR.

[15] A vector containing the nucleic acid according to

[14] above.

[16] A transformed cell containing the vector according to

[15] above.

[17] A method for producing the antibody or antibody fragment according to any one of [1] to

[13] above, comprising culturing the transformed cell according to

[16] above in a culture medium and collecting the antibody or antibody fragment from the culture.

[18] A pharmaceutical composition containing the antibody or antibody fragment according to any one of [1] to

[13] above.

[19] A therapeutic agent for thrombocytopenia or pancytopenia containing the antibody or antibody fragment according to any one of [1] to

[13] above.

[20] The therapeutic agent according to

[19] above, wherein the thrombocytopenia or pancytopenia is at least one of immune thrombocytopenia (ITP), aplastic anemia, chemotherapy-induced thrombocytopenia (CIT), cytopenia after hematopoietic stem cell transplantation, myelodysplastic syndrome (MDS), hematopoietic failure after CAR-T therapy, systemic lupus erythematosus, acute radiation syndrome, and thrombocytopenia associated with liver disease.

[21] A reagent for detecting or measuring human TPOR comprising the antibody or antibody fragment according to any one of [1] to

[13] above.

[0010] The present invention also relates to the following: • A method for treating thrombocytopenia or pancytopenia, comprising administering the antibody or antibody fragment described in [1] above. • Use of the antibody or antibody fragment described in [1] above for the production of a therapeutic agent for thrombocytopenia or pancytopenia. • The antibody or antibody fragment described in [1] above for use as a therapeutic agent for thrombocytopenia or pancytopenia. • A method for detecting or measuring human TPOR using the antibody or antibody fragment described in [1] above. • Use of the antibody or antibody fragment described in [1] above for the production of a reagent for detecting or measuring human TPOR. • The antibody or antibody fragment described in [1] above for use as a reagent for detecting or measuring human TPOR.

[0011] The present invention provides an antibody or antibody fragment that binds to human TPOR and has excellent agonist activity and / or excellent physical properties, a nucleic acid having a nucleotide sequence encoding the antibody or antibody fragment, a vector containing the nucleic acid, transformed cells containing the vector, a method for producing the antibody or antibody fragment, a therapeutic agent and a detection reagent or measurement reagent containing the antibody or antibody fragment.

[0012] Figure 1 shows the increase in the aggregate content of antibodies after low-pH treatment compared to the aggregate content before low-pH treatment. The vertical axis shows the increase in aggregates (%). Figure 2 shows the relationship between the concentration and viscosity of the antibody solution. The vertical axis shows viscosity (cP), and the horizontal axis shows concentration (mg / mL). Figure 3A shows the relationship between the number of human umbilical cord blood-derived HSPCs and antibody concentration. The vertical axis shows the number of cells (cells / well), and the horizontal axis shows the antibody concentration in each well (ng / mL). Figure 3B shows the relationship between the number of human umbilical cord blood-derived HSPCs and antibody concentration. The vertical axis shows the number of cells (cells / well), and the horizontal axis shows the antibody concentration in each well (ng / mL). Figure 4 shows the viscosity of a solution with an antibody concentration of 60 mg / mL. The vertical axis shows viscosity (cP). Figure 5 shows the increase in the aggregate content of antibodies after low-pH treatment compared to the aggregate content before low-pH treatment. The vertical axis shows the increase in aggregates (%). Figure 6A shows the relationship between the number of human umbilical cord blood-derived HSPCs and antibody concentration. The vertical axis shows the number of cells (cells / well), and the horizontal axis shows the antibody concentration in each well (ng / mL). Figure 6B shows the relationship between the number of human umbilical cord blood-derived HSPCs and antibody concentration. The vertical axis shows the number of cells (cells / well), and the horizontal axis shows the antibody concentration in each well (ng / mL). Figure 6C shows the relationship between the number of human umbilical cord blood-derived HSPCs and antibody concentration. The vertical axis shows the number of cells (cells / well), and the horizontal axis shows the antibody concentration in each well (ng / mL). The relationship between the concentration and viscosity of the antibody solution is shown. The vertical axis shows viscosity (cP), and the horizontal axis shows concentration (mg / mL). Figure 8 shows the increase in aggregate content after low pH treatment compared to the aggregate content before low pH treatment. The vertical axis shows the increase in aggregates (%). Figure 9 shows the relationship between the number of human umbilical cord blood-derived HSPCs and antibody concentration. The vertical axis shows the number of cells (cells / well), and the horizontal axis shows the antibody concentration in each well (ng / mL).

[0013] The present invention will be described in detail below, but these are merely examples of preferred embodiments and are not limiting to these. The "~" in numerical ranges indicates a range that includes the numbers before and after it; for example, "0 mass% to 100 mass%" means a range that is 0 mass% or more and 100 mass% or less.

[0014] The antibody or antibody fragment of this embodiment comprises a heavy chain variable region (hereinafter referred to as VH) containing an amino acid sequence in which at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is modified, and a light chain variable region (hereinafter referred to as VL) containing an amino acid sequence in which 0 or 1 or more amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 are modified, and the antibody or antibody fragment binds to human TPOR.

[0015] In this specification, TPOR has an Official Full Name of MPL proto-oncogene, thrombopoietin receptor (Official Symbol: MPL). In this specification, TPOR is also known as c-Mpl, Mpl, MPLV, CD110, THPOR, and THCYT2, and these are used synonymously. In this specification, human TPOR includes, for example, a polypeptide consisting of the amino acid sequence shown in NCBI accession number NP_005364. In addition, human TPOR includes, for example, a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in NCBI accession number NP_005364, and which has the function of human TPOR.

[0016] Polypeptides comprising amino acid sequences having typically 70% or more, preferably 80% or more, and more preferably 90% or more homology to the amino acid sequence shown in NCBI accession number NP_005364, and most preferably 95%, 96%, 97%, 98%, and 99% or more homology, and which have the function of human TPOR, are also included in human TPOR as defined herein.

[0017] Polypeptides having an amino acid sequence in which one or more amino acid residues are deleted, substituted, or added in the amino acid sequence shown in NCBI accession number NP_005364 are subject to site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proceedings of the National Academy of Sciences in USA, 82, 488]. This can be obtained, for example, by introducing site-directed mutations into DNA encoding the amino acid sequence shown in NCBI accession number NP_005364, using

[1985] , etc. The number of amino acids deleted, substituted, or added is not particularly limited, but is preferably 1 to several dozen, for example 1 to 20, and more preferably 1 to several, for example 1 to 5 amino acids.

[0018] Examples of genes encoding human TPOR include the human TPOR nucleotide sequence shown in NCBI accession number NM_005373, and the monkey TPOR nucleotide sequence also shown in NCBI accession number NM_005373. Furthermore, genes comprising DNA encoding a polypeptide having the function of human TPOR, which consists of a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown at NCBI accession number NM_005373, genes comprising DNA encoding a polypeptide having the function of human TPOR, which consists of a nucleotide sequence preferably having 60% or more homology, more preferably 80% or more homology, and even more preferably 95% or more homology, with the nucleotide sequence shown at NCBI accession number NM_005373, and genes comprising DNA encoding a polypeptide having the function of human TPOR, and genes comprising DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence shown at NCBI accession number NM_005373, are also included in the human TPOR encoding genes of this specification.

[0019] DNA that hybridizes under stringent conditions refers to hybridizable DNA obtained by methods such as colony hybridization, plaque hybridization, Southern blot hybridization, or DNA microarray, using a DNA having the base sequence shown in NCBI accession number NM_005373 as a probe. Specifically, examples of DNA that can be identified include DNA derived from hybridized colonies or plaques, or PCR products or oligoDNA containing the sequence, immobilized on filters or slides, and hybridized at 65°C in the presence of 0.7–1.0 mol / L sodium chloride [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987–1997), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995)], followed by washing the filter or slide with a 0.1–2 times concentrated SSC solution (a 1x concentrated SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate) at 65°C. Examples of hybridizable DNA include DNA having preferably 60% or more homology to the base sequence shown in NCBI accession number NM_005373, more preferably 80% or more homology, and even more preferably 95% or more homology.

[0020] Genetic polymorphisms are often observed in the base sequences of proteins-coding genes in eukaryotes. Among the genes used in this embodiment, genes that have undergone small mutations in their base sequence due to such polymorphisms are also included in the human TPOR-coding gene of this embodiment.

[0021] Unless otherwise specified, the homology values ​​in this specification may be values ​​calculated using homology search programs known to those skilled in the art. Examples include values ​​calculated using default parameters in BLAST [J. Mol. Biol., 215, 403 (1990)] for nucleotide sequences, and values ​​calculated using default parameters in BLAST2 [Nucleic Acids Research, 25, 3389 (1997), Genome Research, 7, 649 (1997), http: / / www.ncbi.nlm.nih.gov / Education / BLASTinfo / information3.html] for amino acid sequences.

[0022] The default parameters are: G (Cost to open gap) is 5 for nucleotide sequences and 11 for amino acid sequences; -E (Cost to extend gap) is 2 for nucleotide sequences and 1 for amino acid sequences; -q (Penalty for nucleotide mismatch) is -3; -r (reward for nucleotide match) is 1; -e (expect value) is 10; -W (wordsize) is 11 residues for nucleotide sequences and 3 residues for amino acid sequences; -y [Dropoff(X) for blast extensions in bits] is 20 for blastn and 7 for programs other than blastn; -X (X dropoff value for The value of -Z (final X dropoff value for gapped alignment in bits) is 15 and -Z is 50 for blastn and 25 for programs other than blastn (http: / / www.ncbi.nlm.nih.gov / blast / html / blastcgihelp.html).

[0023] Polypeptides containing a partial sequence of the amino acid sequence at NCBI accession number NP_005364, which is an example of the amino acid sequence of human TPOR, can be prepared by methods known to those skilled in the art. Specifically, they can be prepared by deleting a portion of the DNA encoding the amino acid sequence at NCBI accession number NP_005364 and culturing a transformant into which an expression vector containing this deletion has been introduced. Alternatively, polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence at NCBI accession number NP_005364 can be obtained by the same method as described above. Furthermore, polypeptides consisting of the amino acid sequence at NCBI accession number NP_005364, or polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence at NCBI accession number NP_005364, can also be produced by chemical synthesis methods such as the fluorenylmethyloxycarbonyl (Fmoc) method and the t-butyloxycarbonyl (tBoc) method.

[0024] In this embodiment, deletion, substitution, or addition of amino acid residues is also referred to as modification of amino acid residues.

[0025] The antibodies in this embodiment include polyclonal antibodies, monoclonal antibodies, and oligoclonal antibodies. Polyclonal antibodies refer to a group of antibody molecules secreted by antibody-producing cells of different clones. Monoclonal antibodies are antibodies secreted by antibody-producing cells of a single clone, which recognize only one epitope (also called an antigenic determinant), and whose amino acid sequence (primary sequence) is uniform. Oligoclonal antibodies refer to a group of antibody molecules obtained by mixing multiple different monoclonal antibodies.

[0026] Examples of monoclonal antibodies in this embodiment include antibodies produced by hybridomas, or recombinant antibodies produced by transformants transformed with an expression vector containing an antibody gene.

[0027] An epitope refers to a single amino acid sequence, a three-dimensional structure composed of an amino acid sequence, an amino acid sequence modified by post-translational modification, and a three-dimensional structure composed of the amino acid sequence, etc., which are recognized and bound by a monoclonal antibody.

[0028] Examples of the amino acid sequence modified by post-translational modification include an O-linked sugar chain bound to Tyr and Ser having an OH substituent, an N-linked sugar chain bound to Gln and Asn having an NH ２ substituent, and an amino acid sequence to which a sulfate group bound to Tyr and Ser having an OH substituent is bound, etc.

[0029] That the antibody of this embodiment binds to human TPOR can be confirmed by measuring the binding property of the antibody of this embodiment to human TPOR using ELISA, surface plasmon resonance method, etc. Also, it can be confirmed by combining known immunological detection methods [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)], etc.

[0030] The amino acid residue or epitope of human TPOR to which the antibody of this embodiment binds can be determined by performing a binding experiment of the antibody using a deletion mutant lacking a part of the domain of human TPOR, a mutant substituted with a domain derived from another protein, a partial peptide fragment of human TPOR, etc.

[0031] Alternatively, the amino acid residue or epitope of human TPOR to which the antibody of this embodiment binds can also be determined by adding the antibody of this embodiment to a peptide fragment of human TPOR digested with a proteolytic enzyme and performing epitope mapping using a known mass spectrometry method.

[0032] An antibody molecule is also referred to as an immunoglobulin (hereinafter referred to as Ig). Human antibodies are classified into isotypes of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM according to differences in molecular structure. IgG1, IgG2, IgG3, and IgG4, which have relatively high amino acid sequence homology, are collectively referred to as IgG.

[0033] An antibody molecule is composed of polypeptides called heavy chains (hereinafter also referred to as H chains) and light chains (hereinafter also referred to as L chains). The H chain is composed of VH, the heavy chain constant region (also denoted as CH), and the L chain is composed of VL, the light chain constant region (also denoted as CL) from the N-terminal side. In each subclass, the CH is known as the α, δ, ε, γ, and μ chains. The CH is further composed of CH1 domain, hinge domain, CH2 domain, and CH3 domain from the N-terminal side. A domain refers to a functional structural unit that constitutes each polypeptide of an antibody molecule. Also, the CH2 domain and the CH3 domain are collectively referred to as the Fc region or simply Fc. The CL is known as the C λ chain and the C κ chain.

[0034] The CH1 domain, hinge domain, CH2 domain, CH3 domain, and Fc region in this embodiment can be specified by the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] (hereinafter simply referred to as the EU index) by the numbers of amino acid residues from the N-terminal. Specifically, CH1 is the amino acid sequence of EU index positions 118 to 215, the hinge is the amino acid sequence of EU index positions 216 to 230, CH2 is the amino acid sequence of EU index positions 231 to 340, and CH3 is the amino acid sequence of EU index positions 341 to 447, respectively.

[0035] The antibodies of this embodiment include genetically engineered recombinant mouse antibodies, recombinant rat antibodies, recombinant rabbit antibodies, human-type chimeric antibodies (hereinafter also abbreviated simply as chimeric antibodies), humanized antibodies (also called human-type complementarity-determining region CDR-transplanted antibodies), and human antibodies. The antibodies of this embodiment also include genetically engineered recombinant antibodies produced by recombining the H chain (or VH) and L chain (or VL) from two different types of antibodies. The two different types of antibodies may be hybridoma-derived monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies. Furthermore, the antibodies of this embodiment also include genetically engineered recombinant antibodies in which appropriate amino acid residues have been substituted when producing the above-mentioned genetically engineered recombinant antibodies.

[0036] A chimeric antibody refers to an antibody composed of VH and VL antibodies from a non-human animal and CH and CL antibodies from a human animal. Any non-human animal can be used as the source, such as mice, rats, hamsters, or rabbits, as long as it is possible to create a hybridoma from them.

[0037] A hybridoma is a cell obtained by fusing B cells, acquired by immunizing non-human animals with an antigen, with myeloma cells derived from mice or other animals. It produces monoclonal antibodies with desired antigen specificity. Therefore, the variable region of the antibody produced by the hybridoma consists of the amino acid sequence of the non-human animal antibody.

[0038] Chimeric antibodies can be produced by obtaining cDNA encoding VH and VL of a monoclonal antibody from a hybridoma derived from a non-human animal cell that produces a monoclonal antibody, inserting these cDNAs into an animal cell expression vector containing DNA encoding CH and CL of a human antibody, constructing a human-type chimeric antibody expression vector, and then introducing it into animal cells to express the antibody.

[0039] Humanized antibodies are antibodies in which the amino acid sequences of the CDRs (Cellular Derived Ratios) of the VH and VL (Very Longitudinal) parts of non-human animal antibodies have been transplanted into the corresponding CDRs of the VH and VL parts of human antibodies. The regions of the VH and VL parts other than the CDRs are called framework regions (hereinafter referred to as FR).

[0040] Humanized antibodies can be produced by constructing a cDNA encoding the VH amino acid sequence, which consists of the CDR amino acid sequence of the VH of a non-human animal antibody and the FR amino acid sequence of the VH of any human antibody, and a cDNA encoding the VL amino acid sequence, which consists of the CDR amino acid sequence of the VL of a non-human animal antibody and the FR amino acid sequence of the VL of any human antibody. These cDNAs are then inserted into an animal cell expression vector containing DNA encoding the CH and CL of a human antibody to construct a humanized antibody expression vector, which is then introduced into animal cells for expression.

[0041] Human antibodies originally refer to antibodies that naturally exist in the human body, but recent advances in genetic engineering, cell engineering, and developmental engineering technologies have also led to the creation of human antibody phage libraries and antibodies obtained from human antibody-producing transgenic animals.

[0042] Human antibodies can be obtained by immunizing mice carrying the human immunoglobulin gene (Tomizuka K. et al., Proc Natl Acad Sci US A. 97, 722-7, 2000) with the desired antigen. Furthermore, by using a phase display library in which antibody genes are amplified from human B cells, human antibodies with the desired binding activity can be selected, thereby obtaining human antibodies without immunization (Winter G. et al., Annu Rev Immunol. 12:433-55. 1994). Additionally, by immortalizing human B cells using EB virus, cells that produce human antibodies with the desired binding activity can be created, thereby obtaining human antibodies (Rosen A. et al., Nature 267, 52-54. 1977).

[0043] Antibodies present in the human body can be obtained, for example, by immortalizing lymphocytes isolated from human peripheral blood by infecting them with EB virus or the like, and then cloning them to obtain lymphocytes that produce the antibodies. The antibodies can then be purified from the culture obtained by culturing these lymphocytes.

[0044] A human antibody phage library is a library of phages in which antibody fragments such as Fab and scFv are expressed on the surface by inserting antibody genes prepared from human B cells into phage genes. From this library, phages expressing antibody fragments with desired antigen-binding activity can be recovered, using the binding activity to an antigen-immobilized substrate as an indicator. These antibody fragments can further be converted into human antibody molecules consisting of two complete H chains and two complete L chains using genetic engineering techniques.

[0045] Human antibody-producing transgenic animals are animals in which human antibody genes have been incorporated into the chromosomes of a host animal. Specifically, human antibody-producing transgenic animals can be created by introducing human antibody genes into mouse ES cells, transplanting these ES cells into early-stage embryos of other mice, and then allowing them to develop. Human antibodies can be produced from human antibody-producing transgenic animals by obtaining human antibody-producing hybridomas using the same hybridoma production methods used for mammals other than humans, and then culturing them to produce and accumulate human antibodies in the culture.

[0046] The VH and VL amino acid sequences of the antibody in this embodiment may be any of the VH and VL amino acid sequences of a human antibody, a non-human animal antibody, or a humanized antibody.

[0047] The amino acid sequence of CL in the antibody of this embodiment may be either the amino acid sequence of a human antibody or a non-human animal antibody, but the C of the amino acid sequence of a human antibody may be used. κ or C λ It is preferable.

[0048] The antibodies of this embodiment also include Fc fusion proteins in which Fc and an antibody fragment are bound, Fc fusion proteins (also called immunoadhesins) in which Fc and a naturally occurring ligand or receptor are bound, and Fc fusion proteins in which multiple Fc regions are fused.

[0049] The antibody or antibody fragment of the present embodiment also includes any antibody or antibody fragment containing an amino acid residue modified after translation. Examples of post-translational modifications include, for example, deletion of lysine residues at the C-terminus of the H chain [lysine clipping] or conversion of glutamine residues at the N-terminus of the polypeptide to pyroGlu [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].

[0050] In the present embodiment, the antibody fragment is an antibody fragment that binds to human TPOR, and preferably includes a VH containing an amino acid sequence in which at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is modified, and a part of a VL containing an amino acid sequence in which 0 or one or more amino acid residues are modified in the amino acid sequence represented by SEQ ID NO: 2. More preferably, it includes a VH containing an amino acid sequence in which at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is modified, and a VL containing an amino acid sequence in which 0 or one or more amino acid residues are modified in the amino acid sequence represented by SEQ ID NO: 2.

[0051] In the present embodiment, examples of the antibody fragment include Fab, Fab', F(ab') ２ , scFv, diabody, dsFv, peptides containing peptides containing CDRs, and the like. Fab is a fragment obtained by treating an IgG antibody with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain), and is an antibody fragment having an antigen-binding activity with a molecular weight of about 50,000, in which the N-terminal side of the H chain of about half and the entire L chain are linked by a disulfide bond (S-S bond).

[0052] F(ab') ２ is an antibody fragment having an antigen-binding activity with a molecular weight of about 100,000, which is slightly larger than that obtained by binding Fab through the S-S bond in the hinge region among the fragments obtained by treating IgG with the proteolytic enzyme pepsin (cleaved at the 234th amino acid residue of the H chain). Fab' is the above F(ab') ２This is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, formed by cleaving the S-S bond in the hinge region of the antibody.

[0053] scFv is an antibody fragment having antigen-binding activity, which is a VH-P-VL or VL-P-VH polypeptide formed by linking one VH molecule and one VL molecule using a suitable peptide linker (P), such as a linker peptide consisting of any number of linkers (G4S) composed of four Gly residues and one Ser residue.

[0054] A Diabody is an antibody fragment formed by the dimerization of scFvs with the same or different antigen-binding specificities, and is an antibody fragment that has bivalent antigen-binding activity against the same antigen or specific antigen-binding activity against different antigens.

[0055] dsFv refers to a polypeptide in which one amino acid residue in VH and VL is replaced with a cysteine ​​residue, and these polypeptides are linked together via S-S bonds between the cysteine ​​residues.

[0056] The CDR-containing peptide is composed of at least one region of the VH or VL CDR, preferably containing the three CDRs contained in VH and the three CDRs contained in VL. Multiple CDR-containing peptides can have the CDRs linked directly or via a suitable peptide linker. The modified antibody of this embodiment can be produced by constructing DNA encoding the VH and VL CDRs, inserting this DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryote or eukaryote for expression. Alternatively, the CDR-containing peptide can be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.

[0057] The antibody or antibody fragment of this embodiment comprises VH, which includes an amino acid sequence in which at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is modified, and VL, which includes an amino acid sequence in which 0 or 1 or more amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 are modified, and is an antibody or antibody fragment that binds to human TPOR.

[0058] The antibody or antibody fragment of this embodiment has excellent agonist activity and / or excellent physical properties. In this specification, physical properties include the viscosity when the antibody solution is concentrated to a high level and the amount of aggregate formation after low pH treatment.

[0059] In this specification, high concentration means that the concentration of antibody or antibody fragment in the antibody solution is, for example, 60 mg / mL or higher. Preferably, the high concentration in this specification is 60 mg / mL to 120 mg / mL, and more preferably 60 mg / mL to 100 mg / mL. Viscosity can be measured using, for example, a Viscosizer TD (manufactured by Spectris, Inc.) or a VROC Initium One Plus (manufactured by RheoSense, Inc.). The viscosity (cP) of the antibody or antibody fragment solution in this embodiment can be measured using, for example, a Viscosizer TD by the following procedure: (1) Fill a capillary cell with the antibody under a constant pressure of 1000 mbar at 25°C. (2) The antibody solution is detected at a wavelength of 280 nm or 254 nm as it passes through two windows in the capillary cell, and the time difference is measured by comparing it with the time difference of the corresponding buffer (10 mM glutamic acid, 262 mM D-sorbitol, pH 5.5) or water. (3) The Mooney equation is used to calculate an approximate curve of the concentration-dependent viscosity change of the antibody. The viscosity (cP) of the antibody or antibody fragment solution in this embodiment can be measured, for example, in VROC Initium One Plus by the following procedure: (1) Inject the antibody into a measuring syringe under conditions of 25°C. (2) Flow rate 200 μL / min, shear rate 3700 s －１ The sample is passed through the instrument under the following conditions. (3) The Mooney equation is used to calculate an approximate curve of the viscosity change dependent on the concentration of the antibody.

[0060] In this specification, low pH treatment refers to a process in which, for example, a buffer consisting of 200 mmol / L Citric Acid buffer (pH 2.7) is added to an antibody sample to adjust the pH to 3.5, the solution is heated at 37°C for 60 minutes, and then neutralized by adding 500 mM Phosphate buffer (pH 8.0) to the low pH treatment solution. Alternatively, in this specification, low pH treatment may also refer to a process in which, for example, a buffer consisting of 200 mmol / L Citric Acid buffer (pH 2.7) is added to an antibody sample to adjust the pH to 3.5, the solution is heated at 25°C for 16 hours, and then neutralized by adding 500 mM Phosphate buffer (pH 8.0) to the low pH treatment solution.

[0061] In this specification, the amount of aggregate formation can be confirmed, for example, by measuring it using an ultra-high-performance liquid chromatography system (Shimadzu Corporation) and an ACQUITY UPLC Protein BEH SEC column (200 angstroms, 1.7 μm, 4.6 × 150 mm) (Waters Corporation) under the measurement conditions described below. For example, the measurement conditions are: solvent is 50 mmol / L sodium phosphorate, 500 mmol / L NaCl, 5% EtOH (pH 6.8), flow rate is 0.4 mL / min, detection wavelength is 215 nm, column temperature is 25°C, analysis time is 8 minutes, and 5 μg of antibody is used. Alternatively, a Waters Corporation ultra-high-performance liquid chromatography system can be used. The peaks of antibody monomers, aggregates (high molecular weight), and degradation products (low molecular weight) can be identified, and the aggregate content can be calculated from the area of ​​each peak.

[0062] In this specification, the statement that the antibody or antibody fragment of this embodiment has excellent physical properties means, for example, the following: • In an antibody solution containing the antibody or antibody fragment of the same concentration (in the high concentration range), the viscosity of the solution containing the antibody or antibody fragment of this embodiment is lower than the viscosity of the solution containing the antibody or antibody fragment containing VH, which includes the amino acid sequence represented by SEQ ID NO: 1, and VL, which includes the amino acid sequence represented by SEQ ID NO: 2.

[0063] In high-concentration antibody preparations for subcutaneous injection, if the viscosity of the antibody solution is high, it may not be possible to administer it using a needle of the thickness generally used for subcutaneous injection. In addition, this may lead to a decrease in the stability of the preparation during storage, an increase in the time required for drug injection, and / or an increase in pain during injection. Therefore, it is preferable that the viscosity of the solution containing the antibody or antibody fragment of this embodiment at a high concentration is lower than the viscosity of the solution containing the antibody or antibody fragment containing VH, which contains the amino acid sequence represented by SEQ ID NO: 1, and VL, which contains the amino acid sequence represented by SEQ ID NO: 2, at the same concentration.

[0064] Furthermore, in this specification, the statement that the antibody or antibody fragment of this embodiment has excellent physical properties includes, for example, the following cases: • When the amount of aggregates formed in an antibody solution containing the antibody or antibody fragment of this embodiment after low pH treatment is less than the amount of aggregates formed in a solution containing the antibody or antibody fragment containing VH with the amino acid sequence represented by SEQ ID NO: 1 and VL with the amino acid sequence represented by SEQ ID NO: 2 after low pH treatment.

[0065] In this specification, the statement that the antibody or antibody fragment of this embodiment has excellent physical properties means, more specifically, the following cases: • The content of aggregates after low pH treatment in an antibody solution containing the antibody or antibody fragment of this embodiment is lower than the content of aggregates after low pH treatment in an antibody solution containing the antibody or antibody fragment containing VH with the amino acid sequence represented by SEQ ID NO: 1 and VL with the amino acid sequence represented by SEQ ID NO: 2; or • The increase in aggregates in an antibody solution containing the antibody or antibody fragment of this embodiment, calculated using the following formula, is lower than the increase in aggregates in an antibody solution containing the antibody or antibody fragment containing VH with the amino acid sequence represented by SEQ ID NO: 1 and VL with the amino acid sequence represented by SEQ ID NO: 2. Increase in aggregates (%) = [Agglutination content after low pH treatment (%)] - [Agglutination content before low pH treatment (%)]

[0066] In the manufacturing process of antibody drugs, virus inactivation may be performed by low pH treatment. If aggregates are formed during this process, the stability and productivity of the antibody drug may decrease. Therefore, it is preferable that the amount of aggregates formed after low pH treatment in an antibody solution containing the antibody or antibody fragment of this embodiment is less than the amount of aggregates formed after low pH treatment in a solution containing the antibody or antibody fragment containing VH, which contains the amino acid sequence represented by SEQ ID NO: 1, and VL, which contains the amino acid sequence represented by SEQ ID NO: 2.

[0067] In this specification, agonist activity refers to the function of the antibody or antibody fragment of this embodiment in which it binds to human TPOR on cells that proliferate in a TPOR-dependent manner and activates the signaling mechanism of those cells. Activation of the signaling mechanism of those cells leads to, for example, cell proliferation. In this specification, the strength of agonist activity can be confirmed, for example, by measuring the proliferation rate of cells that express human TPOR and proliferate in a TPOR-dependent manner when cultured together with the antibody or antibody fragment of this embodiment. Specifically, the strength of agonist activity can be measured by the method described later in the examples.

[0068] In this specification, the statement that the antibody or antibody fragment of this embodiment has excellent agonist activity means the following: • At the same antibody concentration, the cell proliferation rate of the antibody or antibody fragment of this embodiment is higher than that of the antibody or antibody fragment containing VH with the amino acid sequence represented by SEQ ID NO: 1 and VL with the amino acid sequence represented by SEQ ID NO: 2; or • The antibody or antibody fragment of this embodiment causes cell proliferation at a lower concentration than the antibody or antibody fragment containing VH with the amino acid sequence represented by SEQ ID NO: 1 and VL with the amino acid sequence represented by SEQ ID NO: 2.

[0069] The antibody or antibody fragment of this embodiment is an antibody or antibody fragment in which the position of the modified amino acid residue is one selected from (a) to (d) below: (a) the 100th amino acid of the amino acid sequence represented by SEQ ID NO: 1 in VH, (b) the 100th amino acid of the amino acid sequence represented by SEQ ID NO: 1 in VH and the 49th amino acid of the amino acid sequence represented by SEQ ID NO: 2 in VL, (c) the 100th amino acid of the amino acid sequence represented by SEQ ID NO: 1 in VH and the 70th amino acid of the amino acid sequence represented by SEQ ID NO: 2 in VH, or (d) the 100th and 70th amino acid of the amino acid sequence represented by SEQ ID NO: 1 in VH and the 49th amino acid of the amino acid sequence represented by SEQ ID NO: 2 in VL.

[0070] In this embodiment, the antibody or antibody fragment is preferably one in which the modification of the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is a substitution with an alanine residue or a glycine residue.

[0071] In this embodiment, the antibody or antibody fragment is more preferably one in which the modification of the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is a substitution with an alanine residue.

[0072] In this embodiment, the antibody or antibody fragment is preferably one in which the modification of the 70th amino acid residue in the amino acid sequence represented by Sequence ID No. 1 in VH is a substitution with a valine residue.

[0073] In this embodiment, the antibody or antibody fragment is preferably one in which the modification of the 49th amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in the VL is a substitution with an alanine residue, a methionine residue, a serine residue, or a threonine residue.

[0074] In this embodiment, the antibody or antibody fragment is more preferably one in which the modification of the 49th amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in the VL is a substitution with an alanine residue or a threonine residue.

[0075] In this embodiment, the antibody or antibody fragment is preferably one in which the modification of the amino acid residue is selected from any one of (A) to (D) below. (A) Two modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with an alanine residue; (B) Two modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with a threonine residue; (C) Three modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue and substitution of the 70th amino acid residue with valine, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with an alanine residue; or (D) Three modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue and substitution of the 70th amino acid residue with valine, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with a threonine residue.

[0076] As for the modification of the above amino acid residue, (C) above is preferred.

[0077] The antibody or antibody fragment of this embodiment is preferably one selected from (1) to (4) below: (1) VH is VH containing the amino acid sequence represented by SEQ ID NO: 3, and VL is VL containing the amino acid sequence represented by SEQ ID NO: 4; (2) VH is VH containing the amino acid sequence represented by SEQ ID NO: 7, and VL is VL containing the amino acid sequence represented by SEQ ID NO: 8; (3) VH is VH containing the amino acid sequence represented by SEQ ID NO: 5, and VL is VL containing the amino acid sequence represented by SEQ ID NO: 6; or (4) VH is VH containing the amino acid sequence represented by SEQ ID NO: 9, and VL is VL containing the amino acid sequence represented by SEQ ID NO: 10.

[0078] In this embodiment, the antibody or antibody fragment comprising (3) above is more preferable.

[0079] The antibody or antibody fragment of this embodiment is preferably one selected from (5) to (8) below: (5) VH is VH consisting of the amino acid sequence represented by SEQ ID NO: 3, and VL is VL consisting of the amino acid sequence represented by SEQ ID NO: 4; (6) VH is VH consisting of the amino acid sequence represented by SEQ ID NO: 7, and VL is VL consisting of the amino acid sequence represented by SEQ ID NO: 8; (7) VH is VH consisting of the amino acid sequence represented by SEQ ID NO: 5, and VL is VL consisting of the amino acid sequence represented by SEQ ID NO: 6; or (8) VH is VH consisting of the amino acid sequence represented by SEQ ID NO: 9, and VL is VL consisting of the amino acid sequence represented by SEQ ID NO: 10.

[0080] In this embodiment, the antibody or antibody fragment comprising (7) above is more preferable.

[0081] The heavy chain constant region (CH) of the antibody in this embodiment is not particularly limited, but subclasses belonging to the IgG class, γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4), are preferred. From the viewpoint of stability and ease of preparation, the heavy chain constant region (CH) of the antibody or antibody fragment in this embodiment is preferably the IgG1 or IgG4 subclass. When the CH of the antibody in this embodiment is the IgG1 subclass, it is preferable that the amino acid sequence of the heavy chain constant region of the antibody includes the amino acid residue substitutions L234A, L235A, and G237A, which are represented by the EU index. When the CH of the antibody in this embodiment is the IgG1 subclass, it is also preferable that the amino acid sequence of the heavy chain constant region of the antibody includes the amino acid residue substitutions L234A, L235A, and P329G, which are represented by the EU index. Furthermore, when the CH of the antibody in this embodiment is an IgG4 subclass, it is preferable that the amino acid sequence of the heavy chain constant region of the antibody includes the amino acid residue substitutions S228P, L235E, and R409K represented by the EU index, and more preferably includes the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14, or consists of the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14.

[0082] The antibody or antibody fragment of this embodiment is preferably an antibody or antibody fragment that includes any one selected from (i) to (v) below: (i) VH containing the amino acid sequence represented by SEQ ID NO: 3, VL containing the amino acid sequence represented by SEQ ID NO: 4, and CH containing the amino acid sequence represented by SEQ ID NO: 13; (ii) VH containing the amino acid sequence represented by SEQ ID NO: 5, VL containing the amino acid sequence represented by SEQ ID NO: 6, and CH containing the amino acid sequence represented by SEQ ID NO: 13; (iii) VH containing the amino acid sequence represented by SEQ ID NO: 7, VL containing the amino acid sequence represented by SEQ ID NO: 8, and CH containing the amino acid sequence represented by SEQ ID NO: 14; (iv) VH containing the amino acid sequence represented by SEQ ID NO: 9, VL containing the amino acid sequence represented by SEQ ID NO: 10, and CH containing the amino acid sequence represented by SEQ ID NO: 14; or (v) VH containing the amino acid sequence represented by SEQ ID NO: 5, VL containing the amino acid sequence represented by SEQ ID NO: 6, and CH containing the amino acid sequence represented by SEQ ID NO: 14.

[0083] The antibody or antibody fragment of this embodiment is more preferably (v) described above.

[0084] The antibody or antibody fragment of this embodiment is preferably an antibody or antibody fragment comprising any one selected from (vi) to (X) below: (vi) VH consisting of the amino acid sequence represented by SEQ ID NO: 3, VL consisting of the amino acid sequence represented by SEQ ID NO: 4, and CH consisting of the amino acid sequence represented by SEQ ID NO: 13; (vii) VH consisting of the amino acid sequence represented by SEQ ID NO: 5, VL consisting of the amino acid sequence represented by SEQ ID NO: 6, and CH consisting of the amino acid sequence represented by SEQ ID NO: 13; (viiii) VH consisting of the amino acid sequence represented by SEQ ID NO: 7, VL consisting of the amino acid sequence represented by SEQ ID NO: 8, and CH consisting of the amino acid sequence represented by SEQ ID NO: 14; (IX) VH consisting of the amino acid sequence represented by SEQ ID NO: 9, VL consisting of the amino acid sequence represented by SEQ ID NO: 10, and CH consisting of the amino acid sequence represented by SEQ ID NO: 14; or (X) VH consisting of the amino acid sequence represented by SEQ ID NO: 5, VL consisting of the amino acid sequence represented by SEQ ID NO: 6, and CH consisting of the amino acid sequence represented by SEQ ID NO: 14.

[0085] The antibody or antibody fragment of this embodiment is more preferably (X) as described above.

[0086] The antibody or antibody fragment of this embodiment may be chemically or genetically engineered to be conjugated with a radioisotope, a low molecular weight drug, a high molecular weight drug, a protein, or an antibody drug, in the form of an antibody or a derivative of the antibody fragment.

[0087] Antibodies or derivatives of antibody fragments can be produced by chemically attaching radioisotopes, low-molecular-weight drugs, high-molecular-weight drugs, immunostimulants, proteins, antibody drugs, or nucleic acid drugs to the N-terminus or C-terminus of the H chain or L chain of the antibody or antibody fragment of this embodiment, or to appropriate substituents, side chains, or sugar chains in the antibody molecule, etc. [Introduction to Antibody Engineering, Chijin Shokan (1994)].

[0088] Furthermore, the antibody of this embodiment can be produced by a genetic engineering method in which the DNA encoding the antibody or antibody fragment of this embodiment is linked to the DNA encoding the protein or antibody drug to be bound, the DNA is inserted into an expression vector, and the expression vector is introduced into a suitable host cell for expression.

[0089] Examples of radioactive isotopes include, １１１ In, １３１ I, １２５ I, ９０ Y, ６４ Cd, ９９ Tc, ７７ Lu or ２１１ Examples include At. Radioactive isotopes can be directly bound to antibodies by methods such as the chloramine T method. Alternatively, a substance that chelates radioactive isotopes may be bound to the antibody. Examples of chelating agents include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).

[0090] For small molecule drugs, known small molecule drugs can be appropriately selected and used depending on the application.

[0091] Methods for conjugating low-molecular-weight drugs with antibodies include, for example, methods that link the amino groups of the drug and the antibody via glutaraldehyde, or methods that link the amino group of the drug with the carboxyl group of the antibody via water-soluble carbodiimide.

[0092] Examples of polymeric drugs include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, or hydroxypropyl methacrylamide. By conjugating these polymeric compounds to antibodies or antibody fragments, effects such as (1) improved stability against various chemical, physical, or biological factors, (2) a significant extension of the blood half-life, or (3) elimination of immunogenicity or suppression of antibody production can be expected [Bioconjugate drugs, Hirokawa Shoten (1993)].

[0093] For example, one method for conjugating PEG with an antibody is to react it with a PEG modification reagent [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)]. Examples of PEG modification reagents include a modifier for the ε-amino group of lysine (Japanese Patent Publication No. 61-178926), a modifier for the carboxyl groups of aspartic acid and glutamic acid (Japanese Patent Publication No. 56-23587), or a modifier for the guanidino group of arginine (Japanese Patent Publication No. 2-117920).

[0094] Immunostimulants can also be natural products known as immunojuvants. Specific examples include immune-enhancing drugs such as β(1→3) glucans (e.g., lentinan or schizophyllan) or α-galactosylceramide (KRN7000).

[0095] Examples of proteins include cytokines, growth factors, or toxic proteins that activate immune cells such as NK cells, macrophages, or neutrophils.

[0096] Examples of cytokines or growth factors include interferon (hereinafter referred to as IFN)-α, IFN-β, IFN-γ, interleukin (hereinafter referred to as IL)-2, IL-12, IL-15, IL-18, IL-21, IL-23, granulocyte colony-stimulating factor (G-CSF), granulocyte / macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF). Examples of toxin proteins include lysine, diphtheria toxin, or ONTAK, and also include protein toxins in which mutations have been introduced into the protein to regulate toxicity.

[0097] Examples of antibody drugs include antibodies against antigens that induce apoptosis upon antibody binding, antigens involved in tumor pathogenesis, antigens that regulate immune function, or antigens involved in angiogenesis at lesion sites.

[0098] Nucleic acid drugs include, for example, pharmaceuticals containing nucleic acids such as small interface ribbonic acid (siRNA) or microRNA that act on living organisms by controlling gene function. For example, conjugate with a nucleic acid drug that suppresses the master transcription factor RORγt of Th17 cells is a possibility.

[0099] When the antibody of this embodiment or a derivative of the antibody fragment is used for the detection and measurement of human TPOR, the agent that binds to the antibody is a label used in conventional immunological detection or measurement methods. Examples of labeling agents include enzymes such as alkaline phosphatase, peroxidase, or luciferase, luminescent substances such as acridinium ester or rofin, or fluorescent substances such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).

[0100] Furthermore, the pharmaceutical composition of this embodiment includes the antibody of this embodiment or a fragment of the antibody. Examples of the form of the pharmaceutical composition include a pharmaceutical composition containing a monoclonal antibody that binds to human TPOR or a fragment of the antibody as an active ingredient. The pharmaceutical composition containing the antibody of this embodiment or a fragment of the antibody may be a therapeutic composition that can be used to treat thrombocytopenia or pancytopenia.

[0101] Furthermore, this embodiment relates to a method for treating thrombocytopenia or pancytopenia, which includes administering the antibody or antibody fragment of this embodiment.

[0102] In this specification, thrombocytopenia refers to a condition in which the number of platelets in the blood is reduced, for example, a condition in which the platelet count is 100,000 / μL or less. In this specification, pancytopenia refers to a condition in which all blood cell components, including red blood cells, white blood cells, and platelets, are reduced, for example, a condition in which a hemoglobin concentration of less than 12.0 g / dL for men and less than 11.0 g / dL for women, a white blood cell count of less than 4,000 / μL, and a platelet count of less than 100,000 / μL are observed simultaneously.

[0103] The antibody or antibody fragment of this embodiment can treat the above-mentioned diseases by the following mechanisms of action: - It increases platelets by binding to human TPOR on megakaryocyte progenitor cells and exerting agonist activity. - It promotes the proliferation and differentiation of megakaryocyte progenitor cells, bone marrow hematopoietic stem cells, and hematopoietic progenitor cells by binding to human TPOR and exerting agonist activity.

[0104] Therefore, it is preferable that the antibody or antibody fragment of this embodiment has strong agonist activity.

[0105] In this specification, thrombocytopenia or pancytopenia refers to, for example, aplastic anemia, myelodysplastic syndrome (MDS), cytopenia after hematopoietic stem cell transplantation (e.g., bone marrow transplantation, umbilical cord blood transplantation, peripheral blood stem cell transplantation), thrombocytopenia associated with autoimmune diseases (e.g., systemic lupus erythematosus, immune thrombocytopenia (ITP), aplastic anemia), chemotherapy-induced thrombocytopenia (CIT), hematopoietic failure after CAR-T therapy, acute radiation syndrome, and thrombocytopenia associated with liver diseases (e.g., cirrhosis, idiopathic portal hypertension).

[0106] The therapeutic composition of this embodiment, which contains the antibody or antibody fragment, may contain only the antibody or antibody fragment as the active ingredient, but it is generally preferable to mix it with one or more pharmacologically acceptable carriers and provide it as a pharmaceutical preparation manufactured by any method known in the art of pharmaceutical formulation.

[0107] The preferred route of administration is the one that is most effective for treatment, and includes oral administration, or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, or intravenous administration, with intravenous administration being preferred. Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.

[0108] The dosage or frequency of administration varies depending on the desired therapeutic effect, method of administration, duration of treatment, age, and weight, but is usually 10 μg / kg to 10 mg / kg per day for adults.

[0109] Examples of the pharmaceutical composition of this embodiment include a reagent for detecting or measuring human TPOR containing an antibody that binds to human TPOR or a fragment of said antibody. This embodiment also relates to a method for detecting or measuring human TPOR using an antibody that binds to human TPOR or a fragment of said antibody. In this embodiment, any known method can be used to detect or measure human TPOR. For example, immunological detection or measurement methods can be used.

[0110] Immunological detection or measurement methods are methods for detecting or measuring the amount of antibody or antigen using labeled antigens or antibodies. Examples of immunological detection or measurement methods include radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay, Western blotting, or physicochemical methods.

[0111] In this embodiment, the biological sample to be used for detecting or measuring human TPOR is not particularly limited, as long as it may contain human TPOR, such as tissue, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0112] The use of this embodiment relates to the use of the antibody or antibody fragment of this embodiment for the manufacture of compositions for the treatment of thrombocytopenia or pancytopenia.

[0113] The following describes in detail the method for producing antibodies and the method for treating diseases according to this embodiment.

[0114] 1. Method for producing the antibody The method for producing the antibody or antibody fragment of this embodiment includes culturing transformed cells containing a vector having a nucleic acid having a base sequence encoding the antibody or antibody fragment of this embodiment in a culture medium, and collecting the antibody or antibody fragment from the culture.

[0115] (1) Preparation of the antigen Human TPOR to be used as an antigen can be obtained by introducing an expression vector containing cDNA encoding the full length or partial length of human TPOR into E. coli, yeast, insect cells, or animal cells. Human TPOR can also be obtained by purifying human TPOR from various human cell lines, human cells, and human tissues that express large amounts of human TPOR. These human cell lines, human cells, and human tissues can also be used as antigens as they are. Furthermore, synthetic peptides having a partial sequence of human TPOR can be prepared by chemical synthesis methods such as the Fmoc method or the tBoc method and used as antigens. Human TPOR or synthetic peptides having a partial sequence of human TPOR may have known tags such as FLAG or His attached to the C-terminus or N-terminus.

[0116] The human TPOR used in this embodiment can be produced by expressing the DNA encoding the human TPOR in a host cell using methods such as those described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) and Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), for example, by the following method.

[0117] First, a recombinant vector is prepared by inserting a full-length cDNA containing the portion encoding human TPOR downstream of the promoter of a suitable expression vector. Instead of the full-length cDNA, a DNA fragment of appropriate length containing the polypeptide-encoding portion, prepared based on the full-length cDNA, may be used. Next, the obtained recombinant vector can be introduced into host cells compatible with the expression vector to obtain a transformant that produces polypeptides.

[0118] Any expression vector can be used as long as it is capable of autonomous replication or integration into the chromosome of the host cell in which it is used, and contains a suitable promoter at a position where the polypeptide-coding DNA can be transcribed. Any host cell capable of expressing the target gene can be used, such as microorganisms belonging to the Escherichia genus, such as E. coli, yeast, insect cells, or animal cells.

[0119] When using prokaryotes such as E. coli as host cells, the recombinant vector is preferably capable of autonomous replication in the prokaryote and contains a promoter, a ribosome-binding sequence, DNA containing a portion encoding human TPOR, and a transcription termination sequence. While a transcription termination sequence is not strictly necessary in the recombinant vector, it is preferable to place it directly below the structural gene. Furthermore, the recombinant vector may also contain a gene that controls the promoter.

[0120] As the recombinant vector, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also called the SD sequence), which is a ribosome-binding sequence, and the start codon has been adjusted to an appropriate distance (for example, 6 to 18 bases).

[0121] Furthermore, the base sequence of the DNA encoding the human TPOR can be modified by substituting bases to create codons that are optimal for expression within the host, thereby improving the production rate of the target human TPOR.

[0122] Any expression vector that can function in the host cells used can be used, for example, pBTrp2, pBTac1, pBTac2 (all from Roche Diagnostics), pKK233-2 (from Pharmacia), pSE280 (from Invitrogen), pGEMEX-1 (from Promega), pQE-8 (from Qiagen), pKYP10 (Japanese Patent Publication No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306]. (1985)], pBluescript II SK(-) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), Japanese Patent Publication No. 60-221091], pGKA2 [Escherichia coli IGKA2 (FERM Examples include BP-6798 (prepared from BP-6798, Japanese Patent Publication No. 60-221091), pTerm2 (U.S. Patent Nos. 4,686,191, 4,939,094, and 160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), or pME18SFL3.

[0123] Any promoter that can function in the host cell being used is acceptable. Examples include promoters derived from E. coli or phages, such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter. Other examples include artificially designed and modified promoters, such as a tandem promoter with two Ptrp promoters in series, the tac promoter, the lacT7 promoter, or the let I promoter.

[0124] Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, or E. coli DH5α.

[0125] Any method for introducing recombinant vectors into host cells that involves introducing DNA into the host cells can be used, such as the method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

[0126] When using animal cells as a host, any expression vector that can function in animal cells can be used, for example, pcDNAI, pCDM8 (Funakoshi Corporation), pAGE107 [Japanese Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Publication No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 1307]. Examples include (1987), pAGE210, pME18SFL3, pKANTEX93 (International Publication No. 97 / 10354), N5KG1val (U.S. Patent No. 6,001,358), INPEP4 (manufactured by Biogen-IDEC), and transposon vectors (International Publication No. 2010 / 143698).

[0127] Any promoter capable of functioning in animal cells can be used, such as the promoter of the cytomegalovirus (CMV) immediate early (IE) gene, the SV40 early promoter, retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters, or Moloney mouse leukemia virus promoters or enhancers. Additionally, the enhancer of the human CMV IE gene may be used in conjunction with the promoter.

[0128] Examples of host cells include human leukemia cells (Namalwa cells), monkey cells (COS cells), and Chinese hamster ovary cells (CHO cells) [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix] I, II (pp. 883-900), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) (CHO / DG44 cells) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), Pro-3, rat myeloma cells YB2 / 3HL. P2. G11.16Ag. Examples include 20 (also known as YB2 / 0), mouse myeloma cells NSO, mouse myeloma cells SP2 / 0-Ag14, Syrian hamster cells BHK or HBT5637 (Japanese Patent Publication No. 63-000299).

[0129] Any method for introducing recombinant vectors into host cells that can introduce DNA into animal cells can be used, such as electroporation [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Publication No. 2-227075), or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

[0130] Human TPOR can be produced by culturing a transformant derived from a microorganism or animal cell, etc., that possesses a recombinant vector incorporating the DNA encoding human TPOR obtained as described above, in a culture medium, generating and accumulating the human TPOR in the culture medium, and then collecting it from the culture medium. The method for culturing the transformant in a culture medium can be carried out according to the usual method used for culturing the host.

[0131] When expressed in eukaryotic cells, human TPOR with added sugars or sugar chains can be obtained.

[0132] When culturing microorganisms transformed with recombinant vectors using inducible promoters, inducers may be added to the culture medium as needed. For example, when culturing microorganisms transformed with recombinant vectors using the lac promoter, isopropyl-β-D-thiogalactopyranoside may be added to the culture medium, and when culturing microorganisms transformed with recombinant vectors using the trp promoter, indoleacrylic acid may be added to the culture medium.

[0133] Examples of culture media for transformants obtained using animal cells as hosts include commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)], or Iscove's Modified Dulbecco's Medium (IMDM) medium, or media to which fetal bovine serum (FBS) or the like has been added. Culture is usually carried out at pH 6-8, 30-40°C, and 5% CO2. ２ The culture is carried out for 1 to 7 days under conditions such as the presence of antibiotics. In addition, antibiotics such as kanamycin or penicillin may be added to the culture medium as needed during the culture.

[0134] Methods for expressing the gene encoding human TPOR include, for example, direct expression, secretory production, or fusion protein expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)].

[0135] Methods for producing human TPOR include, for example, producing it inside a host cell, secreting it outside a host cell, or producing it on the host cell's outer membrane. By changing the host cell used or the structure of the human TPOR produced, an appropriate method can be selected.

[0136] When human TPOR is produced inside or on the host cell membrane, it can be actively secreted outside the host cell by using methods such as those described by Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], Lowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], Japanese Patent Publication No. 05-336963, or International Publication No. 94 / 23021. Furthermore, the production of human TPOR can be increased using gene amplification systems (Japanese Patent Publication No. 2-227075) that utilize the dihydrofolate reductase gene.

[0137] The obtained human TPOR can be isolated and purified, for example, as follows: If human TPOR is expressed in a lysed state within the cells, the cells are collected by centrifugation after the culture is complete, suspended in an aqueous buffer, and then the cells are disrupted using an ultrasonic disruptor, French press, Manton Gaurine homogenizer, or Dynomil to obtain a cell-free extract. From the supernatant obtained by centrifugation of the cell-free extract, a purified standard can be obtained using conventional protein isolation and purification methods, namely solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing, either alone or in combination.

[0138] If human TPOR is expressed in the form of an insoluble form within cells, the cells are collected and lysed as described above, and the insoluble form of human TPOR is recovered as a precipitate fraction by centrifugation. The recovered insoluble form of human TPOR is solubilized with a protein denaturant. After the human TPOR is restored to its normal three-dimensional structure by diluting or dialyzing the solubilized solution, a purified polypeptide preparation can be obtained by the isolation and purification method described above.

[0139] When human TPOR or its glycosylated derivatives are secreted extracellularly, the human TPOR or its glycosylated derivatives can be recovered in the culture supernatant. A soluble fraction can be obtained by processing the culture using methods such as centrifugation as described above, and a purified sample can be obtained from the soluble fraction by using the same isolation and purification method as described above.

[0140] Furthermore, the human TPOR used in this embodiment can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method. It can also be chemically synthesized using peptide synthesizers from companies such as Advanced Chemtech, PerkinElmer, Pharmacia, Protein Technology Instruments, Synthecel-Vega, Perceptive, or Shimadzu Corporation.

[0141] (2) Immunization of animals and preparation of antibody-producing cells for fusion Immunizing animals such as mice, rats, or hamsters between 3 and 20 weeks of age with the antigen obtained in (1), and collecting antibody-producing cells from the spleen, lymph nodes, and peripheral blood of the animals. Alternatively, mouse TPOR knockout mice can be used as immunized animals.

[0142] Immunization is performed by administering the antigen subcutaneously, intravenously, or intraperitoneally to the animal, for example, Freund's complete adjuvant, or an appropriate adjuvant such as aluminum hydroxide gel with Bordetella pertussis vaccine. If the antigen is a partial peptide, a conjugate is prepared with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limpet hemocyanin), and this is used as the immunogen.

[0143] Antigen administration is performed 5 to 10 times at intervals of 1 to 2 weeks after the initial administration. Blood is collected from the retinal venous plexus 3 to 7 days after each administration, and the antibody titer of the serum is measured using enzyme immunoassay [Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] or similar methods. Animals whose serum shows a sufficient antibody titer against the antigen used for immunization are used as the source of antibody-producing cells for fusion.

[0144] Three to seven days after the final administration of the antigen, tissues containing antibody-producing cells, such as the spleen, are excised from the immunized animals, and the antibody-producing cells are collected. When using spleen cells, the spleen is shredded and loosened, then centrifuged, and red blood cells are removed to obtain antibody-producing cells for fusion.

[0145] (3) Preparation of myeloma cells: As myeloma cells, cell lines obtained from mice were used, for example, 8-azaguanine-resistant mouse (BALB / c-derived) myeloma cell lines P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1 / 1-Ag41 (NS-1) [European J. Immunology, 6, 511 (1976)], SP2 / 0-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)] or P3-X63-Ag8 (X63) [Nature, 256, 495 Examples such as (1975) are used.

[0146] The myeloma cells were passaged in normal medium (RPMI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine), then passaged back into normal medium 3-4 days before cell fusion, and 2 x 10⁶ cells were collected on the day of fusion. ７ Ensure a minimum number of cells.

[0147] (4) Preparation of cell fusion and monoclonal antibody-producing hybridomas Wash the antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) thoroughly with Minimum Essential Medium (MEM) medium or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter distilled water, pH 7.2), mix so that the cell count is fusion antibody-producing cells: myeloma cells = 5 to 10:1, centrifuge, and remove the supernatant. After thoroughly loosening the precipitated cell population, add a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium, and dimethyl sulfoxide at 37°C while stirring. Add 1 to 2 mL of MEM medium several times at 1 to 2 minutes intervals, then add MEM medium until the total volume is 50 mL. Centrifuge, and remove the supernatant. After gently loosening the precipitated cell population, the antibody-producing cells for fusion are gently suspended in HAT medium [normal medium supplemented with hypoxanthine, thymidine, and aminopterin]. This suspension is then mixed with 5% CO2. ２ Incubate in an incubator at 37°C for 7 to 14 days.

[0148] After culturing, a portion of the culture supernatant is removed, and a group of cells that react to antigens containing human TPOR but not to antigens that do not human TPOR is selected using hybridoma selection methods such as the binding assay described later. Next, cloning is performed using the limiting dilution method, and those that show stable and strong antibody titers are selected as monoclonal antibody-producing hybridomas.

[0149] (5) Preparation of purified monoclonal antibody Eight-to-ten-week-old mice or nude mice that have been treated with pristane [administered 0.5 mL of 2,6,10,14-tetramethylpentadecane (Pristane) intraperitoneally and reared for two weeks] are injected intraperitoneally with the monoclonal antibody-producing hybridoma obtained in (4). The hybridoma will develop ascites cancer in 10-21 days. Ascites fluid is collected from these mice, centrifuged to remove solids, salted out with 40-50% ammonium sulfate, and purified by caprylic acid precipitation, DEAE-Sepharose column, protein A-column, or gel filtration column to collect the IgG or IgM fraction and obtain the purified monoclonal antibody.

[0150] Alternatively, the monoclonal antibody-producing hybridomas obtained in (4) can be cultured in RPMI1640 medium supplemented with 10% FBS, the supernatant can be removed by centrifugation, and the cells can be suspended in Hybridoma SFM medium and cultured for 3 to 7 days. The resulting cell suspension can be centrifuged, and the supernatant can be purified using a protein A-column or protein G-column to collect the IgG fraction and obtain purified monoclonal antibodies. Note that 5% Daigo GF21 can also be added to the Hybridoma SFM medium.

[0151] Antibody subclass determination is performed by enzyme immunoassay using a subclustering kit. Protein quantity is calculated using the Lowry method or absorbance at 280 nm.

[0152] (6) Selection of monoclonal antibodies The selection of monoclonal antibodies is performed by measuring the binding affinity of the antibody to human TPOR using ELISA, as shown below.

[0153] After dispensing human TPOR into a plate such as a 96-well plate, a test substance such as serum, hybridoma culture supernatant, or purified monoclonal antibody is dispensed as the first antibody and reacted. Next, the plate is thoroughly washed with PBS or the like, and then an anti-immunoglobulin antibody labeled with an enzyme reagent is dispensed as the second antibody and reacted. After that, the plate is thoroughly washed with PBS or the like, and then a substrate is added and the extinction coefficient of each well is measured with a plate reader to select a monoclonal antibody that reacts specifically with human TPOR.

[0154] 2. Production of Recombinant Antibodies The following describes methods for producing human-type chimeric antibodies and humanized antibodies as examples of genetically modified antibodies. Recombinant mouse antibodies, rat antibodies, and rabbit antibodies can also be produced using similar methods.

[0155] (1) Construction of Genetically Modified Antibody Expression Vectors Genetically modified antibody expression vectors are animal cell expression vectors into which DNA encoding the CH and CL of human antibodies is incorporated. These can be constructed by cloning the DNA encoding the CH and CL of human antibodies into the animal cell expression vector, respectively.

[0156] The C region of a human antibody can be any CH and CL from that human antibody. For example, the CH of the γ1 subclass and the CL of the κ class of a human antibody can be used. While cDNA is used for the DNA encoding the CH and CL of the human antibody, chromosomal DNA consisting of exons and introns can also be used. Any expression vector for animal cells that can incorporate and express the gene encoding the C region of a human antibody can be used. For example, pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], or pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)] may be used. Examples of promoters and enhancers among animal cell expression vectors include the initial promoter of SV40 [J. Biochem., 101, 1307 (1987)], Moloney mouse leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)], or the promoter [Cell, 41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)] of immunoglobulin H chains.

[0157] For recombinant antibody expression vectors, a type in which the antibody heavy chain (H) and light chain are present on the same vector (tandem type) [J. Immunol. Methods, 167, 271 (1994)] is used due to its ease of construction, ease of introduction into animal cells, and balance of the expression levels of the antibody heavy chain (H) and light chain (L) within animal cells. However, types in which the antibody heavy chain and light chain are present on separate vectors can also be used. Examples of tandem type recombinant antibody expression vectors include pKANTEX93 (International Publication No. 97 / 10354) and pEE18 [Hybridoma, 17, 559 (1998)].

[0158] (2) Obtaining cDNA encoding the V region of antibodies derived from non-human animals and analyzing the amino acid sequence Obtaining cDNA encoding the VH and VL regions of non-human antibodies and analyzing the amino acid sequence can be done as follows.

[0159] mRNA is extracted from hybridoma cells that produce non-human antibodies, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or plasmid to create a cDNA library. From this library, recombinant phages or recombinant plasmids containing cDNA encoding VH or VL are isolated, respectively, using DNA encoding the C or V region of a mouse antibody as a probe. The complete nucleotide sequences of VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid are determined, respectively, and the complete amino acid sequences of VH or VL are estimated from the nucleotide sequences, respectively.

[0160] Non-human animals used to produce hybridoma cells that produce non-human antibodies include mice, rats, hamsters, or rabbits, but any animal can be used as long as it is capable of producing hybridoma cells.

[0161] Total RNA can be prepared from hybridoma cells using the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], or kits such as the RNA Easy Kit (Qiagen).

[0162] mRNA can be prepared from total RNA using the oligo(dT) immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], or kits such as the Oligo-dT30<Super> mRNA Purification® Kit (Takara Bio Inc.). mRNA can also be prepared from hybridoma cells using kits such as the Fast Track mRNA Isolation® Kit (Invitrogen Inc.) or the QuickPrep mRNA Purification® Kit (Pharmacia Inc.).

[0163] For cDNA synthesis and cDNA library preparation, known methods [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)], or kits such as the SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Invitrogen) or the ZAP-cDNA Synthesis® Kit (Stratagene) are used.

[0164] When preparing a cDNA library, any vector capable of incorporating the cDNA synthesized using mRNA extracted from hybridoma cells as a template can be used. For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPII (Stratagene), λgt10, λgt11 [DNA Cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (Clonetech), λExCell, pT7T3-18U (Pharmacia), pCD2 [Mol. Cell. Biol., 3, 280 (1983)], or pUC18 [Gene, 33, 103 (1985)] are used.

[0165] Any E. coli capable of introducing, expressing, and maintaining a cDNA library constructed by a phage or plasmid vector can be used. For example, XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)], or JM105 [Gene, 38, 275 (1985)] can be used.

[0166] To select cDNA clones encoding VH or VL of non-human antibodies from a cDNA library, colony hybridization using isotopes or fluorescently labeled probes, or plaque hybridization [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], are used.

[0167] Alternatively, cDNA encoding VH or VL can be prepared by preparing primers and using cDNA synthesized from mRNA or a cDNA library as a template to perform Polymerase Chain Reaction (hereinafter referred to as PCR; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)).

[0168] The selected cDNA is cleaved with an appropriate restriction enzyme, cloned into a plasmid such as pBluescript SK(-) (Stratagene), and the base sequence of the cDNA is determined using commonly used sequencing methods. For example, the sequencing method involves performing a reaction such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], followed by the use of an automated sequencing analyzer such as the ABI PRISM3700 (PE Biosystems) or the A.L.F. DNA sequencer (Pharmacia).

[0169] The complete amino acid sequences of VH and VL are estimated from the determined base sequence and compared with the complete amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] to confirm whether the obtained cDNA encodes the complete amino acid sequences of VH and VL of antibodies containing the secretion signal sequence. Regarding the complete amino acid sequences of VH and VL of antibodies containing the secretion signal sequence, the length of the secretion signal sequence and the N-terminal amino acid sequence can be estimated by comparing them with the complete amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and furthermore, the subgroup to which they belong can be identified. In addition, the amino acid sequences of each CDR of VH and VL can also be found by comparing them with the amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].

[0170] Furthermore, the complete amino acid sequences of VH and VL obtained can be used to perform homology searches using methods such as BLAST [J. Mol. Biol., 215, 403 (1990)] against any database, such as SWISS-PROT or PIR-Protein, to confirm whether the complete amino acid sequences of VH and VL are novel.

[0171] (3) Construction of a human chimeric antibody expression vector or a human chimeric antibody variant expression vector A human chimeric antibody expression vector can be constructed by cloning cDNA encoding VH or VL of a non-human antibody upstream of each gene encoding CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1).

[0172] To link the 3' end of the cDNA encoding the VH or VL of a non-human antibody to the 5' end of the CH or CL of a human antibody, cDNAs for VH and VL are constructed, designed so that the nucleotide sequence of the linkage region encodes appropriate amino acids and is a suitable restriction enzyme recognition sequence. The constructed VH and VL cDNAs are then cloned upstream of the respective genes encoding the CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1) so that they are expressed in an appropriate manner, thereby constructing a human chimeric antibody expression vector.

[0173] Alternatively, cDNA encoding non-human antibodies VH or VL can be amplified by PCR using synthetic DNA having appropriate restriction enzyme recognition sequences at both ends, and then cloned into the recombinant antibody expression vector obtained in (1).

[0174] (4) Construction of cDNA encoding the V region of a humanized antibody The cDNA encoding the VH or VL region of a humanized antibody can be constructed as follows.

[0175] Select the amino acid sequence of the VH or VL FR of a human antibody for transplanting the CDR of the VH or VL of a non-human antibody. Any FR amino acid sequence derived from a human antibody can be used. For example, use the FR amino acid sequences of human antibodies registered in databases such as Protein Data Bank, or the common amino acid sequences of each subgroup of human antibody FRs [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. To minimize the decrease in antibody binding activity, select an FR amino acid sequence with as high homology as possible (at least 60%) to the original antibody's VH or VL FR amino acid sequence.

[0176] Next, the amino acid sequence of the CDR of the original antibody is transplanted into the FR amino acid sequence of the VH or VL of the selected human antibody, respectively, to design the VH or VL amino acid sequence of the humanized antibody. The designed amino acid sequence is converted into a DNA sequence, taking into account the frequency of codon usage found in the base sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], to design the DNA sequence that encodes the VH or VL amino acid sequence of the humanized antibody, respectively.

[0177] Based on the designed DNA sequence, several synthetic DNA strands, each approximately 100 base pairs long, are synthesized, and these are used to perform a PCR reaction. In this case, considering the reaction efficiency in the PCR reaction and the length of DNA that can be synthesized, it is preferable to design six synthetic DNA strands each for VH and VL. Furthermore, by introducing appropriate restriction enzyme recognition sequences to the 5' or 3' ends of the synthetic DNA located at both ends, the cDNA encoding the VH or VL of the humanized antibody can be easily cloned into the recombinant antibody expression vector obtained in (1).

[0178] After the PCR reaction, the amplified products are cloned into plasmids such as pBluescript SK(-) (Stratagene), and the base sequences are determined by the same method as described in (2) to obtain plasmids having a DNA sequence encoding the VH or VL amino acid sequence of the desired humanized antibody.

[0179] Alternatively, based on the designed DNA sequence, the full-length VH and VL can each be synthesized as a single long-chain DNA and used in place of the PCR amplification product described above. Furthermore, by introducing appropriate restriction enzyme recognition sequences to both ends of the synthesized long-chain DNA, the cDNA encoding the VH or VL of the humanized antibody can be easily cloned into the recombinant antibody expression vector obtained in (1).

[0180] (5) Modification of the amino acid sequence of the V region of humanized antibodies. When only the CDR of the VH and VL of a non-human antibody is transplanted to the FR of the VH and VL of a human antibody, the antigen-binding activity of the human antibody is reduced compared to the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)]. In humanized antibodies, the reduced antigen-binding activity can be increased by identifying amino acid residues in the amino acid sequences of the VH and VL FR of the human antibody that are directly involved in binding to the antigen, amino acid residues that interact with the amino acid residues of the CDR, and amino acid residues that maintain the three-dimensional structure of the antibody and are indirectly involved in binding to the antigen, and by substituting these amino acid residues with the amino acid residues of the original non-human antibody.

[0181] To identify the amino acid residues of the frequens group (FR) involved in antigen-binding activity, the three-dimensional structure of antibodies can be constructed and analyzed using methods such as X-ray crystallography [J. Mol. Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)]. Furthermore, by repeatedly creating several modified antibodies for each antibody and examining their correlation with their respective antigen-binding activities, a humanized antibody with the required antigen-binding activity can be obtained through trial and error.

[0182] The FR amino acid residues of the VH and VL of human antibodies can be modified by performing the PCR reaction described in (4) using synthetic DNA for modification. The base sequence of the amplified product after the PCR reaction is determined by the method described in (2) to confirm that the desired modification has been made.

[0183] (6) Construction of a humanized antibody expression vector A humanized antibody expression vector can be constructed by cloning the cDNA encoding the VH or VL of the constructed recombinant antibody upstream of the respective genes encoding the CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1).

[0184] For example, when constructing the VH or VL of the humanized antibody obtained in (4) and (5), appropriate restriction enzyme recognition sequences are introduced to the 5' or 3' ends of the synthetic DNA located at both ends, thereby cloning them upstream of the respective genes encoding the CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1) in an appropriate manner.

[0185] Furthermore, when producing genetically modified antibodies such as the chimeric antibodies and humanized antibodies mentioned above, a vector for expressing VL-substituted chimeric antibodies can be constructed by creating an antibody expression vector in which the H chain (or VH) and L chain (or VL) derived from two different antibodies are rearranged.

[0186] (7) Transient expression of recombinant antibodies Transient expression of recombinant antibodies can be performed using the recombinant antibody expression vectors obtained in (3) and (6), or modified expression vectors thereof, to efficiently evaluate the antigen-binding activity of various chimeric antibodies and humanized antibodies produced.

[0187] Any host cell capable of expressing recombinant antibodies can be used as the host cell into which the expression vector is introduced, but for example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651] are used [Methods in Nucleic Acids Res., CRC press, 283 (1991)].

[0188] To introduce expression vectors into COS-7 cells, methods such as the DEAE-dextran method [Methods in Nucleic Acids Res., CRC press (1991)] or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] are used.

[0189] After introducing the expression vector, the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant are measured using enzyme immunoassay [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)].

[0190] (8) Obtaining a transformed cell strain that stably expresses a recombinant antibody and preparing the recombinant antibody A transformed cell strain that stably expresses a recombinant antibody can be obtained by introducing the recombinant antibody expression vector obtained in (3) and (6), or a modified expression vector thereof, into a suitable host cell. For introducing the expression vector into the host cell, electroporation [Japanese Patent Publication No. 2-257891, Cytotechnology, 3, 133 (1990)] or the like can be used.

[0191] Any host cell capable of expressing recombinant antibodies can be used as the host cell into which the recombinant antibody expression vector is introduced. For example, CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), and rat myeloma cells YB2 / 3HL. P2. G11.16Ag. The following cells are used: 20 (ATCC number: CRL1662, also known as YB2 / 0), mouse myeloma cells NS0, mouse myeloma cells SP2 / 0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cells (ATCC number: CRL1580), and CHO cells (CHO / DG44 cells) lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)].

[0192] Furthermore, host cells in which the activity of proteins such as enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose, proteins such as enzymes involved in glycosylation modification in which the 1-position of fucose is α-bonded to the 6-position of N-acetylglucosamine at the reducing end of N-glycosidic complex glycans, or proteins involved in the transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus is reduced or absent, such as CHO cells lacking the α1,6-fucosyltransferase gene (International Publication No. 2005 / 035586, International Publication No. 02 / 31140), or Lec13 cells that have acquired lectin resistance [Somatic Cell and Molecular Genetics, 12, 55 (1986)] can also be used.

[0193] After introducing the expression vector, transformed cells that stably express the recombinant antibody are selected by culturing them in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japanese Patent Publication No. 2-257891).

[0194] For animal cell culture, RPMI1640 medium (Invitrogen), GIT medium (Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (JRH Co., Ltd.), IMDM medium (Invitrogen), or Hybridoma SFM medium (Invitrogen), or any of these media with various additives such as FBS added, can be used. By culturing the resulting transformed cell line in the medium, recombinant antibodies are expressed and accumulated in the culture supernatant. The expression level and antigen-binding activity of the recombinant antibodies in the culture supernatant can be measured by ELISA or other methods. Furthermore, the expression level of recombinant antibodies produced by the transformed cell line can be increased by using a dhfr gene amplification system (Japanese Patent Publication No. 2-257891).

[0195] Recombinant antibodies are purified from the culture supernatant of transformed strains using a Protein A column [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Alternatively, a combination of methods used for protein purification, such as gel filtration, ion exchange chromatography, and ultrafiltration, can be employed.

[0196] The molecular weight of the H chain, L chain, or the entire antibody molecule of purified recombinant antibodies can be measured using polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

[0197] 3. Activity evaluation of purified monoclonal antibodies or antibody fragments The activity of purified monoclonal antibodies or antibody fragments can be evaluated as follows.

[0198] The binding activity of the antibody or antibody fragment of this embodiment to human TPOR can be measured using methods such as ELISA or surface plasmon resonance.

[0199] The strength of the agonist activity of the antibody or antibody fragment of this embodiment can be confirmed, for example, by measuring the proliferation rate of cells that express human TPOR and proliferate in a TPOR-dependent manner when cultured together with the antibody or antibody fragment of this embodiment. Specifically, the strength of the agonist activity can be measured by the method described later in the examples.

[0200] 4. Method of treating diseases using the anti-human TPOR antibody or antibody fragment of this embodiment. The antibody or antibody fragment of this embodiment can be used to treat thrombocytopenia or pancytopenia.

[0201] The therapeutic agent for thrombocytopenia or pancytopenia containing the antibody or antibody fragment of this embodiment may contain only the antibody or antibody fragment as the active ingredient, but is usually provided as a pharmaceutical preparation manufactured by mixing it with one or more pharmacologically acceptable carriers using methods known in the art of pharmaceutical formulation.

[0202] The preferred route of administration is the one that is most effective for treatment, such as oral administration or parenteral administration via the oral cavity, airway, rectum, subcutaneous, intramuscular, or intravenous route. Subcutaneous or intravenous administration is preferred as the route of administration. Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.

[0203] Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, or granules.

[0204] Liquid preparations such as emulsions or syrups are manufactured using water, sugars such as sucrose, sorbitol, or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil, or soybean oil, preservatives such as p-hydroxybenzoic acid esters, or flavors such as strawberry flavor or peppermint as additives.

[0205] Capsules, tablets, powders, or granules are manufactured using excipients such as lactose, glucose, sucrose, or mannitol; disintegrants such as starch or sodium alginate; lubricants such as magnesium stearate or talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, or gelatin; surfactants such as fatty acid esters; or plasticizers such as glycerin as additives.

[0206] Suitable formulations for parenteral administration include injections, suppositories, and sprays. Injections are manufactured using a carrier consisting of a salt solution, a glucose solution, or a mixture of both. Suppositories are manufactured using a carrier such as cocoa butter, hydrogenated fat, or carboxylic acid.

[0207] The spray formulation does not irritate the recipient's oral and respiratory tract mucosa and is manufactured using a carrier that disperses the monoclonal antibody or antibody fragments of this embodiment as fine particles, facilitating absorption. Examples of carriers include lactose or glycerin. It can also be manufactured as an aerosol or dry powder. Furthermore, the above parenteral formulation can also be formulated for oral administration and may contain the ingredients exemplified as additives.

[0208] 5. Method for detecting or measuring human TPOR using the antibody or antibody fragment of this embodiment. Human TPOR can be detected or measured using the antibody or antibody fragment of this embodiment.

[0209] For example, human TPOR present in a patient's body can be detected or measured using immunological methods.

[0210] Immunological methods are techniques for detecting or measuring antibody or antigen levels using labeled antigens or antibodies. Examples include radiolabeled immunoassay, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, Western blotting, or physicochemical methods.

[0211] The radiolabeled immunoassay method involves, for example, reacting an antigen or cells expressing an antigen with the antibody or antibody fragment of this embodiment, and then reacting it with a radiolabeled anti-immunoglobulin antibody or antibody fragment, followed by measurement using a scintillation counter or similar device.

[0212] Enzyme immunoassay involves reacting an antigen or cells expressing an antigen with the antibody or antibody fragment of this embodiment, then reacting it with an anti-immunoglobulin antibody or binding fragment labeled with an enzyme, and finally adding a substrate and measuring the absorbance of the reaction solution with a spectrophotometer. For example, the sandwich ELISA method can be used. As the label used in enzyme immunoassay, the enzyme labels known from [Enzyme Immunoassay, Igaku-Shoin (1987)] can be used.

[0213] For example, alkaline phosphatase labeling, peroxidase labeling, luciferase labeling, or biotin labeling are used. The sandwich ELISA method involves binding an antibody to a solid phase, trapping the antigen to be detected or measured, and then reacting the trapped antigen with a second antibody. In this ELISA method, two types of antibodies or antibody fragments that recognize the antigen to be detected or measured, each with a different antigen recognition site, are prepared. The first antibody or antibody fragment is adsorbed onto a plate (e.g., a 96-well plate) beforehand, and then the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin. After reacting the plate on which the antibodies are adsorbed with cells or their lysates, tissue or its lysates, cell culture supernatant, serum, pleural fluid, ascites, or ocular fluid isolated from a living organism, the labeled monoclonal antibody or antibody fragment is reacted, and a detection reaction is performed according to the labeling substance. The antigen concentration in the test sample is calculated from a calibration curve created by sequentially diluting antigens of known concentration. For the sandwich ELISA method, either a polyclonal antibody or a monoclonal antibody may be used, and the antibody may be Fab, Fab', or F(ab). ２ Antibody fragments such as those mentioned above may also be used. The combination of two antibodies used in the sandwich ELISA method may be a combination of monoclonal antibodies or antibody fragments that recognize different epitopes, or a combination of a polyclonal antibody and a monoclonal antibody or antibody fragment.

[0214] Immunofluorescence assays are performed using methods described in literature such as [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)]. For the label used in immunofluorescence assays, publicly known fluorescent labels [Immuofluorescence Method, Soft Science Co., Ltd. (1983)] can be used. For example, FITC or RITC can be used.

[0215] The luminescence immunoassay is performed using the method described in the literature [Bioluminescence and Chemiluminescence Clinical Tests 42, Hirokawa Shoten (1998)]. Examples of labels used in luminescence immunoassay include well-known luminescent labels such as acridinium esters or rofin.

[0216] Western blotting involves fractionating an antigen or antigen-expressing cells using SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel) [Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)], blotting the gel onto a polyvinylidene fluoride (PVDF) membrane or nitrocellulose membrane, reacting the membrane with an antibody or antibody fragment that recognizes the antigen, and then reacting it with an anti-mouse IgG antibody or conjugated fragment that has been labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin, and finally measuring the result by visualizing the label.

[0217] The physicochemical method involves, for example, forming aggregates by binding the antigen, human TPOR, to the monoclonal antibody or antibody fragment of this embodiment, and then detecting these aggregates. Other physicochemical methods that can be used include the capillary method, one-dimensional immunodiffusion, immunoturbidimetric method, or latex immunoturbidimetric method [Clinical Laboratory Methods Handbook, Kinbara Publishing (1998)]. In the latex immunoturbidimetric method, a carrier such as polystyrene latex with a particle size of about 0.1 to 1 μm, sensitized with an antibody or antigen, is used to induce an antigen-antibody reaction with the corresponding antigen or antibody. When this reaction occurs, the scattered light in the reaction solution increases and the transmitted light decreases. The antigen concentration in the test sample is measured by detecting this change as absorbance or integrating spheroidal turbidity.

[0218] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention.

[0219] The amino acid sequences of VH and VL of the anti-human thrombopoietin receptor antibody 7-10, described in International Publication No. 2007 / 108559, are given as Sequence ID No. 1 and Sequence ID No. 2, respectively.

[0220] In this specification, an antibody comprising VH having the amino acid sequence represented by SEQ ID NO: 1, VL having the amino acid sequence represented by SEQ ID NO: 2, and CH having the amino acid sequence represented by SEQ ID NO: 13 is described as 7-10_4344.

[0221] The amino acid sequence of CH represented by Sequence ID No. 13 (hereinafter referred to as 4344Nullbody type or 4344Nullbody) is an amino acid sequence in which the serine residue at EU index 228 of hIgG4 CH is replaced with a proline residue, the leucine residue at EU index 235 is replaced with a glutamic acid residue, and the arginine residue at EU index 409 is replaced with a lysine residue, and furthermore, it has the same modification of amino acid residues in the upper hinge region as described in Table 1. In addition, CH in which the serine residue at EU index 228 of hIgG4 CH is replaced with a proline residue, the leucine residue at EU index 235 is replaced with a glutamic acid residue, and furthermore, has the same modification of amino acid residues in the upper hinge region as 4344Nullbody described in Table 1 is referred to as 4344_uhm.

[0222]

[0223] In this example, a modified version of 7-10_4344 was prepared by altering at least one amino acid residue in the variable region and the heavy chain constant region (hereinafter referred to as the 7-10 modified version or simply the modified version).

[0224] In this embodiment, the name of the modified product is written in the following order: (heavy chain (abbreviated as hc) or light chain (abbreviated as lc)) (original amino acid residue) (modification location) (modified amino acid residue) _ (abbreviation for heavy chain constant region). The modification location refers to the amino acid numbers assigned sequentially from the N-terminus of the VH or VL amino acid sequence. If the modified product has two amino acid residue modifications, it is written as: (hc or lc) (original amino acid residue) (modification location) (modified amino acid residue) _ (hc or lc) (original amino acid residue) (modification location) (modified amino acid residue) _ (abbreviation for heavy chain constant region).

[0225] Here, the abbreviation for the heavy chain constant region IgG1 is G1, and the abbreviation for 4344 Nullbody is 4344.

[0226] For example, hcT28A_4344 refers to an antibody in which the 28th T from the N-terminus of the VH amino acid sequence represented by SEQ ID NO. 1 of 7-10_4344 is modified to A, and the CH is of the 4344Nullbody type. Similarly, lcI21A_G1 refers to an antibody in which the 21st I from the N-terminus of the VL amino acid sequence represented by SEQ ID NO. 2 of 7-10_4344 is modified to A, and the CH is hIgG1. Note that 7-10_hIgG1, 7-10G4344uhm, 4-49G4344uhm, and 4-49_4344, which will be discussed later, are names that do not follow the naming rules for the above-mentioned modified products.

[0227] [Example 1] Evaluation of physical properties of 7-10 antibody and preparation of modified 7-10 amino acid residues - 1 (1-1) Preparation of 7-10_hIgG1, 7-10_4344, and 4-49_4344 The amino acid sequence of VH of the anti-human thrombopoietin receptor antibody 4-49, described in International Publication No. 2007 / 108559, is described in SEQ ID NO: 11, and the VL is described in SEQ ID NO: 12.

[0228] 7-10hIgG1, 7-10_4344, and 4-49_4344 were prepared using the same method as described in (4-1)(a) below. 7-10hIgG1 is an antibody in which the amino acid sequence of the constant region of 7-10_4344 is replaced with the amino acid sequence of the constant region of hIgG1. 4-49_4344 is an antibody containing VH having the amino acid sequence represented by SEQ ID NO: 11, VL having the amino acid sequence represented by SEQ ID NO: 12, and CH having the amino acid sequence represented by SEQ ID NO: 13.

[0229] (1-2) Evaluation of physical properties The following experiment was performed using the antibodies prepared in (1-1). The increase in antibody aggregates after low pH treatment was measured using the same method as in (8) described later. The results obtained are shown in Figure 1. From Figure 1, the increase in aggregates after low pH treatment was approximately 19% for 7-10_4344, while it was approximately 6% for 4-49_4344. Since the amino acid sequences of CH in 7-10_4344 and 4-49_4344 are the same, it was shown that the formation of a large amount of aggregates in 7-10_4344 after low pH treatment is due to the amino acid sequence of the variable region of 7-10_4344.

[0230] Furthermore, the change in viscosity when the concentration of the antibody solution was increased was measured using the same method as described in (7) below, except that the antibody concentration used for viscosity measurement was changed. Approximate curves of the concentration-dependent viscosity change for each antibody were calculated using the Mooney equation. The obtained results are shown in Figure 2. As shown in Figure 2, the viscosity of 7-10_4344 increased significantly with increasing antibody solution concentration compared to 4-49_4344. Similar results were obtained for 7-10_hIgG1, indicating that the significant increase in viscosity with increasing antibody solution concentration observed in 7-10_4344 is due to the amino acid sequence of the variable region.

[0231] (2) Preparation of 7-10 variants In order to improve the physical properties of 7-10_4344 found in (1-1) and (1-2), 116 variants of 7-10_hIgG1, as shown in Table 2, were prepared by the method described below. The 7-10 variants shown in Table 2 include variants in which amino acid residues other than Al at specific sites are substituted with Al, and variants in which Al at specific sites is substituted with Val.

[0232]

[0233] Specifically, the base sequences encoding each variant listed in Table 2 were artificially synthesized, and antibody expression cassettes were prepared using a procedure similar to that described in Liao et al. (J Virol Methods (2009) 158(1-2): 171-179). Using these cassettes, genes were introduced into Expi293F cells using the Expi293™ Expression System (Thermo Fisher), and the culture supernatant of transiently expressing cells of each variant was obtained and used for subsequent activity evaluation.

[0234] (3) Evaluation of agonist activity The agonist activity of the 7-10 modified cells prepared in (2) above was evaluated. Specifically, a cell proliferation assay was performed using the following method with mouse granulocyte progenitor cell line FDCP cells expressing human MPL (hereinafter referred to as hMPL / FDCP).

[0235] The hMPL / FDCP culture medium was transferred to a 50 mL tube, and cells were collected by centrifugation (340 G, 5 min, 4°C). After removing the culture medium, the cells were washed twice with Iscove's Modified Dulbecco's Medium (hereinafter referred to as IMDM) (hereinafter referred to as IMDM(FBS,P / S)) supplemented with 10% (v / v) FBS and 1% Penicillin-Streptomycin (hereinafter referred to as P / S) (v / v). Subsequently, the cells were washed with fresh IMDM(FBS,P / S) until the cell density reached 1 × 10⁶. ５ The cells were suspended to a concentration of cells / mL. 50 μL of the cell suspension was seeded into each well of a 96-well plate, and then incubated at 37°C under 5% CO2. ２ The cells were incubated for approximately 4 hours under the specified conditions. Antibody solutions were prepared using IMDM (FBS, P / S) and the culture supernatant prepared in (2) above, so that the concentration of each variant was 200 ng / mL. 50 μL of the antibody solution was added to each well (final concentration 100 ng / mL), and incubated at 37°C and 5% CO2. ２ Cells were cultured for two days under the specified conditions. 100 μL of CellTiter-Glo 2.0 reagent was added to each well. After shaking in a shaker for two minutes, the 96-well plate was allowed to stand at room temperature for 10 minutes. Finally, the luminescence signal was measured using the EnVision microplate reader. The luminescence signal of each sample was subtracted from the luminescence signal of the IMDM (FBS, P / S) well without added cells, and the result was divided by the luminescence signal of the untreated sample to calculate the cell proliferation rate as a percentage.

[0236] A higher cell proliferation rate indicates stronger agonist activity.

[0237] Based on the results obtained, among the 7-10 variants that had cell proliferation rates equal to or better than 7-10_hIgG1, the variants listed in Table 3 were selected, and the relationship between physical properties and activity was evaluated in subsequent experiments (Table 3). Of these, hcL100A_G1 and lcY49A_G1 showed a significantly increased cell proliferation rate compared to 7-10_hIgG1. Therefore, it was shown that hcL100A_G1 and lcY49A_G1 have significantly stronger agonist activity than 7-10_hIgG1.

[0238]

[0239] Furthermore, among the 7-10 variants that showed a lower cell proliferation rate than 7-10_hIgG1, the variants listed in Table 4 were also evaluated in subsequent experiments to assess the relationship between their physical properties and activity.

[0240]

[0241] (4-1) Examination of the trend of improvement in physical properties To predict viscosity, affinity-capture self-interaction nanoparticle spectrum scopy (hereinafter referred to as AC-SINS) was performed. For evaluation by AC-SINS, modified compounds were prepared in which the CH of the 7-10 modified compounds described in Tables 3 and 4 was replaced with the 4344 Nullbody type.

[0242] (a) Preparation of modified compounds Specifically, first, the expression plasmids of each modified compound were introduced into host cells Expi293 in a flask by lipofection and transiently expressed. Subsequently, each modified compound was purified from the culture supernatant using a protein A column.

[0243] (b) Measurement of AC-SINS The gold colloid solution used for measuring AC-SINS was prepared by the following method. 200 μL of 0.4 mg / mL Goat IgG (ChromPure Goat IgG, whole molecular / Jackson ImmunoResearch / Cat. #005-000-003) dissolved in 20 mmol / L sodium acetate buffer (pH 4.3), 800 μL of 0.4 mg / mL Goat Anti-Human IgG (AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific / Jackson ImmunoResearch / Cat. #109-005-098), and Colloidal Gold (ted The mixture was divided into 9 mL portions of pella / Cat. #15705-20 and incubated in a 25°C bath for 2 hours. A syringe was attached to a 0.22 μm PVDF filter (MILLEX GV / MILLEX), and the gold colloid solution was passed through the filter to adsorb the gold colloid onto the filter. A new syringe was attached to the filter, and PBS was passed through the filter until the gold colloid concentration after buffer exchange was approximately 5 to 10 times, thereby eluting the gold colloid.

[0244] AC-SINS measurements were performed using the following method: The measurement sample diluted to 50 μg / mL with PBS or 90 μL / well of PBS was added to a 384-well plate (Fisherbrand), followed by the addition of 10 μL / well of gold colloid solution, and then mixed by pipetting. The mixture was incubated in a 25°C bath for 1.5 to 3 hours. A multiscan was performed using an Enspire (PerkinElmer) at wavelengths from 450 nm to 620 nm. The wavelength at which the absorbance was maximum was used, and the difference between the maximum absorbance wavelength of the measurement sample and the maximum absorbance wavelength of the PBS sample was calculated as Δλmax. A higher value of Δλmax was considered to indicate a higher viscosity risk.

[0245] (4-2) Evaluation of agonist activity The modified 7-10 prepared in (4-1)(a) above was subjected to a cell proliferation assay in the same manner as described in (3), and the agonist activity of each modified and 7-10_4344 was compared.

[0246] (4-3) Results Based on the results of (4-1) and (4-2) above, of the modified compounds evaluated, only two (lcY49A_4344 and hcL100A_4344) showed the desired effect of having a lower viscosity risk than 7-10_4344 and agonist activity equal to or greater than 7-10_4344. lcY49A_4344 and hcL100A_4344 showed significantly stronger agonist activity than 7-10_4344.

[0247] Other variants either (a) had a viscosity risk similar to or higher than 7-10_4344, or (b) had lower agonist activity than 7-10_4344, and did not exhibit the desired effect. The two amino acid residue modifications that produced the desired effect were subjected to further evaluation.

[0248] (5) Investigation of effects on human umbilical cord blood-derived cells lcY49A_4344 and hcL100A_4344, as well as a modified version hcL100A_lcY49A_4344 combining amino acid residue modifications of these two antibodies, were prepared in the same manner as in (4-1)(a) of Example 1. A negative control antibody (indicated as Negative Control in the figure) was prepared as an anti-dinitrophenyl (DNP) antibody with a nullbody CH (Motoki et al., Clin Cancer Res. 2005; 11: 3126-3135). Using human umbilical cord blood-derived hematopoietic stem / progenitor cell (HSPC), the following cell proliferation assays were performed to evaluate the agonist activity of 7-10_4344 and its modified versions.

[0249] Human umbilical cord blood-derived HSPCs are being processed by StemSpan. ＴＭ 5 × 10⁶ cells in Serum-Free Expansion Medium II (hereinafter referred to as SFEMII) medium (L-Gln, P / S). ４The cells were suspended to a concentration of cells / mL and seeded in a 96-well flat-bottom plate at 100 μL / well. The test substance, prepared to twice the final concentration in SFEMII medium (L-Gln, PS), was added at 100 μL / well and mixed. After static incubation at 37°C for 7 days, the number of HSPCs was counted. A higher count indicated stronger agonist activity. The results are shown in Figures 3A and 3B.

[0250] Figures 3A and 3B show that HSPCs proliferated at lower concentrations with hcL100A_4344, lcY49A_4344, and hcL100A_lcY49A_4344 than with 7-10_4344. This confirms that the agonist activity of hcL100A_4344, lcY49A_4344, and hcL100A_lcY49A_4344 is significantly enhanced compared to 7-10_4344 when evaluated using human umbilical cord blood-derived HSPC cells. In other words, it was confirmed that the amino acid residue modifications of hcL100A and lcY49A contribute to an increase in agonist activity.

[0251] (6) Examination of the trend of physical property improvement AC-SINS was measured for 7-10_4344 and three modified products (hcL100A_4344, lcY49A_4344, and hcL100A_lcY49A_4344) in the same manner as in (4-1)(b) of Example 1. As a result, it was confirmed that hcL100A_4344, lcY49A_4344, and hcL100A_lcY49A_4344 all had a lower viscosity risk than 7-10_4344.

[0252] (7) Physical Property Evaluation - 1 (Viscosity) To confirm the effect of improving the physical properties of the prepared modified product, the viscosity at an antibody concentration of 60 mg / mL was measured. Specifically, the viscosity (cP) of hcL100A_4344, lcY49A_4344, and hcL100A_lcY49A_4344 was measured using a Viscosizer TD (manufactured by Spectris Co., Ltd.). Under conditions of 25°C, the antibody was packed into a capillary cell under a constant pressure of 1000 mbar, and the antibody solution was detected at a wavelength of 280 nm or 254 nm as it passed through two windows in the capillary cell. The time difference at that time was measured by comparing it with the time difference of the corresponding buffer (10 mM glutamic acid, 262 mM D-sorbitol, pH 5.5) or water. The obtained results are shown in Figure 4.

[0253] As shown in Figure 4, as predicted from the AC-SINS evaluation results in (6) above, it was confirmed that all of the modified compounds had a lower viscosity than 7-10_4344. Therefore, it was confirmed that in 7-10_4344, the substitution of the 100th L residue from the N-terminus of the VH amino acid sequence with an A residue, and the substitution of the 49th Y residue from the N-terminus of the VL amino acid sequence with an A residue, are both amino acid residue modifications that contribute to the improvement of physical properties, namely the reduction in viscosity. It was also confirmed that a similar effect can be obtained by combining the two modifications.

[0254] (8) Physical property evaluation - 2 (Stability after low pH treatment) Next, the stability of the antibody after low pH treatment was evaluated. Specifically, a buffer consisting of 200 mmol / L Citric Acid buffer (pH 2.7) was added to 35 μL of antibody sample to adjust the pH to 3.5. After heating at 37°C for 60 minutes, the low pH treatment solution was neutralized by adding 500 mM Phosphate buffer (pH 8.0). The aggregate content after low pH treatment was measured using an ultra-high-performance liquid chromatography system (Shimadzu Corporation) and an ACQUITY UPLC Protein BEH SEC column (200 angstroms, 1.7 μm, 4.6 × 150 mm) (Waters). The measurement conditions were as follows: solvent was 50 mmol / L sodium phosphorate, 500 mmol / L NaCl, 5% EtOH (pH 6.8), flow rate was 0.4 mL / min, detection wavelength was 215 nm, column temperature was 25°C, and analysis time was 8 minutes. 5 μg of antibody was used. The elution times for aggregates (high molecular weight), monomers, and degradation products (low molecular weight) were approximately 2.0–3.3 minutes, 3.3–4.4 minutes, and 4.4–5.2 minutes, respectively. The peaks for antibody monomers, aggregates (high molecular weight), and degradation products (low molecular weight) were identified, and the aggregate content was calculated from the peak area of ​​each. Subsequently, the increase in aggregates after low pH treatment was calculated using the following formula. The obtained results are shown in Figure 5. Increase in aggregates (%) = [Agglutination content after low pH treatment (heating at 37°C for 60 minutes) (%)] - [Agglutination content before low pH treatment (%)]

[0255] As shown in Figure 5, the increase in aggregates after low pH treatment was approximately 23% for 7-10_4344 and lcY49A_4344, while it decreased to approximately 8% for hcL100A_4344 and hcL100A_lcY49A_4344. From the above, it was confirmed that in 7-10_4344, the substitution of the 100th L residue from the N-terminus to the A residue in the VH amino acid sequence suppresses the formation of aggregates after low pH treatment. In other words, the above amino acid residue modification is shown to be an amino acid residue modification that contributes to improving the physical property of the antibody, namely its stability after low pH treatment.

[0256] [Example 2] Preparation of 7-10 Modified Products - 2 Modified products of the 4344 Nullbody type (hcA23T_4344, hcA40V_4344, hcI70V_4344, hcL118T_4344, lcV104L_4344) were prepared in the same manner as in (4-1)(a) of Example 1.

[0257] The above modified product was evaluated using AC-SINS in the same manner as in Example 1 (4-1) (b) to confirm viscosity risk. Furthermore, a cell proliferation assay was performed in the same manner as in Example 1 (3) to confirm agonist activity.

[0258] AC-SINS measurements confirmed that all the modified compounds prepared in Example 2 had a viscosity risk equivalent to or higher than 7-10_4344. On the other hand, the agonist activity of all the modified compounds prepared in Example 2 was equivalent to or better than 7-10_4344, and among them, hcI70V_4344 showed enhanced agonist activity compared to 7-10_4344. Therefore, from the viewpoint of enhancing agonist activity, the 70th I residue of VH was selected as a promising modification site.

[0259] [Example 3] Optimization of the types of amino acid residues in the modified product (1) Preparation of the modified product With the aim of optimizing the types of amino acid residues after modification, substitution of amino acid residues other than A residues was considered for the amino acid residues after substitution of Y49 in VL and L100 in VH.

[0260] Specifically, in 7-10_4344, a modified compound in which the 49th Y residue of VL was replaced with a G, M, S, T, D, E, N, Q, R, K, H, or P residue (hereinafter also referred to as the lcY49 modified compound), and a modified compound in which the 100th L residue of VH was replaced with a G, M, S, T, D, E, N, Q, R, K, H, Y, or P residue (hereinafter also referred to as the hcL100 modified compound) were prepared by the method described in (4-1)(a) of Example 1.

[0261] (2) Evaluation of Agonist Activity Similar to (3) in Example 1, the agonist activity of the modified compounds was evaluated by a cell proliferation assay. All hcL100 modified compounds showed agonist activity equivalent to or less than 7-10_4344. hcL100G_4344 was used as a representative example of a modified compound showing agonist activity equivalent to 7-10_4344 for subsequent evaluations. For the lcY49 modified compounds, lcY49M_4344, lcY49S_4344, and lcY49T_4344, which showed stronger agonist activity than 7-10_4344, were used for subsequent evaluations. The other lcY49 modified compounds showed agonist activity equivalent to or less than 7-10_4344.

[0262] (3) Examination of the trend of physical property improvement AC-SINS was measured for the four modified compounds found in (2), lcY49M_4344, lcY49S_4344, lcY49T_4344, and hcL100G_4344, using the method described in (4-1)(b) of Example 1. As a result, it was confirmed that only hcL100G_4344 was a modified compound with a lower viscosity risk than 7-10_4344.

[0263] [Example 4] Optimization of combinations (1) Preparation of modified compounds From the results of the modified compound screening so far, it has become clear that in 7-10_4344, the physical properties can be improved by changing Y49 of VL to A, or by substituting L100 of VH with an A or G residue, and that agonist activity is enhanced by substituting Y49 of VL with an A, M, S or T residue, substituting L100 of VH with an A residue, and substituting I70 of VH with a V residue.

[0264] To investigate all possible combinations of these modification patterns, 7-10 variants were created by combining modifications to three amino acid residues: five residues at position 49 of VL (Y (original residue), A, M, S, and T residues), three residues at position 100 of VH (L (original residue), A, and G residues), and two residues at position 70 of VH (I (original residue), and V residue). For the constant region, two types were investigated: the 4344 Nullbody type and the Nullbody type (an amino acid sequence in which the serine residue at EU index 228 of hIgG4's CH amino acid sequence is replaced with a proline residue, the leucine residue at EU index 235 is replaced with a glutamic acid residue, and the arginine residue at 409 is replaced with a lysine residue). By combining all of these, 60 variants of the 7-10 were created. The variants were expressed in the same manner as in Example 1(2) and purified using a proteinA column.

[0265] (2) Evaluation of Agonist Activity The agonist activity of the prepared variants was evaluated using a cell proliferation assay with a human TPOR (hereinafter also referred to as "hTPOR")-dependent cell line, in the same manner as described in (3) of Example 1. After confirming improvements in physical properties (viscosity when antibody solution concentration is increased and aggregate formation after low pH treatment), modification of the 100th amino acid residue of VH was made mandatory, and the five variants with the highest agonist activity were selected. The names of the five variants, the 49th amino acid residue of VL, the 70th and 100th amino acid residues of VH, the name of CH, and the sequence numbers of each variable region are shown in Table 5.

[0266]

[0267] [Example 5] Higher-order evaluation The five modified compounds 3-29, 3-26, 3-50, 3-53, and 3-59 prepared in Example 4 were evaluated according to the following (1) to (4).

[0268] (1) Binding ability: The binding ability of each variant to hTPOR was evaluated by the SPR method. Specifically, Human Fab binder (containing the reagents from the Human Fab Capture Kit, anti-human kappa antibody, and anti-human lamdba antibody) was immobilized onto Sensor Chip CM5 using the Amine Coupleling Kit and Human Fab Capture Kit (Human Fab binder flow rate: 10 μL / min, addition time: 420 seconds). Next, 2 μg / mL of 7-10_4344, 3-29, 3-26, 3-50, 3-53, or 3-59 was added (flow rate: 10 μL / min, addition time: 60 seconds). A buffer solution without antibody was flowed through the control flow cell. Finally, 0.5–81 nmol / L (hereafter, mol / L will be denoted as M) of hTPOR-His was added as analyte (flow rate 30 μL / min, addition time 120 seconds, dissociation time 600 seconds). Regeneration Solution (reagent from Human Fab Capture Kit) was used to regenerate the sensor chip (addition time: 30 μL / min, addition time 90 seconds). Kinetic constants (ka and kd) and affinity (KD) were calculated using BIAcore T-200 evaluation software. In the analysis, the response of the control flow cell was subtracted from the response of the flow cell that captured the antibody, and the response with only buffer added was subtracted from the response with analyte added. Since hTPOR-His is a monomer, fitting was performed using a 1:1 binding model. HBS-EP+buffer was used as the buffer. The results are shown in Table 6.

[0269]

[0270] As shown in Table 6, all five variants showed improved binding affinity to hTPOR compared to 7-10_4344.

[0271] (2) Evaluation of agonist activity Similar to (5) in Example 1, a cell proliferation assay was performed using human umbilical cord blood-derived HSPCs to evaluate the agonist activity of each variant. The results obtained are shown in Figures 6A, 6B, and 6C. From these figures, it was confirmed that in the evaluation using human umbilical cord blood-derived HSPCs, all five variants showed significantly increased agonist activity compared to 7-10_4344.

[0272] As shown in Table 5, antibodies 3-29 and 3-59 are modified versions in which the amino acid sequences of the variable regions of the heavy and light chains are identical, with only the amino acid sequence of CH differing. Since both antibodies exhibit significantly enhanced agonist activity compared to 7-10_4344, this effect was demonstrated to be due to the amino acid sequences of the variable regions of the heavy and light chains, regardless of the amino acid sequence of CH.

[0273] (3) Physical property evaluation - 1 (viscosity) For each modified compound, the change in viscosity with increasing antibody solution concentration was measured in the same manner as in (7) of Example 1, except that the antibody concentration used when measuring viscosity was changed. Approximate curves of the concentration-dependent viscosity change for each antibody were calculated using the Mooney equation. The concentrations were adjusted to approximately 25-140 mg / mL. The obtained results are shown in Figure 7.

[0274] As shown in Figure 7, at concentrations of approximately 60 mg / mL or higher, all five variants showed a reduced increase in viscosity with increasing antibody solution concentration compared to 7-10_4344.

[0275] Compounds 3-29 and 3-59 had similar viscosities. As shown in Table 5, compounds 3-29 and 3-59 have identical amino acid sequences in the variable regions of the heavy and light chains, with only the CH amino acid sequence differing. Therefore, the decrease in viscosity of each compound at high concentrations was shown to be due to the difference in the amino acid sequences of the variable regions of the heavy and light chains, regardless of the CH amino acid sequence. Furthermore, compounds 3-26, 3-59, and 3-29 showed lower viscosities compared to compounds 3-50 and 3-53. This indicates that in the 7-10 compound, substituting the 49th amino acid residue of VL with an A residue reduces viscosity more than substituting it with a T residue.

[0276] (4) Physical property evaluation - 2 (Stability after low pH treatment) The stability of each antibody after low pH treatment was evaluated in the same manner as in (8) of Example 1, except that the temperature and time during heating for the low pH treatment were changed. The conditions for the low pH treatment were to adjust the pH to 3.5 and then heat at 25°C for 16 hours. The increase in aggregates after low pH treatment was calculated using the following formula: Increase in aggregates (%) = [Agglutination content after low pH treatment (heating at 25°C for 16 hours) (%)] - [Agglutination content before low pH treatment (%)]

[0277] The results obtained are shown in Figure 8. From Figure 8, the increase in aggregates after low pH treatment was 8.9% for 7-10_4344, while the increase was less than 5% for all five modified compounds: 3-29, 3-26, 3-50, 3-53, and 3-59. Furthermore, compounds 3-29 and 3-59, which differed only in the amino acid sequence of CH, showed similar increases in aggregates.

[0278] From the above, it was shown that all five variants showed suppressed aggregate formation after low pH treatment and improved stability compared to 7-10_4344, and that this effect is due to differences in the amino acid sequences of the variable regions of the heavy and light chains, regardless of the amino acid sequence of CH.

[0279] [Example 6] Higher-order evaluation The agonist activity and physical properties of 7-10_4344, the modified compound 3-59 prepared in Example 4, were compared with the anti-human thrombopoietin receptor antibodies 7-10G4344uhm and 4-49G4344uhm described in International Publication No. 2007 / 108559. The sequence numbers for the VH and VL amino acid sequences of 7-10G4344uhm and 4-49G4344uhm are shown in Table 7, respectively. Furthermore, the amino acid sequence of CH in 7-10G4344uhm and 4-49G4344uhm is the amino acid sequence represented by sequence number 79 described in the specification of International Publication No. 2007 / 108559.

[0280]

[0281] (1) Preparation of 7-10G4344uhm and 4-49G4344uhm 7-10G4344uhm and 4-49G4344uhm were prepared using the same method as in Example 1 (4-1) (a). CHO cells were used as the host cells.

[0282] (2) Activity evaluation Similar to (5) in Example 1, a cell proliferation assay was performed using human umbilical cord blood-derived HSPCs, and the agonist activity of 3-59 prepared in Example 4 was compared with that of 7-10G4344uhm, 4-49G4344uhm, and 7-10_4344. As a result, as shown in Figure 9, it was confirmed that 3-59 had significantly higher agonist activity than 7-10G4344uhm, 4-49G4344uhm, and 7-10_4344.

[0283] (3) Physical property evaluation - 1 (viscosity) For 7-10G4344uhm, 7-10_4344, and 3-59, the change in viscosity of the antibody solution in the range of antibody concentrations from approximately 50 mg / mL to 120 mg / mL was measured by the following method. First, each antibody was diluted with a buffer solution (10 mM glutamic acid, 262 mM D-sorbitol, pH 5.5) to prepare the antibody solution. Then, using a B05 tip and VROC Initium One Plus (manufactured by RheoSense Inc.), the temperature was set at 25°C, a flow rate of 200 μL / min, and a shear rate of 3700 s. －１ The viscosity (cP) of each antibody solution was measured under the specified conditions. Viscosity measurements were repeated five times for each antibody. As a result, in the antibody concentration range of approximately 50 mg / mL to 120 mg / mL, 7-10G4344uhm and 7-10_4344 showed a similar concentration-dependent increase in viscosity, while 3-59 showed a more gradual concentration-dependent increase in viscosity than 7-10G4344uhm and 7-10_4344. Therefore, compared to 7-10G4344uhm and 7-10_4344, 3-59 showed suppressed viscosity increase at high concentrations.

[0284] (4) Physical property evaluation - 2 (Stability after low pH treatment) The stability of 7-10G4344uhm, 7-10_4344, and 3-59 after low pH treatment (pH 3.5, 25°C, 16 hours) was evaluated in the same manner as in Example 1 (8), except for the following points. The changes from Example 1 (8) are described below. - The heating conditions during low pH treatment were changed from "37°C for 60 minutes" to "25°C for 16 hours". - The ultrahigh-performance liquid chromatography apparatus was changed to one manufactured by Waters. - The elution times for aggregates (high molecular weight), monomers, and decomposition products (low molecular weight) were approximately 2.0 to 2.8 minutes, 2.8 to 3.8 minutes, and 3.8 to 4.3 minutes, respectively.

[0285] The monomer, aggregate (high molecular weight), and degradation product (low molecular weight) peaks of the antibody obtained by ultra-high-performance liquid chromatography were identified, and the aggregate content was calculated from the peak area of ​​each peak. Subsequently, the increase in aggregates after low pH treatment was calculated using the following formula: Increase in aggregates (%) = [Agglutination content after low pH treatment (heated at 25°C for 16 hours) (%)] - [Agglutination content before low pH treatment (%)]

[0286] Based on the calculated values, the increase in aggregates after low pH treatment was lowest for 3-59. This result indicates that 3-59 has higher stability after low pH treatment than 7-10G4344uhm and 7-10_4344.

[0287] It is clear to those skilled in the art that various modifications or alterations can be conceived within the scope of the claims, and these will naturally fall within the technical scope of the present invention. Furthermore, the components of the above embodiments may be combined in any way without departing from the spirit of the invention.

[0288] This application is based on the Japanese Patent Application No. 2024-184159 filed on October 18, 2024, the contents of which are incorporated by reference within this application.

[0289] SEQ ID NO: 1: Amino acid sequence of VH 7-10 SEQ ID NO: 2: Amino acid sequence of VL 7-10 SEQ ID NO: 3: Amino acid sequence of VH 3-26 SEQ ID NO: 4: Amino acid sequence of VL 3-26 SEQ ID NO: 5: Amino acid sequence of VH 3-29 and 3-59 SEQ ID NO: 6: Amino acid sequence of VL 3-29 and 3-59 SEQ ID NO: 7: Amino acid sequence of VH 3-50 SEQ ID NO: 8: Amino acid sequence of VL 3-50 SEQ ID NO: 9: Amino acid sequence of VH 3-53 SEQ ID NO: 10: Amino acid sequence of VL 3-53 SEQ ID NO: 11: Amino acid sequence of VH 4-49 SEQ ID NO: 12: Amino acid sequence of VL 4-49 SEQ ID NO: 13: Amino acid sequence of 4344 Nullbody (CH) SEQ ID NO: 14: Amino acid sequence of Nullbody (CH)

Claims

1. An antibody or antibody fragment comprising a heavy chain variable region (hereinafter referred to as VH) containing an amino acid sequence in which at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is modified, and a light chain variable region (hereinafter referred to as VL) containing an amino acid sequence in which zero or one or more amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 are modified, and which binds to the human thrombopoietin receptor (hereinafter referred to as human TPOR).

2. The antibody or antibody fragment according to claim 1, wherein the position of the modified amino acid residue is one selected from (a) to (d) below: (a) the 100th amino acid sequence represented by SEQ ID NO: 1 in the VH; (b) the 100th amino acid sequence represented by SEQ ID NO: 1 in the VH and the 49th amino acid sequence represented by SEQ ID NO: 2 in the VL; (c) the 100th amino acid sequence represented by SEQ ID NO: 1 in the VH and the 70th amino acid sequence represented by SEQ ID NO: 2 in the VH; or (d) the 100th and 70th amino acid sequences represented by SEQ ID NO: 1 in the VH and the 49th amino acid sequence represented by SEQ ID NO: 2 in the VL.

3. The antibody or antibody fragment according to claim 1 or 2, wherein the modification of the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in the VH is a substitution with an alanine residue or a glycine residue.

4. The antibody or antibody fragment according to any one of claims 1 to 3, wherein the modification of the 100th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in the VH is a substitution with an alanine residue.

5. The antibody or antibody fragment according to any one of claims 1 to 4, wherein the modification of the 70th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in the VH is a substitution with a valine residue.

6. The antibody or antibody fragment according to any one of claims 1 to 5, wherein the modification of the 49th amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in the VL is a substitution with an alanine residue, a methionine residue, a serine residue, or a threonine residue.

7. The antibody or antibody fragment according to any one of claims 1 to 6, wherein the modification of the 49th amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in the VL is a substitution with an alanine residue or a threonine residue.

8. The antibody or antibody fragment according to any one of claims 1 to 7, wherein the modification of the amino acid residue is one selected from (A) to (D) below. (A) Two modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with an alanine residue; (B) Two modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with a threonine residue; (C) Three modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue and substitution of the 70th amino acid residue with valine, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with an alanine residue; or (D) Three modifications: substitution of the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH with an alanine residue and substitution of the 70th amino acid residue with valine, and substitution of the 49th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with a threonine residue.

9. The antibody or antibody fragment according to any one of claims 1 to 7, wherein the modification of the amino acid residues is three modifications: substitution of the 100th amino acid residue with an alanine residue and substitution of the 70th amino acid residue with valine in the amino acid sequence represented by SEQ ID NO: 1 in VH, and substitution of the 49th amino acid residue with an alanine residue in the amino acid sequence represented by SEQ ID NO: 2 in VL.

10. An antibody or antibody fragment according to any one of claims 1 to 7, which is one selected from (1) to (4) below: (1) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 3, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO: 4; (2) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 7, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO: 8; (3) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 5, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO: 6; or (4) The VH is a VH containing the amino acid sequence represented by SEQ ID NO: 9, and the VL is a VL containing the amino acid sequence represented by SEQ ID NO:

10.

11. The antibody or antibody fragment according to any one of claims 1 to 10, wherein the subclass of the heavy chain constant region of the antibody is IgG4, and the amino acid sequence of the heavy chain constant region of the antibody includes the amino acid residue substitutions S228P, L235E, and R409K, which are represented by the EU index.

12. The antibody or antibody fragment according to any one of claims 1 to 11, wherein the heavy chain constant region of the antibody is a heavy chain constant region comprising the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO:

14.

13. The antibody fragments are Fab, Fab', F(ab') ２ The antibody fragment according to any one of claims 1 to 12, which is one selected from a peptide comprising a single-chain antibody (scFv), a dimerized V region (diabody), a disulfide-stabilized V region (dsFv), and a CDR.

14. A nucleic acid having a base sequence encoding an antibody or antibody fragment according to any one of claims 1 to 13.

15. A vector containing the nucleic acid described in claim 14.

16. Transformed cells comprising the vector according to claim 15.

17. A method for producing an antibody or antibody fragment according to any one of claims 1 to 13, comprising culturing the transformed cells described in claim 16 in a culture medium and collecting an antibody or antibody fragment from the culture.

18. A pharmaceutical composition comprising an antibody or an antibody fragment according to any one of claims 1 to 13.

19. A therapeutic agent for thrombocytopenia or pancytopenia, comprising an antibody or antibody fragment according to any one of claims 1 to 13.

20. The therapeutic agent according to claim 19, wherein the thrombocytopenia or pancytopenia is at least one of immune thrombocytopenia (ITP), aplastic anemia, chemotherapy-induced thrombocytopenia (CIT), cytopenia after hematopoietic stem cell transplantation, myelodysplastic syndrome (MDS), hematopoietic failure after CAR-T therapy, systemic lupus erythematosus, acute radiation syndrome, and thrombocytopenia associated with liver disease.

21. A reagent for detecting or measuring human TPOR, comprising the antibody or antibody fragment described in any one of claims 1 to 13.

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