Anti-FGF23 antibody or antibody fragment

Abstract

This invention provides a novel anti-FGF23 antibody in which antibody degradation at low pH levels is suppressed. [Solution] The present invention relates to an antibody or antibody fragment comprising a heavy chain variable region (hereinafter referred to as VH) containing a specific amino acid sequence and a light chain variable region (hereinafter referred to as VL) containing a specific amino acid sequence, wherein the amino acid sequence at a specific position is substituted with another amino acid residue, and the antibody binds to FGF23.

Description

[Technical Field]

[0001] This invention relates to an anti-FGF23 antibody or a fragment of the antibody. [Background technology]

[0002] Fibroblast growth factor (FGF) forms a structurally similar group of polypeptide families and has been reported to have various effects, including not only fibroblast proliferation activity but also proliferation of mesoderm and neuroectoderm, angiogenesis, and limb bud formation during development. In adults, it also functions as a homeostatic factor, involved in tissue maintenance, repair, regeneration, and metabolism (Non-Patent Literature 1).

[0003] In mammals, 22 types of proteins belonging to the FGF family are known. In humans, 22 types have been identified, from FGF1 to FGF23, excluding FGF15. The human FGF family consists of approximately 150-300 amino acids, with the core sequence of approximately 120 amino acids being identical at a rate of about 30-60%. The FGF family is classified into those expressed as secretory proteins that act on receptor tyrosine kinases and those expressed as intracellular proteins that act on voltage-gated sodium channels and other molecules (Non-Patent Literature 2).

[0004] FGF23 is a secreted protein identified from mice using a database search and PCR method that utilizes homology with FGF15, and subsequently identified by homology search. Human FGF23 is a polypeptide composed of 251 amino acid residues, with the N-terminal 24 residues functioning as a secretion signal, and is known to be cleaved during the protein's maturation process (Non-Patent Literature 3).

[0005] Hypophosphatemic diseases caused by excessive production of FGF23 are known and can be broadly classified into diseases with identified causative genes and acquired diseases (Non-Patent Literature 4). Among FGF23-related hypophosphatemic diseases with identified causative genes, X-linked hypophosphatemic rickets (XLH), caused by phosphate-regulating endopeptidase homolog, X-linked (PHEX) mutations, is the most frequent, and numerous PHEX gene mutations have been reported to date (Non-Patent Literature 5).

[0006] PHEX is a single-pass transmembrane protein and is known to be highly expressed in cartilage, osteoblasts, and odontoblasts (Non-Patent Literature 6 and Non-Patent Literature 7). XLH is said to occur in approximately 1 in 20,000 people (Non-Patent Literature 8). Examples of acquired diseases include tumor-induced osteomalacia (TIO).

[0007] For these FGF23-related hypophosphatemic disorders, active vitamin D3 preparations and oral phosphate preparations have traditionally been used as symptomatic treatment. However, long-term administration could potentially lead to complications such as hypercalcemia and urinary tract infections, nephrocalcification, and persistent hyperparathyroidism (Non-Patent Documents 9 and 10).

[0008] Brosumab, an FGF23 neutralizing antibody, is known as a treatment for the above-mentioned diseases, which are thought to be caused by the overproduction of FGF23.

[0009] In addition, other known human FGF23 neutralizing antibodies include the antibody described in Patent Document 1. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2008 / 099969 [Non-patent literature]

[0011] [Non-Patent Document 1] Wiley Interdiscip Rev Dev Biol. 2015 May-Jun;4(3):215-66 [Non-Patent Document 2] J Biochem. 2011 Feb;149(2):121-30 [Non-Patent Document 3] Biochem Biophys Res Commun. 2000 Oct 22;277(2):494-8 [Non-Patent Document 4] Endocrinology. 2011 Jan;152(1):4-10 [Non-Patent Document 5] Hum Mutat. 2000;16(1):1-6. [Non-Patent Document 6] J Clin Invest. 2008 Feb 1; 118(2): 722-734 [Non-Patent Document 7] J Clin Invest. 1997 Mar 15; 99(6): 1200-1209. [Non-Patent Document 8] Orphanet J Rare Dis. 2019; 14: 58 [Non-Patent Document 9] Lancet. 2019 Jun 15;393(10189):2416-2427 [Non-Patent Document 10] J Bone Miner Res. 2011 Jul;26(7):1381-8 [Overview of the project] [Problems that the invention aims to solve]

[0012] As a result of intensive studies on the physical properties of the anti-human FGF23 antibody described in International Publication No. 2008 / 099969, the present inventors found that the antibody has reduced stability and is degraded at low pH, and that this degradation is due to cleavage between the 99th D and the 100th I in the amino acid sequence of the heavy chain variable region of the antibody. Generally, it is known that antibodies tend to aggregate when the antibody formulation is concentrated, and that increasing the absolute value of the charge of a protein in a protein solution can reduce the likelihood of protein aggregation (Arch Pharm Res Vol 35, No 11, 1871-1886, 2012). Therefore, when concentrating an antibody formulation, lowering the pH of the formulation is sometimes used as one method of suppressing antibody aggregation. Accordingly, when it is necessary to lower the pH of an antibody formulation, an antibody with a wider range of pH values that can be considered is more desirable.

[0013] Therefore, an object of the present invention is to provide a novel anti-FGF23 antibody in which antibody degradation at low pH is suppressed as compared with the anti-human FGF23 antibody described in International Publication No. 2008 / 099969.

Means for Solving the Problems

[0014] As a result of intensive studies on the above problems, the present inventors found that the above problems can be solved by an anti-FGF23 antibody in which the 100th or 105th amino acid residue of the VH of the anti-FGF23 antibody described in International Publication No. 2008 / 099969 is substituted, and completed the present invention.

[0015] 1. An antibody or an antibody fragment that binds to FGF23, wherein at least the 100th or 105th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in the heavy chain variable region (hereinafter referred to as VH) and the 100th or 105th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in the light chain variable region (hereinafter referred to as VL) are substituted with other amino acid residues. 2. The antibody or antibody fragment described in 1 above, wherein the 100th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in VH is replaced with one amino acid residue selected from alanine, asparagine, glycine, tyrosine, arginine, aspartic acid, histidine, tryptophan, and methionine residues. 3. The antibody or antibody fragment according to 1 or 2, wherein the 100th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in VH is substituted with an alanine residue or a tyrosine residue. 4. The antibody or antibody fragment according to any one of 1 to 3 above, wherein the 105th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in VH is substituted with one amino acid residue selected from alanine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, valine, tryptophan, tyrosine, threonine, asparagine, and serine residues. 5. The antibody or antibody fragment according to any one of 1 to 4, wherein the antibody further comprises one substitution selected from (a1) to (a4) below. (a1) At least one substitution selected from the following: substitution of the 50th amino acid residue with a leucine residue in the amino acid sequence represented by Sequence ID No. 1 in VH, substitution of the 54th amino acid residue with a tryptophan residue, substitution of the 55th amino acid residue with a histidine residue, substitution of the 57th amino acid residue with a threonine residue, and substitution of the 58th amino acid residue with a phenylalanine residue. (a2) At least one substitution selected from the following: substitution of the 91st amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in VL with a methionine or leucine residue, substitution of the 92nd amino acid residue with a tyrosine residue, substitution of the 94th amino acid residue with an aspartic acid residue, and substitution of the 96th amino acid residue with an asparagine or aspartic acid residue. (a3) At least one substitution selected from the following: substitution of the 28th amino acid residue to an aspartic acid residue in the amino acid sequence represented by Sequence ID No. 2 in VL, substitution of the 29th amino acid residue to a valine residue, substitution of the 31st amino acid residue to a threonine residue, and substitution of the 34th amino acid residue to a leucine residue, (a4) At least one substitution selected from the substitution of the 92nd amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in VL with a tyrosine or tryptophan residue, the 94th amino acid residue with an aspartic acid residue, and the 96th amino acid residue with an aspartic acid residue. 6. The antibody or antibody fragment described in any one of items 1 to 5 above, wherein the antibody is selected from (c1) to (c10) below. (c1) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 39 and VL containing the amino acid sequence represented by SEQ ID NO: 2 (c2) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 47 and VL containing the amino acid sequence represented by SEQ ID NO: 2, (c3) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 44, (c4) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO. 6 and VL containing the amino acid sequence represented by SEQ ID NO. 44, (c5) Antibodies containing VH, which contains the amino acid sequence represented by SEQ ID NO: 3, and VL, which contains the amino acid sequence represented by SEQ ID NO: 45. (c6) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO. 6 and VL containing the amino acid sequence represented by SEQ ID NO. 45, (c7) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 46, (c8) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO: 46, (c9) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 58, and (c10) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO: 59. 7. The antibody or antibody fragment according to any one of 1 to 6 above, wherein the antibody subclass is IgG1, IgG2, IgG3, or IgG4. 8. The antibody or antibody fragment according to any one of 1 to 7 above, wherein the Fc region of the antibody is 1 selected from (d1) to (d5) below. (d1) Fc region including substitution of the 252nd amino acid residue of the EU index with a tyrosine residue, substitution of the 254th amino acid residue with a threonine residue, and substitution of the 256th amino acid residue with a glutamic acid residue, (d2) Fc region including substitution of the 428th amino acid residue of the EU index with a leucine residue, and substitution of the 434th amino acid residue with a serine residue, (d3) Fc region including substitution of the 308th amino acid residue of the EU index with a proline residue, (d4) Fc region including substitution of the 250th amino acid residue of the EU index with a glutamine residue, and substitution of the 428th amino acid residue with a leucine residue, and (d5) Fc region containing the substitution of the 434th amino acid residue of the EU index with an alanine residue. 9. The antibody or antibody fragment according to any one of 1 to 8, wherein the heavy chain constant region of the antibody comprises an amino acid sequence represented by SEQ ID NOs: 48, 49, 50, 51, or 52. 10. The antibody fragment according to any one of 1 to 9, wherein the antibody fragment is selected from peptides comprising Fab, Fab', (Fab')2, scFv, Diabody, dsFv, and CDR. 11. A nucleic acid having a base sequence encoding an antibody or an antibody fragment as described in any one of items 1 to 10 above. 12. A vector containing the nucleic acid described in item 11 above. 13. Transformed cells containing the vector described in item 12 above. 14. A method for producing an antibody or antibody fragment according to any one of 1 to 10, comprising culturing the transformed cells described in 13 in a culture medium and collecting an antibody or antibody fragment from the culture. 15. A composition comprising the antibody or antibody fragment described in any one of items 1 to 10 above. 16. A therapeutic agent for human FGF23-related disease comprising the antibody or antibody fragment described in any one of items 1 to 10 above. 17. A method for treating human FGF23-related disease comprising the antibody or antibody fragment described in any one of items 1 to 10 above. [Effects of the Invention]

[0016] The anti-FGF23 antibody of the present invention exhibits superior stability, with suppressed degradation at low pH, compared to the anti-FGF23 antibody described in International Publication No. 2008 / 099969. According to the present invention, it is possible to provide an anti-FGF23 antibody or an antibody fragment, 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, and a composition containing the antibody or antibody fragment. [Modes for carrying out the invention]

[0017] The present invention relates to an antibody that binds to fibroblast growth factor 23 (hereinafter referred to as FGF23) as described in International Publication No. 2008 / 099969 (hereinafter referred to as an antibody comprising a heavy chain variable region (hereinafter referred to as VH) containing the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region (hereinafter referred to as VL) containing the amino acid sequence represented by SEQ ID NO: 2), wherein at least the 100th or 105th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted with another amino acid residue, or to a fragment of said antibody (hereinafter referred to as the antibody of the present invention).

[0018] FGF23 is a type of fibroblast growth factor and a hormone derived from osteocytes. Human FGF is a protein consisting of 251 amino acids, with a 24-amino acid secretion signal at its N-terminus, which is cleaved during the protein's maturation process. The functions of FGF23 include suppressing phosphorus reabsorption and vitamin D activation in the kidney, primarily through the FGF receptor 1 (hereinafter also referred to as FGFR1) / α-Klotho complex on the proximal tubular cells of the kidney.

[0019] In the present invention, examples of human FGF23 include polypeptides containing the amino acid sequence of NCBI accession number NP_065689, polypeptides consisting of amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of NCBI accession number NP_065689 and having the function of human FGF23, and polypeptides consisting of amino acid sequences having 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology to the amino acid sequence of NCBI accession number NP_065689 and having the function of human FGF23.

[0020] Polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence indicated by NCBI accession number NP_065689 can be subjected 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), Proc. Natl. Acad. Sci. USA, 82, 488]. It can be obtained by introducing site-directed mutations into DNA encoding a polypeptide containing the amino acid sequence indicated by, for example, NCBI accession number NP_065689, using (1985) and other methods.

[0021] 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.

[0022] The nucleotide sequence with NCBI accession number NM_020638 is an example of a gene encoding human FGF23. The genes encoding human FGF23 of the present invention also include genes comprising a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence of NM_020638 and containing DNA encoding a polypeptide having the function of human FGF23, a nucleotide sequence comprising at least 60% homology to the nucleotide sequence of NM_020638, preferably 80% homology, more preferably 90% homology, and most preferably 95% homology, and containing DNA encoding a polypeptide having the function of human FGF23, or genes comprising DNA that hybridizes under stringent conditions with DNA containing the nucleotide sequence of NM_020638 and encoding a polypeptide having the function of human FGF23.

[0023] 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 DNA containing the NM_020638 base sequence as a probe.

[0024] Specifically, DNA that can be identified can be found by performing a hybridization method [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)] at 65°C in the presence of 0.7-1.0 mol / L sodium chloride, using a filter or slide glass immobilized with DNA from hybridized colonies or plaques, or PCR products or oligo DNA containing the said sequence. This is followed by washing the filter or slide glass 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.

[0025] Examples of DNA that can be hybridized include DNA having at least 60% homology to the base sequence of NM_020638, preferably DNA having 80% or more homology, and more preferably DNA having 95% or more homology.

[0026] Genetic polymorphisms are often observed in the base sequences of genes encoding eukaryotic proteins. Among the genes used in this invention, genes that have undergone small mutations in their base sequence due to such polymorphisms are also included in the human FGF23 encoding gene of this invention.

[0027] Unless otherwise specified, the homology values ​​in this invention 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 Res., 25, 3389 (1997), Genome Res., 7, 649 (1997)] for amino acid sequences.

[0028] The default parameters are as follows: 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 (word size) 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 other programs; -X (X dropoff value for gapped alignment in bits) is 15; and -Z (final X dropoff value for gapped alignment in bits) is 50 for blastn and 25 for other programs.

[0029] Polypeptides containing a partial sequence of the amino acid sequence of NCBI accession number NP_065689 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 of NP_065689 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 of NCBI accession number NP_065689 can be obtained by the same method as described above. Furthermore, polypeptides consisting of the amino acid sequence of NCBI accession number NP_065689, or polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of NCBI accession number NP_065689, can also be produced by chemical synthesis methods such as the fluorenylmethyloxycarbonyl (Fmoc) method and the t-butyloxycarbonyl (tBoc) method.

[0030] In this invention, the deletion, substitution, or addition of amino acids is also referred to as modification of amino acids.

[0031] The antibodies of the present invention encompass 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.

[0032] Examples of monoclonal antibodies in the present invention include antibodies produced by hybridomas, or recombinant antibodies produced by transformants transformed with an expression vector containing an antibody gene.

[0033] An epitope is a single amino acid sequence, a three-dimensional structure consisting of an amino acid sequence, an amino acid sequence modified by post-translational modification, or a three-dimensional structure consisting of said amino acid sequence, which is recognized and bound by a monoclonal antibody.

[0034] Examples of amino acid sequences modified by post-translational modification include O-linked glycans attached to Tyr and Ser having OH substituents, N-linked glycans attached to Gln and Asn having NH2 substituents, and amino acid sequences to which sulfate groups attached to Tyr and Ser having OH substituents are linked.

[0035] The binding of the antibody of the present invention to human FGF23 can be confirmed by measuring the binding affinity of the antibody to human FGF23 using methods such as ELISA and surface plasmon resonance. It can also 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)].

[0036] The amino acid residues or epitopes of human FGF23 to which the antibody of the present invention binds can be determined by performing antibody binding experiments using a knockout molecule in which some domains of human FGF23 are deleted, a mutant in which domains derived from other proteins are substituted, and a partial peptide fragment of human FGF23.

[0037] Alternatively, the amino acid residues or epitopes of human FGF23 to which the antibody of the present invention binds can also be determined by adding the antibody of the present invention to a peptide fragment of human FGF23 digested with a proteolytic enzyme, and performing epitope mapping using a known mass spectrometry method.

[0038] Antibody molecules are also called immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into isotypes 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.

[0039] Antibody molecules are composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (hereinafter referred to as L chains). The H chain is composed of VH and the constant region (also denoted as CH) from the N-terminus, while the L chain is composed of VL and the constant region (also denoted as CL) from the N-terminus. Within each subclass, α, δ, ε, γ, and μ chains are known for the CH. The CH is further composed of the CH1 domain, hinge domain, CH2 domain, and CH3 domain from the N-terminus. A domain is a functional structural unit that constitutes each polypeptide of an antibody molecule. The CH2 and CH3 domains together are called the Fc region or simply Fc. CL is C λ Chain and C κ Chains are known.

[0040] The CH1 domain, hinge domain, CH2 domain, CH3 domain, and Fc region in this invention can be identified by the number of amino acid residues from the N-terminus using 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). Specifically, CH1 is identified as the amino acid sequence corresponding to EU index 118-215, the hinge as the amino acid sequence corresponding to EU index 216-230, CH2 as the amino acid sequence corresponding to EU index 231-340, and CH3 as the amino acid sequence corresponding to EU index 341-447.

[0041] The antibodies of the present invention 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 the present invention also include genetically engineered recombinant antibodies produced by recombining the H chain (or VH) and L chain (or VL) from two different antibodies. The two different antibodies may be hybridoma-derived monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies. Furthermore, the antibodies of the present invention also include genetically engineered recombinant antibodies in which appropriate amino acid residues have been substituted when producing the above-mentioned genetically engineered recombinant antibodies.

[0042] 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 a hybridoma, such as mice, rats, hamsters, or rabbits.

[0043] 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.

[0044] 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.

[0045] Humanized antibodies are antibodies in which the amino acid sequences of the CDRs (Cellular Derived Ratios) of the VH and VL (Very Longitudinal) regions of non-human animal antibodies have been transplanted into the corresponding CDRs of the VH and VL regions of human antibodies. The regions of the VH and VL regions other than the CDRs are called framework regions (hereinafter referred to as FR).

[0046] 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, respectively, to construct a humanized antibody expression vector, which is then introduced into animal cells for expression.

[0047] 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.

[0048] 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 phage 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).

[0049] 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.

[0050] 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.

[0051] 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.

[0052] The VH and VL amino acid sequences of the antibody of the present invention may be any of the VH and VL amino acid sequences of a human antibody, a non-human animal antibody, or a humanized antibody.

[0053] The amino acid sequence of CL in the antibody of the present invention 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.

[0054] The CH of the antibody of the present invention may be any immunoglobulin, but subclasses belonging to the IgG class, γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4), are preferred.

[0055] The antibodies of the present invention 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.

[0056] The antibodies or antibody fragments of the present invention include antibodies or antibody fragments containing any post-translationally modified amino acid residues. Examples of post-translational modifications include the deletion of a lysine residue at the C-terminus of the H chain [lysine clipping] or the conversion of a glutamine residue at the N-terminus of a polypeptide to pyroglutamine (pyroGlu) [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].

[0057] In the present invention, an antibody fragment is an antibody fragment that binds to human FGF23 in a way that suppresses degradation at low pH compared to antibodies containing a VH containing the amino acid sequence represented by SEQ ID NO: 1 and a VL containing the amino acid sequence represented by SEQ ID NO: 2. Examples of antibody fragments in the present invention include Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (Diabody), disulfide-stabilized V region (dsFv), or peptides containing multiple CDRs. Fab is an antibody fragment obtained by treating an IgG antibody with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain), in which approximately half of the N-terminal side of the H chain and the entire L chain are linked by disulfide bonds (SS bonds), and has antigen-binding activity with a molecular weight of approximately 50,000.

[0058] F(ab')2 is an antibody fragment with antigen-binding activity and a molecular weight of approximately 100,000, obtained by treating IgG with the proteolytic enzyme pepsin (cleaved at the 234th amino acid residue of the H chain). Fab is slightly larger than the one formed when Fab is linked via a disulfide bond in the hinge region. Fab' is an antibody fragment with antigen-binding activity and a molecular weight of approximately 50,000, obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0059] scFv is an antibody fragment with antigen-binding activity, consisting of 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 and one Ser residues.

[0060] 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.

[0061] 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 disulfide bonds between the cysteine ​​residues.

[0062] A peptide containing a CDR is composed of at least one region of the VH or VL CDR. Multiple CDR-containing peptides can be linked together directly or via a suitable peptide linker. The modified antibody of the present invention 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.

[0063] One embodiment of the antibody of the present invention is the antibody or antibody fragment described in (i) and (ii) below. (i) An antibody or antibody fragment that binds to FGF23 comprising VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, wherein at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is replaced with one amino acid residue selected from alanine, asparagine, glycine, tyrosine, arginine, aspartic acid, histidine, tryptophan, and methionine residues. (ii) An antibody or antibody fragment that binds to FGF23 comprising VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, wherein at least the 105th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted with one amino acid residue selected from alanine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, valine, tryptophan, tyrosine, threonine, asparagine, and serine residues.

[0064] Compared to antibodies 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, this antibody exhibits higher stability at low pH and can suppress antibody degradation. Preferably, this antibody is an antibody that binds to FGF23 in which, in an antibody 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, at least the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted with an alanine residue or a tyrosine residue.

[0065] In this invention, low pH refers to a weakly acidic or acidic solution with a pH of less than 6, and examples include, but are not particularly limited to, pH 5, pH 4.5, pH 4, etc.

[0066] Compared to antibodies 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, the antibodies of the present invention exhibit suppressed degradation at low pH and superior stability. In the present invention, antibody degradation can be measured by size exclusion chromatography (SEC) or SDS-PAGE (SDS-polyacrylamide gel electrophoresis).

[0067] The fact that the antibody of the present invention exhibits suppressed degradation of the antibody at low pH compared to antibodies 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 can be confirmed, for example, by a method including the following steps (I) to (III), but is not limited to this method. (I) Prepare an antibody solution by replacing the solvent of an antibody solution containing the antibody of the present invention, or an antibody containing VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, with a suitable solvent of pH 4, 4.5, or 5 using a column such as NAP(trademark) 25 (GE Healthcare Life Sciences). (II) After allowing the antibody solution prepared in (I) above to stand at 40°C for 1 month or 2 weeks, or at 25°C for 3 months, the peak (%) corresponding to the antibody degradation product is detected using SEC. Alternatively, the antibody solution prepared in (I) above is allowed to stand at 40°C for one month or two weeks, or at 25°C for three months, and then SDS-PAGE is performed under reducing conditions to detect a band around 40 kDa (e.g., 35 kDa to 45 kDa) corresponding to the degradation products of the antibody. (III) If the peak (%) corresponding to the degradation product of the antibody detected by SEC in (II) above is lower for the antibody of the present invention than for the antibody containing VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, or if the intensity of the band around 40 kDa in SDS-PAGE is lower for the antibody of the present invention than for the antibody containing VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, then it can be confirmed that antibody degradation at low pH is suppressed for the antibody of the present invention compared to the antibody containing VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2.

[0068] Instead of the SDS-PAGE described in (II) above, a Bioanalyzer electrophoresis system (Agilent Technologies, Inc.) and the Agilent Protein 230 kit (Agilent Technologies, Inc.) may be used. In this case, if the peak area percentage (%) of the band corresponding to the antibody degradation product detected at a size approximately 10-20 kDa smaller than the peak of the entire H chain (e.g., 40-60 kDa) in the resulting electropherogram is lower for the antibody of the present invention than for the antibody containing VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, then it can be confirmed that antibody degradation at low pH is suppressed for the antibody of the present invention compared to the antibody containing VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2.

[0069] In other words, examples of embodiments that demonstrate the stability of the antibody of the present invention at low pH include the following. Compared to antibodies 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, the intensity of the band around 40 kDa in SDS-PAGE is reduced after standing at low pH (such as pH 4, 4.5, or 5) and 40°C for 1 month or 2 weeks, or at 25°C for 3 months. The band intensity can be evaluated visually. Compared to antibodies 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, the peak (%) corresponding to the degradation product of the antibody detected by SEC is reduced after standing for one month or two weeks at a low pH such as pH 4, 4.5, or 5 and 40 degrees Celsius, or for three months at 25 degrees Celsius. Preferably, the peak (%) is reduced by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to antibodies 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. Compared to antibodies 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, the peak area percentage (%) of the band corresponding to the antibody degradation product detected at a size approximately 10-20 kDa smaller than the peak of the full-length H chain (e.g., 40-60 kDa) is reduced in electropherograms obtained using a Bioanalyzer electrophoresis system (Agilent Technologies, Inc.) and the Agilent Protein 230 kit (Agilent Technologies, Inc.) after standing for 1 month or 2 weeks at a low pH such as pH 4, 4.5, or 5 and 40 degrees Celsius, or for 3 months at 25 degrees Celsius. Preferably, the peak area percentage (%) is reduced by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to antibodies 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.

[0070] One embodiment of the antibody of the present invention is the antibody further comprising one substitution selected from (a1) to (a4) below. (a1) At least one substitution selected from the following: substitution of the 50th amino acid residue with a leucine residue in the amino acid sequence represented by Sequence ID No. 1 in VH, substitution of the 54th amino acid residue with a tryptophan residue, substitution of the 55th amino acid residue with a histidine residue, substitution of the 57th amino acid residue with a threonine residue, and substitution of the 58th amino acid residue with a phenylalanine residue. (a2) At least one substitution selected from the following: substitution of the 91st amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in VL with a methionine or leucine residue, substitution of the 92nd amino acid residue with a tyrosine residue, substitution of the 94th amino acid residue with an aspartic acid residue, and substitution of the 96th amino acid residue with an asparagine or aspartic acid residue. (a3) At least one substitution selected from the following: substitution of the 28th amino acid residue to an aspartic acid residue in the amino acid sequence represented by Sequence ID No. 2 in VL, substitution of the 29th amino acid residue to a valine residue, substitution of the 31st amino acid residue to a threonine residue, and substitution of the 34th amino acid residue to a leucine residue, (a4) At least one substitution selected from the substitution of the 92nd amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in VL with a tyrosine or tryptophan residue, the 94th amino acid residue with an aspartic acid residue, and the 96th amino acid residue with an aspartic acid residue.

[0071] Furthermore, it is preferable that the antibodies of the present invention have all of the amino acid residues described above in (a1) to (a4) substituted.

[0072] The antibody of the present invention can have its binding activity to FGF23 and its FGF23 neutralizing activity improved by the substitution of one amino acid residue selected from (a1) to (a4) above. FGF23 neutralizing activity refers to the activity of inhibiting the signal produced when FGF23 binds to its receptor. An example of an FGF23 receptor is the complex of FGFR1 and αKlotho.

[0073] The FGF23 neutralizing activity of the antibody of the present invention can be confirmed by reporter assays (also called promoter assays) as described in Nature 2006 Dec 7;444(7120). Furthermore, the FGF23 neutralizing activity of the antibody of the present invention can also be confirmed by reporter assays using αKlotho stable-expressing HEK293 cells transformed with a luciferase expression vector having a promoter derived from the mouse Egr1 gene.

[0074] Specific examples of the antibodies of the present invention include any one antibody selected from (b1) to (b5) below.

[0075] (b1) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, wherein the 50th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is replaced with a leucine residue, the 54th amino acid residue with a tryptophan residue, the 55th amino acid residue with a histidine residue, the 57th amino acid residue with a threonine residue, the 58th amino acid residue with a phenylalanine residue, and the 100th amino acid residue with an alanine or tyrosine residue.

[0076] (b2) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, wherein the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is replaced with an alanine residue or a tyrosine residue, and the 91st amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL is replaced with a methionine residue, the 92nd amino acid residue is replaced with a tyrosine residue, the 94th amino acid residue is replaced with an aspartic acid residue, and the 96th amino acid residue is replaced with an asparagine residue.

[0077] (b3) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, wherein the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is replaced with an alanine residue or a tyrosine residue, and the 28th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL is replaced with an aspartic acid residue, the 29th amino acid residue is replaced with a valine residue, the 31st amino acid residue is replaced with a threonine residue, and the 34th amino acid residue is replaced with a leucine residue.

[0078] (b4) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, wherein the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is replaced with an alanine residue or a tyrosine residue, and the 91st amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL is replaced with a leucine residue, the 92nd amino acid residue is replaced with a tyrosine residue, the 94th amino acid residue is replaced with an aspartic acid residue, and the 96th amino acid residue is replaced with an aspartic acid residue, and

[0079] (b5) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 1 and VL containing the amino acid sequence represented by SEQ ID NO: 2, wherein the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is replaced with an alanine residue or a tyrosine residue, and the 92nd amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL is replaced with a tyrosine residue or a tryptophan residue, the 94th amino acid residue is replaced with an aspartic acid residue, and the 96th amino acid residue is replaced with an aspartic acid residue.

[0080] Furthermore, specific examples of the antibody of the present invention include antibodies selected from (c1) to (c10) below, with (c1), (c8), (c9), or (c10) being preferred, and (c1) or (c9) being more preferred. (c1) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 39 and VL containing the amino acid sequence represented by SEQ ID NO: 2 (c2) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 47 and VL containing the amino acid sequence represented by SEQ ID NO: 2, (c3) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 44, (c4) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO. 6 and VL containing the amino acid sequence represented by SEQ ID NO. 44, (c5) Antibodies containing VH, which contains the amino acid sequence represented by SEQ ID NO: 3, and VL, which contains the amino acid sequence represented by SEQ ID NO: 45. (c6) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO. 6 and VL containing the amino acid sequence represented by SEQ ID NO. 45, (c7) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 46, (c8) Antibodies comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO: 46, (c9) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 58, and (c10) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO: 59.

[0081] The antibody of the present invention may also utilize an Fc region in which amino acid residues have been substituted to control its binding affinity to FcRn, for the purpose of controlling its half-life in the blood.

[0082] Examples of Fc regions in which amino acid residues have been substituted to improve the antibody's binding affinity to FcRn include any one of the following Fc regions (d1) to (d5). Among these, the Fc region (d1) is preferred. (d1) Fc region including substitution of the 252nd amino acid residue of the EU index with a tyrosine residue, substitution of the 254th amino acid residue with a threonine residue, and substitution of the 256th amino acid residue with a glutamic acid residue, (d2) Fc region including substitution of the 428th amino acid residue of the EU index with a leucine residue, and substitution of the 434th amino acid residue with a serine residue, (d3) Fc region including substitution of the 308th amino acid residue of the EU index with a proline residue, (d4) Fc region including substitution of the 250th amino acid residue of the EU index with a glutamine residue, and substitution of the 428th amino acid residue with a leucine residue, and (d5) Fc region containing the substitution of the 434th amino acid residue of the EU index with an alanine residue.

[0083] Furthermore, in the present invention, specific examples of amino acid sequences in the heavy chain constant region, which includes an Fc region in which amino acid residues have been substituted to improve the antibody's binding affinity to FcRn, include the amino acid sequences represented by SEQ ID NOs: 48, 49, 50, 51, and 52. Among these, the amino acid sequence represented by SEQ ID NO: 48 is preferred.

[0084] The monoclonal antibody or antibody fragment of the present invention includes antibodies or derivatives of the antibody fragment obtained by chemically or genetically engineering a radioisotope, a low molecular weight drug, a high molecular weight drug, a protein, or an antibody drug, etc., to a monoclonal antibody or antibody fragment that binds to human FGF23 of the present invention.

[0085] Antibodies or derivatives of the antibody fragment 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-terminal or C-terminal side of the H or L chain of the monoclonal antibody or antibody fragment that binds to human FGF23 according to the present invention, or to appropriate substituents, side chains, or sugar chains in the antibody molecule [Introduction to Antibody Engineering, Chijin Shokan (1994)].

[0086] Furthermore, it can be produced by a genetic engineering method in which DNA encoding a monoclonal antibody or an antibody fragment that binds to human FGF23 of the present invention is ligated to DNA encoding a protein or antibody drug to be bound, inserted into an expression vector, and the expression vector is introduced into an appropriate host cell for expression.

[0087] Examples of the radioisotope include 111 In, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 At and the like. The radioisotope can be directly bound to the antibody by a method such as the chloramine T method. Alternatively, a substance that chelates the radioisotope may be bound to the antibody. Examples of the chelating agent include 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) and the like.

[0088] Examples of the low-molecular-weight drugs include anticancer drugs such as alkylating agents, nitrosourea agents, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, M-phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy Society (1996)], steroid agents such as hydrocortisone or prednisone, non-steroid agents such as aspirin or indomethacin, immunomodulating agents such as gold thiomalate or penicillamine, immunosuppressive agents such as cyclophosphamide or azathioprine, or anti-inflammatory agents such as antihistamines such as chlorpheniramine maleate or clemastine [Inflammation and Anti-Inflammatory Therapy, Medical Dental Publishing Co., Ltd. (1982)] and the like.

[0089] Examples of anticancer drugs include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechloretamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), epirubicin, gemcitabine (gemzar), daunorubicin, procarbazine, and mitoma. Icin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol), docetaxel (Taxothea), aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, 10- Hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea, idarubicin, mesna, irinotecan (CPT-11), nogitecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, hydroxycarbamide, plicamycin, mitotane, pegasparagase, pentostatin, pipobromane, tamoxifen, goserelin Leuprorenin, flutamide, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone, methylprednisolone, vindesine, nimustine, semustine, capecitabine, tomdex, azacitidine, UFT, oxaloplatin, gefitinib (Iressa), imatinib (STI571), erlotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitors, Vascular Endothelial Growth Factor receptor (VEGFR) inhibitors, Fibroblast Growth Factor Receptor (FGFR) inhibitors, Epidermal Growth Factors such as Iressa or TarcevaReceptor (EGFR) inhibitors, radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrin, all-trans retinoic acid, thalidomide, lenalidomide, anastrozole, fadrozol, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (targretin), tamoxifen, dexamethasone, progestins, est Examples include chlorogenic acids, anastrozole (Arimidex), leuprin, aspirin, indomethacin, celecoxib, penicillamine, gold thiomarate, chlorpheniramine maleate, chloropheniramine, clemasitin, tretinoin, bexarotene, arsenic, bortezomib, allopurinol, calicheamicin, ibritumomab tiuxetan, targretin, ozogamin, clarithromycin, leucovorin, ketoconazole, aminoglutethimide, suramin or meitansinoids or their derivatives.

[0090] 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.

[0091] 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)].

[0092] For example, one method for conjugating PEG with an antibody is to react it with a PEGylation modification reagent [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)]. Examples of PEGylation 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).

[0093] Immunostimulants can also be natural products known as immunojuvants. Specific examples include immune-enhancing drugs such as β(1→3) glucan (e.g., lentinan or schizophyllan) or α-galactosylceramide (KRN7000).

[0094] Examples of proteins include cytokines, growth factors, or toxic proteins that activate immune cells such as NK cells, macrophages, or neutrophils.

[0095] 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.

[0096] 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.

[0097] Antigens that induce apoptosis upon antibody binding include, for example, cluster of differentiation (hereinafter referred to as CD) 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA)-Class II, or epidermal growth factor receptor (EGFR).

[0098] Antigens involved in tumor pathogenesis or antibodies that modulate immune function include, for example, CD4, CD40, CD40 ligand, B7 family molecules (e.g., CD80, CD86, CD274, B7-DC, B7-H2, B7-H3 or B7-H4), ligands of B7 family molecules (e.g., CD28, CTLA-4, ICOS, PD-1 or BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF) receptor family molecules (e.g., DR4, DR5, TNFR1 or TNFR2), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecules, receptor family of TRAIL family molecules (e.g., TRAIL-R1, TRAIL-R2, TRAIL-R3 or TRAIL-R4), and receptor activator of nuclear factor kappa B Examples include ligands (RANK), RANK ligands, CD25, folate receptors, cytokines [e.g., IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF)β or TNFα, etc.] or receptors for these cytokines, or chemokines (e.g., SLC, ELC, I-309, TARC, MDC or CTACK, etc.) or receptors for these chemokines.

[0099] Antigens of antibodies that inhibit angiogenesis at lesion sites include, for example, Vascular Endothelial Growth Factor (VEGF), angiopoietin, Fibroblast Growth Factor (FGF), EGF, Hepatocyte Growth Factor (HGF), Platelet-Derived Growth Factor (PDGF), Insulin-like Growth Factor (IGF), erythropoietin (EPO), TGFβ, IL-8, ephrin, or SDF-1, or their receptors.

[0100] Nucleic acid drugs include, for example, pharmaceuticals containing nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA that act on living organisms by controlling gene function. For example, conjugate with nucleic acid drugs that suppress the master transcription factor RORγt of Th17 cells is a possibility.

[0101] When the antibody or a derivative of the antibody fragment of the present invention is used for the detection and measurement of human FGF23 and for the diagnosis of human FGF23-related diseases, the agent that binds to the antibody may be 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).

[0102] Furthermore, one embodiment of the present invention is a composition comprising the antibody of the present invention or a fragment of the antibody. Examples of compositional forms include compositions containing a monoclonal antibody that binds to human FGF23 or a fragment of the antibody as an active ingredient. A composition comprising the antibody of the present invention or a fragment of the antibody can be used to treat human FGF23-related diseases. As one embodiment of the present invention, a therapeutic agent for human FGF23-related diseases comprising the antibody of the present invention is provided.

[0103] The present invention also relates to a method for treating human FGF23-related diseases, comprising administering a monoclonal antibody or a fragment of that antibody that binds to human FGF23.

[0104] Human FGF23-related diseases include any disease involving human FGF23 or its receptor, such as tumor-induced osteomalacia (TIO), autosomal overt hypophosphatemic rickets / osteomalacia (ADHR), X-linked hypophosphatemia (XLH), fibrous dysplasia, McCune-Albright syndrome, autosomal recessive hypophosphatemic rickets / osteomalacia (ARHR), osteoporosis, rickets (including hypophosphatemic rickets and vitamin D-resistant rickets), hypercalcemia, hypocalcemia, ectopic calcification, osteosclerosis, Paget's disease, hyperparathyroidism, hypoparathyroidism, pruritus, and diseases associated with renal failure or hemodialysis in cases of renal failure, such as renal tubular dysfunction.

[0105] The present invention also includes a therapeutic agent containing a monoclonal antibody or an antibody fragment that binds to human FGF23 as an active ingredient, for treating symptoms such as hypophosphatemia, bone mineralization disorders, bone pain, muscle weakness, skeletal deformities, growth disorders, and hypo-1,25 vitamin D deficiency seen in diseases such as TIO, ADHR, XLH, fibrous dysplasia, McCune-Albright syndrome, and ARHR.

[0106] The therapeutic agent comprising the antibody or antibody fragment of the present invention may consist solely of the antibody or antibody fragment as an active ingredient, but it is generally preferable to provide it as a pharmaceutical preparation manufactured by mixing it with one or more pharmacologically acceptable carriers and using 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] One embodiment of the composition of the present invention is a reagent for the detection or measurement of human FGF23, containing a monoclonal antibody or a fragment of the antibody that binds to human FGF23. The present invention also relates to a method for the detection or measurement of human FGF23 using a monoclonal antibody or a fragment of the antibody that binds to human FGF23. Any known method can be used as the method for detecting or measuring human FGF23 in the present invention. 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), luminescent immunoassay, Western blotting, or physicochemical methods.

[0111] One embodiment of the composition of the present invention is a diagnostic agent for FGF23-related diseases comprising a monoclonal antibody or an antibody fragment that binds to human FGF23. The present invention also relates to a diagnostic method for FGF23-related diseases, comprising detecting or measuring FGF23 using a monoclonal antibody or an antibody fragment that binds to human FGF23. By detecting or measuring human FGF23 using the monoclonal antibody or an antibody fragment of the present invention in accordance with the above method, diseases related to human FGF23 can be diagnosed.

[0112] In the present invention, the biological sample to be used for detecting or measuring human FGF23 is not particularly limited, as long as it may contain human FGF23, such as tissue, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0113] The diagnostic agent containing the monoclonal antibody or its antibody fragment according to the present invention may include a reagent for performing an antigen-antibody reaction and a reagent for detecting the reaction, depending on the diagnostic method to be used. Examples of reagents for performing an antigen-antibody reaction include buffers and salts. Examples of detection reagents include a labeled secondary antibody that recognizes the monoclonal antibody or its antibody fragment, or a substrate corresponding to the label, which are reagents used in conventional immunological detection or measurement methods.

[0114] One embodiment of the present invention relates to the use of an anti-human FGF23 monoclonal antibody or a fragment of the antibody for the manufacture of a therapeutic or diagnostic agent for FGF23-related disease. Another embodiment of the present invention relates to a method for treating or diagnosing FGF23-related disease.

[0115] The following describes in detail the method for producing the antibody of the present invention, the method for treating the disease, and the method for diagnosing the disease.

[0116] 1. Method for producing antibodies (1) Preparation of antigen Human FGF23, which serves as an antigen, can be obtained by introducing an expression vector containing cDNA encoding the full length or a partial length of human FGF23 into E. coli, yeast, insect cells, or animal cells. Alternatively, human FGF23 can be obtained by purifying it from various human cell lines, human cells, and human tissues that express large amounts of human FGF23. These human cell lines, human cells, and human tissues can also be used directly as antigens. Furthermore, synthetic peptides containing partial sequences of human FGF23 can be prepared using chemical synthesis methods such as the Fmoc method or the tBoc method and used as antigens. Human FGF23 or synthetic peptides containing partial sequences of human FGF23 may have known tags such as FLAG or His attached to their C-terminus or N-terminus.

[0117] The human FGF23 used in this invention can be produced by expressing the DNA encoding the human FGF23 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.

[0118] First, a recombinant vector is prepared by inserting a full-length cDNA containing the portion encoding human FGF23 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 transformants that produce polypeptides.

[0119] 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 that can express the target gene can be used, such as microorganisms belonging to the Escherichia genus, such as E. coli, yeast, insect cells, or animal cells.

[0120] 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 FGF23, 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.

[0121] As the recombinant vector, it is preferable to use a plasmid in which the distance between the ribosome-binding sequence, the Shine-Dalgarno sequence (also called the SD sequence), and the start codon has been adjusted to an appropriate distance (for example, 6 to 18 bases).

[0122] Furthermore, the base sequence of the DNA encoding human FGF23 can be modified to include codons optimized for expression within the host, thereby improving the production rate of the target human FGF23.

[0123] 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 E. coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [prepared from E. coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from E. coli IGHA2 (FERM BP-400), Japanese Patent Publication No. 60-221091], pGKA2 [prepared from E. coli IGKA2 (FERM BP-6798), Japanese Patent Publication No. 60-221091], pTerm2 (US Patent Nos. 4,686,191, 4,939,094, and 160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Examples include Bacteriol., 172, 2392 (1990), pGEX (Pharmacia), pET system (Novagen), or pME18SFL3.

[0124] 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.

[0125] 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.

[0126] Any method for introducing recombinant vectors into host cells that involves introducing DNA into the host cells can be used, for example, the method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

[0127] When using animal cells as hosts, 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).

[0128] 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 the promoter or enhancer of Moloney's mouse leukemia virus. Additionally, the enhancer of the human CMV IE gene may be used in conjunction with the promoter.

[0129] 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, Examples include 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.20 (also called YB2 / 0), mouse myeloma cells NSO, mouse myeloma cells SP2 / 0-Ag14, Syrian hamster cells BHK or HBT5637 (Japanese Patent Publication No. 63-000299).

[0130] 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)].

[0131] Human FGF23 can be produced by culturing a transformant derived from a microorganism or animal cell, which possesses a recombinant vector incorporating the DNA encoding human FGF23 obtained as described above, in a culture medium, allowing the human FGF23 to be generated and accumulated 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.

[0132] When expressed in eukaryotic cells, human FGF23 with added sugars or sugar chains can be obtained.

[0133] 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.

[0134] Examples of culture media for transformants obtained using animal cells as hosts include commonly used RPMI1640 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 for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40°C, and in the presence of 5% CO2. Antibiotics such as kanamycin or penicillin may also be added to the culture medium as needed during the culture.

[0135] Methods for expressing the gene encoding human FGF23 include, for example, direct expression, secretory production, or fusion protein expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)].

[0136] Methods for producing human FGF23 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 FGF23 being produced, an appropriate method can be selected.

[0137] When human FGF23 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 FGF23 can be increased using gene amplification systems (Japanese Patent Publication No. 2-227075) that utilize the dihydrofolate reductase gene, etc.

[0138] The obtained human FGF23 can be isolated and purified, for example, as follows: If human FGF23 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. By centrifuging the cell-free extract, the supernatant obtained can be used to obtain purified samples from conventional protein isolation and purification methods, such as 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, chromatographic focusing, or electrophoresis such as isoelectric focusing, either alone or in combination.

[0139] If human FGF23 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 FGF23 is recovered as a precipitate fraction by centrifugation. The recovered insoluble form of human FGF23 is solubilized with a protein denaturant. After the human FGF23 is restored to its normal three-dimensional structure by diluting or dialyzing the solubilized solution, a purified polypeptide can be obtained by the isolation and purification method described above.

[0140] When human FGF23 or its glycosylated derivatives are secreted extracellularly, the human FGF23 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.

[0141] Furthermore, the human FGF23 used in this invention 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.

[0142] (2) Preparation of antibody-producing cells for animal immunization and fusion Mice, rats, or hamsters aged 3 to 20 weeks are immunized with the antigen obtained in (1), and antibody-producing cells are collected from the spleen, lymph nodes, and peripheral blood of the animals. Alternatively, mouse FGF23 knockout mice can be used as immunized animals.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] (3) Preparation of myeloma cells For myeloma cells, mouse cell lines are used, such as the 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 (1975)].

[0147] 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 × 10⁶ cells were collected on the day of fusion. 7 Ensure a minimum number of cells.

[0148] (4) Preparation of cell fusion and monoclonal antibody-producing hybridomas Wash the fusion antibody-producing cells obtained in (2) and the myeloma cells obtained in (3) thoroughly with Minimum Essential Medium (MEM) 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 them so that the cell ratio of fusion antibody-producing cells to myeloma cells is 5-10:1, 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 1-2 mL of MEM medium several times every 1-2 minutes, then add MEM medium until the total volume is 50 mL. After centrifugation, remove the supernatant. After gently loosening the precipitated cell population, gently suspend the fusion antibody-producing cells in HAT medium [normal medium with hypoxanthine, thymidine, and aminopterin added]. This suspension is incubated in a 5% CO2 incubator at 37°C for 7 to 14 days.

[0149] After culturing, a portion of the culture supernatant is removed, and a group of cells that react to antigens containing human FGF23 but not to antigens that do not contain human FGF23 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.

[0150] (5) Preparation of purified monoclonal antibodies 8-10 week old mice or nude mice that have been treated with pristane [0.5 mL of 2,6,10,14-tetramethylpentadecane (Pristane) is administered intraperitoneally and kept for 2 weeks] are injected intraperitoneally with the monoclonal antibody-producing hybridoma obtained in (4). The hybridoma will develop ascites malignancy 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.

[0151] 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.

[0152] 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.

[0153] (6) Selection of monoclonal antibodies The selection of monoclonal antibodies is performed by measuring the antibody's binding affinity to human FGF23 using ELISA, as shown below.

[0154] After dispensing human FGF23 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 similar, 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 similar, 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 specifically reacts with human FGF23.

[0155] 2. Production of genetically modified antibodies As an example of the production of genetically modified antibodies, the methods for producing human-type chimeric antibodies and humanized antibodies are shown below. Genetically modified mouse antibodies, rat antibodies, and rabbit antibodies can also be produced using the same methods.

[0156] (1) Construction of vectors for recombinant antibody expression Genetically modified antibody expression vectors are animal cell expression vectors into which DNA encoding the CH and CL of human antibodies is incorporated. These vectors can be constructed by cloning the DNA encoding the CH and CL of human antibodies into the animal cell expression vector, respectively.

[0157] 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 expression vectors for animal cells include the initial promoter of SV40 [J. Biochem., 101, 1307 (1987)], Moloney mouse leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)], or promoters and enhancers of immunoglobulin H chains [Cell, 41, 479 (1985)] and [Cell, 33, 717 (1983)].

[0158] For recombinant antibody expression vectors, tandem vectors [J.Immunol. Methods, 167, 271 (1994)] are used because they are easy to construct, easy to introduce into animal cells, and ensure a balanced expression level of antibody heavy and light chains within animal cells. However, vectors in which the antibody heavy and light chains reside on separate vectors can also be used. Examples of tandem recombinant antibody expression vectors include pKANTEX93 (International Publication No. 97 / 10354) and pEE18 [Hybridoma, 17, 559 (1998)].

[0159] (2) Obtaining cDNA encoding the V region of antibodies derived from non-human animals and analyzing their amino acid sequences. The cDNA encoding VH and VL of non-human antibodies and the analysis of their amino acid sequences can be performed as follows.

[0160] 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.

[0161] 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.

[0162] For the preparation of total RNA from hybridoma cells, the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] or kits such as the RNA easy kit (Qiagen) can be used.

[0163] For mRNA preparation from total RNA, the oligo(dT)-immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] or Oligo-dT30 <super>Kits such as the mRNA Purification (registered trademark) Kit (manufactured by Takara Bio Inc.) can be used. Alternatively, mRNA can be prepared from hybridoma cells using kits such as the Fast Track mRNA Isolation (registered trademark) Kit (manufactured by Invitrogen Inc.) or the QuickPrep mRNA Purification (registered trademark) Kit (manufactured by Pharmacia Inc.).

[0164] 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.

[0165] When constructing 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)] can be used.

[0166] Any E. coli organism 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.

[0167] 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.

[0168] 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 the Polymerase Chain Reaction method [hereinafter referred to as PCR method, 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)].

[0169] The selected cDNA is cleaved with an appropriate restriction enzyme, cloned into a plasmid such as pBluescript SK(-) (Stratagene), and the nucleotide 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 nucleotide sequence analyzer such as ABI PRISM3700 (PE Biosystems) or ALFDNA sequencer (Pharmacia).

[0170] 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)].

[0171] 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.

[0172] (3) Construction of human chimeric antibody expression vectors or human chimeric antibody variant expression vectors 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).

[0173] To link the 3' end of the cDNA encoding VH or VL of a non-human antibody to the 5' end of 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 cloned upstream of the respective genes encoding CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1) so that they are expressed in the appropriate manner, thereby constructing a human chimeric antibody expression vector.

[0174] 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).

[0175] (4) Construction of cDNA encoding the V region of a humanized antibody The cDNA encoding the VH or VL of a humanized antibody can be constructed as follows:

[0176] Select the amino acid sequence of the VH or VL FR of a human antibody for transplanting the CDR amino acid sequence of the VH or VL of a non-human antibody. Any FR amino acid sequence derived from a human antibody can be used for selection. For example, use the FR amino acid sequences of human antibodies registered in databases such as the Protein Data Bank, or the common amino acid sequences of each subgroup of FR of human antibodies [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 the highest possible homology (at least 60%) to the VH or VL FR amino acid sequence of the original antibody.

[0177] 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.

[0178] 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, based on 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 strands 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).

[0179] After the PCR reaction, the amplified products are cloned into plasmids such as pBluescript SK(-) (Stratagene), and their base sequences are determined by the same method as described in (2) to obtain plasmids containing DNA sequences encoding the VH or VL amino acid sequence of the desired humanized antibody.

[0180] 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).

[0181] (5) Modification of the amino acid sequence of the V region of humanized antibodies Humanized antibodies exhibit reduced antigen-binding activity compared to the original non-human antibody when only the CDRs of the VH and VL of non-human antibodies are transplanted into the FRs of the VH and VL of human antibodies [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 FRs of human antibodies that are directly involved in antigen binding, 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 antigen binding, and then substituting these amino acid residues with those of the original non-human antibody.

[0182] To identify the amino acid residues of 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.

[0183] 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.

[0184] (6) Construction of humanized antibody expression vectors 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).

[0185] 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 human antibody CH or CL in the recombinant antibody expression vector obtained in (1) in an appropriate manner.

[0186] 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.

[0187] (7) Transient expression of recombinant antibodies By using the recombinant antibody expression vectors obtained in (3) and (6), or modified expression vectors thereof, recombinant antibodies can be transiently expressed, and the antigen-binding activity of various chimeric antibodies and humanized antibodies produced can be efficiently evaluated.

[0188] 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)].

[0189] 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.

[0190] 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)].

[0191] (8) Obtaining transformant strains that stably express recombinant antibodies and preparing recombinant antibodies By introducing the recombinant antibody expression vectors obtained in (3) and (6), or modified expression vectors thereof, into a suitable host cell, a transformed cell that stably expresses recombinant antibodies can be obtained. Methods such as electroporation [Japanese Patent Publication No. 2-257891, Cytotechnology, 3, 133 (1990)] are used to introduce expression vectors into host cells.

[0192] Any host cell capable of expressing a recombinant antibody 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), rat myeloma cells YB2 / 3HL.P2.G11.16Ag.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) and others are used.

[0193] Furthermore, host cells in which the activity of proteins such as enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose, proteins involved in the α-linking of the 1-position of fucose 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.

[0194] After introducing the expression vector, transformant cells that stably express the recombinant antibody are selected by culturing them in an animal cell culture medium containing drugs such as G418 sulfate (hereinafter referred to as G418) (Japanese Patent Publication No. 2-257891).

[0195] 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 using a dhfr gene amplification system (Japanese Patent Publication No. 2-257891).

[0196] 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, methods used for protein purification, such as gel filtration, ion exchange chromatography, and ultrafiltration, can be combined.

[0197] 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)].

[0198] 3. Activity evaluation of purified monoclonal antibodies or antibody fragments. The activity of the purified monoclonal antibody or antibody fragment of the present invention can be evaluated as follows.

[0199] The binding activity of the antibody or antibody fragment of the present invention to human FGF23 can be measured using methods such as ELISA or surface plasmon resonance.

[0200] The human FGF23 neutralizing activity of the antibody or antibody fragment of the present invention can be measured using the reporter assay described above.

[0201] 4. Method for treating diseases using the anti-human FGF23 monoclonal antibody or antibody fragment of the present invention The monoclonal antibody or antibody fragment of the present invention can be used to treat diseases associated with human FGF23.

[0202] The therapeutic agent containing the monoclonal antibody or an antibody fragment of the present invention may contain only the antibody or the 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.

[0203] Routes of administration include, for example, oral administration, or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, or intravenous administration. Forms of administration include, for example, sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.

[0204] Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, or granules.

[0205] 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 or peppermint as additives.

[0206] 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.

[0207] 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.

[0208] 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 the present invention 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 the components exemplified as additives may be added.

[0209] 5. A method for diagnosing a disease using the anti-human FGF23 monoclonal antibody or antibody fragment of the present invention. Human FGF23-related diseases can be diagnosed by detecting or measuring human FGF23 using the monoclonal antibody or antibody fragment of the present invention.

[0210] The diagnosis of human FGF23-related disease can be made, for example, by detecting or measuring human FGF23 present in the patient's body using immunological methods.

[0211] 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.

[0212] The radiolabeled immunoassay method involves, for example, reacting an antigen or cells expressing an antigen with the antibody or antibody fragment of the present invention, and then reacting it with a radiolabeled anti-immunoglobulin antibody or antibody fragment, followed by measurement using a scintillation counter or similar device.

[0213] Enzyme immunoassay involves reacting an antigen or cells expressing an antigen with the antibody or antibody fragment of the present invention, followed by a reaction with an anti-immunoglobulin antibody or binding fragment labeled with an enzyme, and then 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 labeling described in the publicly known [Enzyme Immunoassay, Igaku-Shoin (1987)] can be used.

[0214] 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 above 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 polyclonal or monoclonal antibodies may be used, and antibody fragments such as Fab, Fab', or F(ab)2 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.

[0215] 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 use in immunofluorescence assays, known fluorescent labels [Immuofluorescence Method, Soft Science Co., Ltd. (1983)] can be used. For example, FITC or RITC can be used.

[0216] The luminescence immunoassay is performed using the method described in literature such as [Bioluminescence and Chemiluminescence, Clinical Laboratory 42, Hirokawa Shoten (1998)]. Known luminescent labels can be used in luminescence immunoassay, including acridinium esters or rofin.

[0217] 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) 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. The results are then measured by visualizing the labels.

[0218] Physicochemical methods are performed, for example, by forming aggregates by binding the antigen, human FGF23, to the monoclonal antibody or antibody fragment of the present invention, and then detecting these aggregates. Other physicochemical methods that can be used include capillary methods, one-dimensional immunodiffusion, immunoturbidimetric methods, or latex immunoturbidimetric methods [Clinical Laboratory Methods Handbook, Kinbara Publishing (1998)]. In latex immunoturbidimetric methods, 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, and when an antigen-antibody reaction is induced with the corresponding antigen or antibody, the scattered light in the reaction solution increases and the transmitted light decreases. By detecting this change as absorbance or integrating spheroidal turbidity, the antigen concentration in the test sample is measured.

[0219] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. [Examples]

[0220] [Example 1] Detection of degradation products of antibody A The amino acid sequence of the heavy chain variable region of the anti-human FGF23 antibody described in International Publication No. 2008 / 099969 is described as SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is described as SEQ ID NO: 2. Hereinafter, human IgG1 antibodies 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, will be referred to as antibody A.

[0221] Antibody A was prepared in the same manner as in Example 2, and the solvent in the antibody solution was replaced with a pH 4 solvent containing 10 mM Sodium L-glutamate, 262 mM D-Sorbitol, and 0.05 mg / mL Polysorbate 80. The resulting antibody solution was allowed to stand at 40°C for 1 month (hereinafter referred to as the 40°C 1M sample) or at -80°C for 1 month, after which it was thawed (hereinafter referred to as the initial sample).

[0222] Subsequently, SEC analysis and SDS-PAGE under reducing conditions were performed on the 40°C 1M sample and the initial sample. The SEC analysis results showed that the initial sample showed peaks corresponding to antibody degradation products at approximately 3%, while the 40°C 1M sample showed peaks at 9.32%.

[0223] Furthermore, SDS-PAGE results showed that, compared to the initial sample, the 40°C 1M sample exhibited a significantly darker band (band A) located approximately 10 kDa smaller than the H chain around 50 kDa, and correlated with a lighter H chain band. Therefore, band A is a band of H chain degradation products, suggesting that the 40°C 1M sample is degraded at the H chain. Analysis of the N-terminal amino acid sequence of band A confirmed that it is cleaved at position 99 (D) and position 100 (I) in the heavy chain variable region of antibody A.

[0224] [Example 2] Preparation of I100 modified antibody For antibody A, an antibody was prepared by substituting the 100th amino acid residue, I, of the amino acid sequence represented by Sequence ID No. 1 in VH with the amino acid residue listed in Table 1, using the method described below. Hereinafter, some or all of the antibodies listed in Table 1 will also be referred to as I100 modified antibodies.

[0225] [Table 1]

[0226] The gene fragments corresponding to the nucleotide sequences encoding the VH amino acid sequences of each antibody listed in Table 1 were introduced into expression vectors using seamless cloning (commissioned to Fasmac Corporation) to construct the necessary plasmids. The pCI-OtCAG_hG1 vector, containing both a signal sequence and a human γ-chain constant region sequence, was used as the H-chain expression vector. In all clones, human IgG1 was used for the H-chain constant region.

[0227] Furthermore, the amino acid sequence of the VL of the I100 modified antibody was the same as that of the VL of the A antibody (SEQ ID NO: 2). As the L-chain expression vector, a pCI-OtCMV_hK vector containing a signal sequence and a human κ-chain constant region sequence was used. The completed plasmids were prepared in large quantities using the QIAGEN Plasmid Maxi Kit (QIAGEN).

[0228] Next, each antibody was transiently expressed using the Expi293 Expression System Kit (Life Technologies). Plasmid introduction was performed according to the instructions in the accompanying documentation. The light chain expression vector and heavy chain expression vector were mixed in a 1:2 ratio and introduced. After plasmid introduction, cells were cultured for 3 days under conditions of 37°C, 5% CO2, and 125 rpm. Subsequently, the cell culture suspension was centrifuged, and the culture supernatant was collected through a 0.2 μm filter (Thermo Scientific). Purified antibodies were obtained from the culture supernatant by affinity purification using MabSelect SuRe (GE Healthcare).

[0229] Specifically, the resin packed into the column was equilibrated with PBS, the culture supernatant was added to the column, washed twice with PBS, washed once each with Wash Buffer 1 (PBS with 1M NaCl) and Wash Buffer 2 (20mM citrate, 50mM NaCl, pH 5.0), and then the antibody was eluted using elution buffer (20mM citrate, 50mM NaCl, pH 3.4).

[0230] The obtained antibody solution was neutralized by adding 1 / 10 volume of neutralizing buffer (1M phosphate-NaOH, pH 7.0), and the solvent of the antibody solution was replaced with PBS using NAP25 (GE Healthcare). The antibody solution after buffer replacement was concentrated by ultrafiltration using Amicon Ultra-4 Centrifugal Filter Units (Millipore), and the absorbance A280 was measured using Nanodrop (Thermo Scientific) to measure and adjust the concentration of the antibody solution. The extinction coefficient was calculated from the amino acid sequence of each humanized antibody according to the method of CNPace et al. (1995, Prot. Sci.4:2411-2423).

[0231] [Example 3] Evaluation of antigen-binding activity of I100 modified antibody The binding activity of the I100 modified antibody and A antibody obtained in Example 2 to Recombinant Human FGF-23 (R&D Systems, Cat No. 2604-FG-025 / CF) was measured as follows.

[0232] Using the Human Antibody Capture Kit (Global Life Science Technologies Japan Co., Ltd., Cat. No. BR-1008-39), anti-human IgG antibody was immobilized onto a CM5 sensor chip (Global Life Science Technologies Japan Co., Ltd., Cat. No. BR100530) according to the attached protocol.

[0233] Anti-human IgG antibody, prepared to a concentration of 1 μg / mL, was added to a flow cell immobilized with anti-human IgG antibody at a flow rate of 10 μL / min for 30 seconds.

[0234] Next, Recombinant Human FGF-23, prepared in five stepwise concentrations by 2-fold dilution from 130.5 ng / mL, was administered at a flow rate of 30 μL / min. The binding reaction was monitored for 2 minutes, and the dissociation reaction for 10 minutes. The measurement was performed using a single-cycle method. The acquired sensorgrams were analyzed using Bia Evaluation Software (Global Life Science Technologies Japan Co., Ltd.), and the kinetic constants of each antibody were calculated. The calculated binding rate constant (ka), dissociation rate constant (kd), and dissociation constant [kd1 / ka1=K] for each antibody were determined. D Some of the results are shown in Tables 2 and 3. In Table 3, A_1, A_2, and A_3 all refer to antibody A.

[0235] [Table 2]

[0236] [Table 3]

[0237] Tables 2 and 3 show that almost all I100 modified antibodies, including I100A and I100Y antibodies, exhibited lower antigen-binding activity compared to the I100A antibody.

[0238] Furthermore, it was confirmed that the antigen-binding activity of antibodies I100W, I100S, I100K, and I100E was lower than that of antibody A, while antibodies I100T, I100Q, I100M, and I100F had antigen-binding activity comparable to that of antibody A.

[0239] [Example 4] Confirmation of the degradation inhibitory effect of the modified I100 antibody For the I100 modified antibody and A antibody obtained in Example 2, the solvent in the antibody solution was replaced with a pH 4 solvent containing 10 mM Sodium L-glutamate and 262 mM D-Sorbitol using NAP25 (GE Healthcare). The resulting antibody solutions were allowed to stand at 40°C for 1 month or 2 weeks, and then SDS-PAGE was performed under reducing conditions. Table 4 shows the results of comparing the intensity of the band around 40 kDa, which corresponds to the degradation products of the antibody, for each antibody and the A antibody. When the intensity of the band was lower in the modified antibody compared to the A antibody, it was determined that degradation was suppressed in the modified antibody compared to the A antibody.

[0240] The numerical values ​​listed in the "Degradation Inhibition Effect" column of Table 4 were set as follows: Antibodies whose degradation was more inhibited than antibody A were marked with 1, antibodies whose degradation was similar to antibody A were marked with 2, and antibodies whose degradation was more accelerated than antibody A were marked with 3.

[0241] [Table 4]

[0242] As shown in Table 4, I100A, I100Y, I100W, I100R, I100N, I100M, I100H, I100G, and I100D showed less degradation than antibody A. I100L, I100V, I100T, I100S, I100K, I100F, and I100E were degraded to a similar extent as antibody A. Furthermore, I100Q showed more accelerated degradation than antibody A.

[0243] [Example 5] Evaluation of the thermal stability of antibody A and modified antibody I100 The thermal stability of each antibody domain (Fab, CH2, CH3) of antibody A and modified I100 antibody was evaluated using differential scanning calorimetry (DSC).

[0244] The measurement sample was prepared at a concentration of 0.5 mg / mL in D-PBS buffer. For the measurement, a Micro Cal VP-Capillary DSC system (Spectris Co., Ltd.) was used. Also, the measurement was carried out with a program of heating at a rate of 1 degree per minute from 25 degrees to 100 degrees. The obtained results are shown in Table 5 and Table 6.

[0245]

Table 5

[0246]

Table 6

[0247] From Table 5 and Table 6, it was found that for I100A, I100G, I100N, and I100R, the peaks of Fab and CH2 overlapped and were detected, and the thermal stability of Fab was improved by 3 to 8 degrees compared to antibody A. From the above, it was confirmed that the structural stability of I100A, I100G, I100N, and I100R was higher than that of antibody A.

[0248] [Example 6] Preparation of D105-modified antibody In the same manner as in Example 2, an antibody in which the 105th D in the amino acid sequence of VH containing the amino acid sequence represented by SEQ ID NO: 1 of antibody A was substituted with the amino acid residues shown in Table 7 (hereinafter, a part or all of the antibody is also referred to as a D105-modified antibody) was prepared.

[0249]

Table 7

[0250] [Example 7] Evaluation of antigen-binding activity of D105-modified antibody The binding activity of the D105 modified antibody obtained in Example 6 to Recombinant Human FGF-23 (R&D Systems, Cat No. 2604-FG-025 / CF) was measured using the same method as in Example 3. The results are shown in Tables 8 and 9. In Table 8, A_1 and A_2 are both measurement results for antibody A.

[0251] [Table 8]

[0252] [Table 9]

[0253] Tables 8 and 9 confirm that the D105 modified antibodies all exhibited lower antigen-binding activity than the A antibody.

[0254] [Example 8] Confirmation of the degradation inhibitory effect of the D105 modified antibody The D105-modified antibody obtained in Example 6 was used to confirm its antibody degradation inhibitory effect in the same manner as in Example 4. The antibody solution was left to stand at 40°C for two weeks.

[0255] The results obtained are shown in Table 10. In the column for degradation inhibition effect in Table 10, a value of 1 indicates an antibody whose degradation was more inhibited than antibody A, a value of 2 indicates an antibody whose degradation was similar to antibody A, and a value of 3 indicates an antibody whose degradation was more accelerated than antibody A.

[0256] [Table 10]

[0257] Table 10 shows that the degradation of the 17 D105 modified antibodies, excluding D105E, was suppressed compared to antibody A. In all cases, the intensity of the approximately 40 kDa band corresponding to antibody degradation products was significantly reduced in SDS-PAGE compared to antibody A, confirming that antibody degradation was significantly suppressed.

[0258] [Example 9] Measurement of Neutralizing Activity of I100 Modified Antibody and D105 Modified Antibody against Human FGF23 For the 24 types of I100 modified antibodies and D105 modified antibodies (I00A, I100N, I100G, I100Y, I100R, I100D, I100H, D105A, D105F, D105G, D105H, D105I, D105K, D105L, D105M, D105P, D105Q, D105R, D105V, D105W, D105Y, D105T, D105N, and D105S) in which antibody degradation was suppressed more than that of antibody A in Examples 4 and 8, the neutralizing activity against human FGF23 was measured by the method described below.

[0259] For the measurement of neutralizing activity, a promoter assay using αKlotho stable expression HEK293 cells (hereinafter referred to as mEgr1 / αKL / HEK293) transformed with a luciferase expression vector having a promoter derived from the mouse Egr1 gene was used. mEgr1 / αKL / HEK293 was prepared by the same method as described in Nature 2006 Dec 7;444(7120).

[0260] In the promoter assay, when FGF23 binds to αKlotho on mEgr1 / αKL / HEK293, a signal flows into the cells and the luciferase gene is expressed, and the fluorescence of the luciferase can be detected. When FGF23 is neutralized by the presence of a FGF23 neutralizing antibody, the fluorescence intensity decreases.

[0261] The antibody was diluted to 100 μg / ml with a buffer containing 10 mM SodiumL-glutamate and 262 mM D-Sorbitol to prepare a stock solution. As the standard culture medium for mEgr1 / αKL / HEK293, DMEM medium (Thermofisher) supplemented with 10 (vol%) FetalBovine Serum (Thermofisher) and 1 (vol%) penicillin / streptomycin was used.

[0262] The antibody was diluted 1000-fold in the standard culture medium described above to obtain a maximum concentration of 100 ng / ml. Eight dilution series were prepared using √10-fold dilutions and used for evaluation. The evaluation was performed using a 384-well plate. The cell density was 2000 cells / well, and the FGF23 concentration added was 4 ng / ml. The FGF23 used was the same as in Example 3, diluted in standard culture medium.

[0263] After adding each antibody to the cells, they were cultured for 24 hours, and then FGF23 was added and the cells were cultured for another 4 hours. The fluorescence intensity of luciferase was measured using a Bright-Glo® Luciferase Assay System (Promega) and a multi-plate reader Envision (Perkinelmer).

[0264] For the obtained results, the fluorescence intensity with FGF23 added was set to 100%, and the fluorescence intensity without FGF23 added was set to 0%. The percentage of fluorescence intensity with each antibody added was calculated, and the IC50 values ​​(ng / ml) are shown in Table 11.

[0265] [Table 11]

[0266] As shown in Table 11, all modified antibodies exhibited lower FGF23 neutralizing activity than antibody A.

[0267] [Example 10] Preparation of a modified heavy chain constant region antibody of antibody A An amino acid-modified antibody of the heavy chain constant region of antibody A (hereinafter referred to as CH-modified antibody) was prepared by the method described below.

[0268] CH-modified antibodies are derived from the CH (human IgG1) of antibody A, and are derived from human IgG2, IgG2AAAS (a human IgG2-modified antibody in which the Fc of human IgG2 is modified by substituting valine at EU index 234 with alanine, glycine at EU index 237 with alanine, and proline at EU index 331 with serine) (Michael, S., et al., J.Immunol., 1997, 159: 3613; J.Immunol. 2000, 164:4178-4184), or IgG4PE_R409K (human Regarding the Fc region of IgG4, we created a total of 23 antibodies by adding the following Fc amino acid modifications, known to improve binding activity to human and monkey FcRn, to the Fc region of each of the three types of isotype antibodies of a modified human IgG4 antibody (International Publication No. 2006 / 033386) in which serine at EU index 228 was replaced with proline, leucine at 235 with glutamic acid, and arginine at 409 with lysine, or A antibody or three types of isotype antibodies.

[0269] <Fc amino acid modification> (10-1) Amino acid modification by substituting M at EU index 252 with Y, S at 254 with T, and T at 256 with E (J. Biol. Chem. 2006b;281:23514-23524) (10-2) Amino acid modification by substituting T at EU index 250 with Q and N at EU index 428 with L (J. Biol. Chem. 2004;279:6213-6216) (10-3) Amino acid modification by substituting N with A at EU index position 434 (J. Immunol. 1997;158:2211-2217), (10-4) Amino acid modification by substituting V with P at EU index position 308 (Drug. Metab. Dispos. 2012a;40:1545-1555) (10-5) Amino acid modification by substituting M at EU index 428 with L and N at EU index 434 with S (Nat. Biotechnol. 2010;28:157-159)

[0270] Hereafter, a CH-modified antibody in which the CH of antibody A is replaced with the CH of human IgG2 will be referred to as A_G2 antibody. CH-modified antibodies with substitutions to other subclasses will be referred to similarly.

[0271] Hereinafter, CH-modified antibodies in which the amino acid modifications described in (10-1) to (10-5) above have been added to the Fc region of antibody A will be referred to as A_YTE antibody, A_QL antibody, A_A antibody, A_P antibody, and A_LS antibody, respectively. Similarly, CH-modified antibodies in which the amino acid modifications described in (10-1) to (10-5) above have been added to antibody A_G2, antibody A_G2AAS, and antibody A_G4PE_R409K will also be referred to in the same manner.

[0272] A plasmid containing the nucleotide sequences encoding the amino acid sequences of the variable and constant regions of the H chain of antibody A, prepared in Example 2, was subjected to restriction enzyme treatment with NheI and BamHI to prepare a vector fragment with the constant region removed. Based on this plasmid fragment, Fasmac synthesized gene fragments containing the nucleotide sequences encoding the amino acid sequences of the 23 types of CH-modified antibodies mentioned above, and introduced them into appropriate expression vectors to prepare the necessary plasmids. Antibodies were prepared using the obtained plasmids in the same manner as in Example 2.

[0273] [Example 11] Measurement of the binding activity of antibody A and CH-modified antibody of antibody A to FcRn The binding activity of the A antibody and the CH-modified A antibody prepared in Examples 2 and 10 to human and monkey FcRn was measured using Biacore by the method described below.

[0274] HBS-EP+ (Global Life Science Technologies Japan, Cat. No. BR-1006-69) was diluted to pH 6 by adding 1M hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 083-01095). Each antibody was diluted with the above pH 6 HBS-EP+ buffer using NAP25. Human FcRn and monkey FcRn were also diluted with pH 6 HBS-EP+. The human FcRn and monkey FcRn used in the experiment were prepared as follows.

[0275] DNA sequences encoding human FcRn (using only amino acids 1-297 of the full-length amino acid sequence of human FcRn for expression as a soluble molecule), a human FcRn-His tag (amino acid sequence: SEQ ID NO: 54) with six histidines added to its C-terminus, and human β2-microglobulin (amino acid sequence: SEQ ID NO: 55) were inserted downstream of the CMV promoter in a mammalian cell expression plasmid. This plasmid was transiently expressed using the Expi293 Expression System Kit (Life Technologies) in the same manner as described in Example 2.

[0276] Monkey FcRn (using only amino acids 1-297 of the full-length amino acid sequence of monkey FcRn to express it as a soluble molecule), monkey FcRn-His tag (amino acid sequence: SEQ ID NO: 56) with six histidines added to its C-terminus, and monkey β2 microglobulin (amino acid sequence: SEQ ID NO: 57) were also transiently expressed using the same method as human FcRn-His tag.

[0277] After obtaining the respective culture supernatants, human and monkey FcRn were purified using Ni-NTA Agarose (QIAGEN, Cat. No. 30210). The purification method followed the standard procedure described in the Ni-NTA Agarose manual.

[0278] Biacore measurements were performed under the following conditions: Tetra His Antibody BSA Free (QIAGEN) was immobilized on a CM5 sensor chip (Global Life Science Technologies Japan Co., Ltd., Cat. No. BR100530). Human FcRn and monkey FcRn, prepared to 10 μg / mL, were added to the flow cell immobilized with Tetra His Antibody BSA Free at a flow rate of 10 μL / min for 120 seconds. Next, each Fc-modified antibody, prepared to five concentrations by stepwise dilutions of 3-fold from 450 μg / mL, was added at a flow rate of 30 μL / min, and the binding reaction was monitored for 2 minutes and the dissociation reaction for 5 minutes.

[0279] Measurements were performed using the multi-cycle method. The acquired sensorgrams were analyzed using Bia Evaluation Software (Global Life Science Technologies Japan Co., Ltd.) to calculate the dissociation constant [kd1 / ka1=KD] for each antibody. The results are shown in Tables 12 and 13.

[0280] [Table 12]

[0281] [Table 13]

[0282] As shown in Tables 12 and 13, the CH-modified antibodies, which have the amino acid modifications described in Examples 10(1) to (5) introduced into Fc, showed improved binding activity to human FcRn and monkey FcRn compared to the A antibody and its isotypes (A2_G2, A_G2AAAS, and A_IgG4PE_R409K antibody).

[0283] [Example 12] Preparation of CH-modified antibodies of I100 and D105 modified antibodies In Examples 4 and 8, it was confirmed that antibody degradation was suppressed more effectively with the I100 and D105 modified antibodies than with antibody A. CH modified antibodies were then prepared by adding the amino acid modifications described below (12-1) to (12-5) to each of the D105N, I100A, I100H, and I100Y antibodies using the method described below.

[0284] (12-1) Amino acid modification by substituting M at EU index 252 with Y, S at 254 with T, and T at 256 with E

[0285] (12-2) Amino acid modification by substituting CH from human IgG1 to human IgG2, substituting M at EU index 252 with Y, S at 254 with T, and T at 256 with E.

[0286] (12-3) Amino acid modification by substituting CH from human IgG1 to human IgG2, substituting M at EU index 428 with L, and N at EU index 434 with S.

[0287] (12-4) Amino acid modification: Replace CH with human IgG4PE_R409K from human IgG1, and replace V at EU index 308 with P.

[0288] (12-5) Amino acid modification: Replace CH with human IgG4PE_R409K from human IgG1, replace M at EU index 428 with L, and replace N at EU index 434 with S.

[0289] Hereinafter, CH-modified antibodies that have undergone the amino acid modifications described in (12-1) to (12-5) above for the D105N antibody will be referred to as D105N_YTE, D105N_G2_YTE, D105N_G2_LS, D105N_G4PE_R409K_P, and D105N_G4PE_R409K_LS antibodies, respectively. The same notation will be applied to other antibodies. The amino acid sequences of CH in the CH-modified antibodies that have undergone the amino acid modifications described in (12-1) to (12-5) above are shown in SEQ ID NOs. 48, 49, 50, 51, and 52, respectively.

[0290] The plasmid prepared in Example 10 was restricted with Bstz17I and NheI to prepare a vector fragment from which the variable region had been removed. Based on this plasmid fragment, Fasmac synthesized gene fragments containing the nucleotide sequences encoding the VH amino acid sequences of the D105N, I100A, I100H, and I100Y antibodies, and introduced them into appropriate expression vectors to prepare the required plasmids. Using the obtained plasmids, each antibody was prepared in the same manner as in Example 2.

[0291] [Example 13] Measurement of FGF23 neutralizing activity of heavy chain constant region modified antibody The FGF23 neutralizing activity of the 20 CH-modified antibodies prepared in Example 12 was measured using the same method as in Example 9. The results are shown in Table 14.

[0292] [Table 14]

[0293] As shown in Table 14, antibodies with the same amino acid sequence in the variable region exhibited similar levels of FGF23 neutralizing activity even when the amino acid sequence in the constant region was altered. Therefore, it was confirmed that amino acid modifications in the heavy chain constant region of each antibody did not affect the antibody's FGF23 neutralizing activity.

[0294] [Example 14] Production of a modified antibody with improved antigen-binding activity To improve the antigen-binding activity of the I100A and I100Y antibodies as described in Tables 2 and 3, affinity maturation was performed using phage display at Abwiz Bio, and the amino acid sequence information for (14-1) to (14-9) below was obtained.

[0295] (14-1) VH containing the amino acid sequence represented by SEQ ID NO: 1, in which the 51st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is replaced with V, the 54th amino acid with F, the 55th amino acid with W, the 57th amino acid with R, the 58th amino acid with W, and the 100th amino acid with A, is obtained as VH (H2B11_A, amino acid sequence: SEQ ID NO: 38).

[0296] (14-2) VH containing the amino acid sequence represented by SEQ ID NO: 1, in which the 50th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is replaced with L, the 54th amino acid with W, the 55th amino acid with H, the 57th amino acid with T, the 58th amino acid with F, and the 100th amino acid with A (VH (J2H2B9_A, amino acid sequence: SEQ ID NO: 39))

[0297] (14-3) VH containing the amino acid sequence represented by SEQ ID NO: 1, in which the 50th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is replaced with V, the 54th amino acid with F, the 55th amino acid with C, the 57th amino acid with F, the 58th amino acid with V, and the 100th amino acid with A (J2H2E9_A, amino acid sequence: SEQ ID NO: 40)

[0298] (14-4) In a VH containing the amino acid sequence represented by SEQ ID NO: 1, the 51st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is replaced with L, the 54th amino acid with W, the 55th amino acid with T, the 57th amino acid with Y, the 58th amino acid with R, and the 100th amino acid with A (VH(2H2E1_A, amino acid sequence: SEQ ID NO: 41)

[0299] (14-5) In a VH containing the amino acid sequence represented by SEQ ID NO: 1, the 54th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is replaced with W, the 55th amino acid with V, the 57th amino acid with R, the 58th amino acid with A, and the 100th amino acid with A, resulting in VH (2H2E5_A, amino acid sequence: SEQ ID NO: 42).

[0300] (14-6) In a VH containing the amino acid sequence represented by SEQ ID NO: 1, the 51st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is replaced with V, the 54th amino acid with Y, the 55th amino acid with R, the 57th amino acid with K, the 58th amino acid with W, and the 100th amino acid with Y, resulting in VH (2H2E8_Y, amino acid sequence: SEQ ID NO: 43).

[0301] (14-7) In a VL containing the amino acid sequence represented by SEQ ID NO: 2, the 91st amino acid of the amino acid sequence represented by SEQ ID NO: 2 is replaced with M, the 92nd amino acid with Y, the 94th amino acid with D, and the 96th amino acid with N in a VL (L3G12, amino acid sequence: SEQ ID NO: 44).

[0302] (14-8) In a VL containing the amino acid sequence represented by SEQ ID NO: 2, the 28th amino acid of the amino acid sequence represented by SEQ ID NO: 2 is replaced with D, the 29th amino acid with V, the 31st amino acid with T, and the 34th amino acid with L (VL (L1H8, amino acid sequence: SEQ ID NO: 45)).

[0303] (14-9) In a VL containing the amino acid sequence represented by SEQ ID NO: 2, the 91st amino acid of the amino acid sequence represented by SEQ ID NO: 2 is replaced with L, the 92nd amino acid with Y, the 94th amino acid with D, and the 96th amino acid with D (VL (2L3H7, amino acid sequence: SEQ ID NO: 46)).

[0304] [Example 15] Preparation of modified antibody Using the amino acid sequence information obtained in Example 14 and the amino acid sequence of VH described in (15-1), modified antibodies A-1 to A-8, as shown in Table 15, were prepared.

[0305] (15-1) VH containing the amino acid sequence represented by SEQ ID NO: 1, in which the 50th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is replaced with L, the 54th amino acid with W, the 55th amino acid with H, the 57th amino acid with T, the 58th amino acid with F, and the 100th amino acid with Y, resulting in VH (J2H2B9_Y, amino acid sequence: SEQ ID NO: 47)

[0306] [Table 15]

[0307] Plasmids containing the nucleotide sequences encoding the amino acid sequences of each antibody were prepared using the same method as in Example 12, and antibodies were prepared from these plasmids using the same method as in Example 2.

[0308] [Example 16] Evaluation of modified antibody The antigen-binding activity, FGF23 neutralizing activity, and thermal stability of the eight modified antibodies prepared in Example 15, along with antibody A as a control, were evaluated. For each modified antibody, the buffer used was D-PBS (Nacalai Tesque Co., Ltd., Code 14249-24), and the concentration was adjusted to 0.5 mg / mL. The samples were then diluted as needed for each measurement.

[0309] 16-1) Measurement of antigen-binding activity of modified antibodies The binding activity to human FGF23 was measured using the same method as in Example 3. Instead of immobilizing the Anti-human IgG antibody on a CM5 sensor chip, a ProteinA sensor chip (Global Life Science Technologies Japan Co., Ltd., Cat. No. 29127555) was used. Table 16 shows the binding rate constant (ka), dissociation rate constant (kd), and the resulting dissociation constant [kd1 / ka1=KD] for each antibody.

[0310] [Table 16]

[0311] Table 16 confirms that all antibodies A-1 through A-8 possess antigen-binding activity comparable to that of antibody A.

[0312] In Example 3, the antigen-binding activity of the I100A antibody and the I100Y antibody was lower than that of the A antibody. Furthermore, in all antibodies from A-1 to A-8, the 100th amino acid residue of VH is either A or Y.

[0313] Based on the above, it was confirmed that substituting the 100th amino acid residue of VH in antibody A with A or Y reduces the antigen-binding activity of the antibody, but further substitutions of amino acid residues in antibodies A-1 to A-8 improve the antigen-binding activity of the antibody to a level comparable to that of antibody A.

[0314] 16-2) Measurement of human FGF23 neutralizing activity The human FGF23 neutralizing activity of the modified antibodies was measured using the same method as in Example 9. The results are shown in Table 16. From Table 16, it was confirmed that all of the modified antibodies had FGF23 neutralizing activity comparable to that of antibody A from the same measurement lot.

[0315] In Example 9, the FGF23 neutralizing activity of the I100A and I100Y antibodies was lower than that of the A antibody. Furthermore, in all antibodies A-1 through A-8, the 100th amino acid residue of VH is either A or Y.

[0316] Based on the above, it was confirmed that substituting the 100th amino acid residue of VH in antibody A with A or Y reduces the antibody's FGF23 neutralizing activity, but further amino acid residue substitutions in antibodies A-1 to A-8 improve the neutralizing activity of the antibodies to a level comparable to that of antibody A.

[0317] 16-3) Confirmation of isoelectric point The isoelectric points of each antibody were determined using the iCE3 system (Protein Simple). Pharmalyte 3-10 for IEF (Global Life Science Technologies Japan, Cat. No. 17-0456-01) was used as the measurement carrier, pI Marker 5.12 (Protein Simple, Cat. No. 102224) was used as the acid marker, and pI Marker 9.77 (Protein Simple, Cat. No. 102219) was used as the basic marker. The measurement method followed a standard protocol.

[0318] The results obtained are shown in Table 16. From Table 16, it was confirmed that all modified antibodies showed a lower pI than antibody A.

[0319] 16-4) Confirmation of antibody degradation inhibition rate The degradation inhibition rate of antibodies A-1 to A-8 was confirmed using the same method as in Example 4. In this example, each antibody was allowed to stand at 40°C for 2 weeks, and antibody degradation was confirmed using a bioanalyzer electrophoresis system (Agilent Technologies, Inc.) and the Agilent Protein 230 kit (Agilent Technologies, Inc., Cat. 5067-1517). Sample preparation and electrophoresis conditions were carried out according to the protocol included with the kit. In the obtained electropherogram, the H chain band of the antibody was detected at 63 kDa, and the band corresponding to the antibody degradation product was detected at approximately 55 kDa. The peak area percentage (%) of the band corresponding to the antibody degradation product and the ratio (%) of the peak area percentage of the degradation band of each antibody to antibody A are shown in Tables 17 and 18.

[0320] [Table 17]

[0321] [Table 18]

[0322] As shown in Tables 17 and 18, it was confirmed that degradation was suppressed in all antibodies from A-1 to A-8 compared to antibody A.

[0323] 16-5) Confirmation of thermal stability The thermal stability of six antibodies—A antibody, A_YTE antibody, A-1 antibody, A-3 antibody, A-5 antibody, and A-8 antibody—was evaluated in the same manner as described in Example 5. The results are shown in Table 19.

[0324] [Table 19]

[0325] As shown in Table 19, there was no change in the thermal stability of each part of the antibody between antibody A and antibody A_YTE. On the other hand, antibodies A-1 and A-3, which have the same heavy chain constant region amino acid sequence as antibody A_YTE, showed approximately 3 degrees higher thermal stability of Fab compared to antibody A and antibody A_YTE.

[0326] [Example 17] Preparation of A-9 antibody and A-10 antibody The A-9 antibody and A-10 antibody listed in Table 20 were prepared using the same method as in Example 2 (hereinafter, the A-9 antibody and A-10 antibody may also be referred to as modified antibodies).

[0327] For the A-9 antibody, the light chain expression vector used was the pcDNA3.4 vector (Invitrogen), rather than the pCI vector used in Example 2. Furthermore, for both the A-9 and A-10 antibodies, the light chain expression vector and the heavy chain expression vector were mixed in a 1:1 ratio and introduced into cells.

[0328] [Table 20]

[0329] For the VL of the A-9 antibody, affinity maturation using phage display was performed on the I100A antibody at Abwiz Bio, and the following amino acid sequence information was obtained.

[0330] In a VL containing the amino acid sequence represented by SEQ ID NO: 2, the 92nd amino acid of the amino acid sequence represented by SEQ ID NO: 2 is replaced with W, the 94th amino acid with D, and the 96th amino acid with D (amino acid sequence: SEQ ID NO: 58).

[0331] The following VL (Very Large Volume) was designed and used for the A-10 antibody.

[0332] In a VL containing the amino acid sequence represented by SEQ ID NO: 2, the 92nd amino acid of the amino acid sequence represented by SEQ ID NO: 2 is replaced with Y, the 94th amino acid with D, and the 96th amino acid with D (amino acid sequence: SEQ ID NO: 59).

[0333] [Example 18] Evaluation of modified antibody The antigen-binding activity, FGF23 neutralizing activity, and thermal stability of the two modified antibodies prepared in Example 17, along with antibody A as a control, were evaluated. For all modified antibodies, the buffer used was D-PBS (Nacalai Tesque Co., Ltd., Code 14249-24), and the concentration was adjusted to 1 mg / mL. Samples were then diluted as needed for each measurement.

[0334] (18-1) Measurement of antigen-binding activity of modified antibodies The binding activity to human FGF23 was measured using the same method as in Example 16. The binding rate constant (ka), dissociation rate constant (kd), and dissociation constant [kd1 / ka1=KD] calculated from each antibody are shown in Table 21.

[0335] [Table 21]

[0336] Table 21 confirms that the A-9 antibody has twice the antigen-binding activity of the A antibody, and the A-10 antibody has similar antigen-binding activity to the A antibody. In Example 3, the I100A antibody and I100Y antibody showed lower antigen-binding activity than the A antibody. In addition, the 100th amino acid residue of the VH group in the A-9 antibody and the A-10 antibody is A and Y, respectively. Based on the above, it was confirmed that substituting the 100th amino acid residue of VH in antibody A with A or Y reduces the antigen-binding activity of the antibody, but further amino acid residue substitutions in A-9 antibody and I100Y antibody improve the antibody binding activity to a level equal to or greater than that of antibody A.

[0337] (18-2) Measurement of human FGF23 neutralizing activity The human FGF23 neutralizing activity of the modified antibodies was measured using the same method as in Example 9. The results are shown in Table 21. From Table 21, it was confirmed that antibody A-9 had three times the FGF23 neutralizing activity of antibody A, and antibody A-10 had twice the FGF23 neutralizing activity of antibody A.

[0338] In Example 9, I100A and I100Y showed lower neutralizing activity than antibody A. Furthermore, in antibodies A-9 and A-10, the 100th amino acid residue of VH is A and Y, respectively.

[0339] Based on the above, it was confirmed that substituting the 100th amino acid residue of VH in antibody A with A or Y reduces the antibody's FGF23 neutralizing activity, but further amino acid residue substitutions in antibodies A-9 and A-10 improve their FGF23 neutralizing activity to a level exceeding that of antibody A.

[0340] [Example 19] Confirmation of the degradation inhibition rate of the modified antibody We used four modified antibodies—A-1 antibody, A-3 antibody, A-5 antibody, and A-8 antibody—along with antibody A as a control to confirm the degradation inhibition rate.

[0341] In this example, the antibody was obtained by inserting the target gene sequence into a mammalian expression vector, introducing it into CHO cells, and then performing affinity purification and cation exchange chromatography from the supernatant of mammalian cell culture medium. The antibody was then replaced with pH 4 and pH 4.5 solvents containing 10 mM Sodium L-glutamate, 262 mM D-Sorbitol, and 0.05 mg / mL Polysorbase 80 using NAP25 (Global Life Science Technologies Japan Co., Ltd.), and the protein concentration was adjusted to 1 mg / mL.

[0342] The obtained antibody solution was allowed to stand at 40°C for 1 month and at 25°C for 3 months, and then frozen at -80°C. After thawing the antibody solution, antibody degradation was confirmed using the PA800Plus biologics analysis system (SCIEX). Basic conditions for sample preparation and analysis were carried out according to the method of Oscar Salas-Solano et al. (2006, Anal. Chem.:6583-6594), with modifications as appropriate.

[0343] The peak area percentage (%) of the band corresponding to the degradation product of the antibody is shown in Table 22 for pH 4.5 and in Table 23 for pH 5.0. Samples frozen at -80°C immediately after solvent transfer were labeled "Initial," samples left to stand at 40°C for 1 month and then frozen at -80°C were labeled "40°C 1M," and samples left to stand at 25°C for 3 months and then frozen at -80°C were labeled "25°C 3M." All samples were thawed under the same conditions before measurement.

[0344] [Table 22]

[0345] [Table 23]

[0346] As shown in Tables 22 and 23, similar to Example 16, even with buffer conditions of pH 4.5 and pH 5.0 and storage conditions of 40°C for 1 month and 25°C for 3 months, it was confirmed that the degradation of antibodies A-1, A-3, A-5, and A-8 was suppressed compared to antibody A.

[0347] [Example 20] Confirmation of the degradation inhibition rate of the modified antibody The degradation inhibition rates were confirmed using the two modified antibodies prepared in Example 17, along with antibody A and antibody A-1 as controls.

[0348] In this example, the A antibody and A-1 antibody were obtained by inserting the target gene sequence into a mammalian expression vector, introducing it into CHO cells, and then obtaining purified antibodies from the supernatant of mammalian cell culture medium by affinity purification using MabSelect SuRe (Global Life Science Technologies Japan Co., Ltd.) and cation exchange chromatography. The solvent of the antibody solution was replaced with solvents at pH 4, pH 4.5, and pH 5 containing 10 mM Sodium L-glutamate, 262 mM D-Sorbitol, and 0.05 mg / mL Polysorbate using NAP25 (Global Life Science Technologies Japan Co., Ltd.), and the protein concentration was adjusted to 1 mg / mL.

[0349] The obtained antibody solution was allowed to stand at 40°C for one month, and then antibody degradation was confirmed using the LabChip microchip electrophoresis system (PerkinElmer) and the Protein Clear Reagent kit (PerkinElmer, Cat.CLS960014). Sample preparation and electrophoresis conditions were carried out according to the protocol included with the kit.

[0350] The peak area percentage (%) of the band corresponding to the degradation product of the antibody in the measurement results under reducing conditions is shown in Table 24 for pH 4.0, Table 25 for pH 4.5, and Table 26 for pH 5.0.

[0351] [Table 24]

[0352] [Table 25]

[0353] [Table 26]

[0354] As shown in Tables 24, 25, and 26, it was confirmed that the degradation of A-9 antibody and A-10 antibody was suppressed compared to antibody A under buffer conditions of pH 4.0, pH 4.5, or pH 5.0 and storage conditions of 40 degrees Celsius for one month.

[0355] [Example 21] To confirm the duration of the pharmacological effect after a single subcutaneous administration of the test antibody to male cynomolgus monkeys, serum inorganic phosphorus concentrations (mg / dL) were measured. A8 antibody (1.8 mg / kg) was administered subcutaneously as the test antibody. Blood samples were collected at time points for 56 days after administration, and the inorganic phosphorus concentration in the serum was measured at each time point. Phosphorus concentration was measured using the PNP-XDH method with a clinalyzer (JCA-BM6070).

[0356] The results obtained are shown in Table 27. After administration of A-8 antibody, serum inorganic phosphorus concentration increased from day 3 post-administration and remained higher than baseline inorganic phosphorus concentration (phosphorus concentration on Day 0 in Table 27) even on day 56 post-administration.

[0357] [Table 27]

[0358] On the other hand, after a single subcutaneous administration of 3 mg / kg of brosumab (KRN23, Crysvita) to cynomolgus monkeys, serum inorganic phosphorus levels increased from day 3 post-administration, and by day 42 post-administration, inorganic phosphorus levels decreased to levels equivalent to those of the vehicle-administered group and baseline (day 0 of administration) (Crysvita Subcutaneous Injection Drug Interview Form, December 2021 Revision (5th Edition), p. 44).

[0359] Therefore, the A-8 antibody was shown to have a longer duration of inorganic phosphorus concentration in serum in cynomolgus monkeys compared to brosumab.

[0360] Furthermore, A-1 antibody and A-5 antibody were subcutaneously administered to cynomolgus monkeys at a dose of 3 mg / kg, and serum inorganic phosphorus concentrations were measured using the same method as described above. As a result, similar to burosumab, these antibodies increased serum inorganic phosphorus concentrations from day 3 after administration, and decreased to baseline levels by day 42 after administration.

[0361] The amino acid sequence of the heavy chain constant region of burosumab is represented by SEQ ID NO: 53, while the amino acid sequences of the heavy chain constant region of antibodies A-1, A-5, and A-8 are represented by SEQ ID NO: 48. Of the A-1, A-5, and A-8 antibodies, which have identical heavy chain constant region amino acid sequences, only the A-8 antibody sustained serum inorganic phosphorus concentrations longer than burosumab in cynomolgus monkeys.

[0362] Therefore, the longer duration of inorganic phosphorus concentration in serum in cynomolgus monkeys with A-8 antibody compared to burosumab, A-1 antibody, and A-5 antibody is suggested to be due to differences in the amino acid sequence of the variable region, rather than differences in the amino acid sequence of the heavy chain constant region.

[0363] [Example 22] The inorganic phosphorus concentration (mg / dL) in serum was measured using the same method as in Example 21. A-9 antibody and A-10 antibody (3.0 mg / kg) were administered subcutaneously as test antibodies. Blood samples were collected at various time points for 57 days after administration, and the inorganic phosphorus concentration in serum was measured at each time point.

[0364] The results obtained are shown in Table 28.

[0365] [Table 28]

[0366] As shown in Table 28, similar to the A-8 antibody in Example 22, after administration of the A-9 antibody and A-10 antibody, serum inorganic phosphorus concentrations increased from day 3 post-administration and remained higher than baseline inorganic phosphorus concentrations (phosphorus concentration on Day 0 in Table 28) even on day 57 post-administration.

[0367] The amino acid sequences of the heavy chain constant region of antibodies A-9 and A-10 are the same as those of antibodies A-1, A-5, and A-8, represented by sequence number 48.

[0368] Therefore, it was suggested that the longer duration of inorganic phosphorus concentration in serum in cynomolgus monkeys for A-9 and A-10 antibodies compared to brosumab, A-1, and A-5 antibodies is due not to differences in the amino acid sequence of the heavy chain constant region, but rather to differences in the amino acid sequence of the variable region, similar to the A-8 antibody.

[0369] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2022-128011 filed on 10 August 2022 and Japanese Patent Application No. 2022-164256 filed on 12 October 2022, which are incorporated herein by reference in their entirety. All references incorporated herein are incorporated as a whole. [Sequence Listing Free Text]

[0370] SEQ ID NO: 1: Amino acid sequence of VH of antibody A SEQ ID NO: 2: Amino acid sequence of VL of antibody A SEQ ID NO: 3: Amino acid sequence of VH of I100A antibody SEQ ID NO: 4: Amino acid sequence of VH of I100L antibody SEQ ID NO: 5: Amino acid sequence of VH of I100V antibody SEQ ID NO: 6: Amino acid sequence of VH of I100Y antibody SEQ ID NO:7: Amino acid sequence of VH of I100W antibody SEQ ID NO: 8: Amino acid sequence of VH of I100T antibody SEQ ID NO: 9: Amino acid sequence of VH of I100S antibody SEQ ID NO: 10: Amino acid sequence of VH of I100R antibody SEQ ID NO: 11: Amino acid sequence of VH of I100Q antibody SEQ ID NO: 12: Amino acid sequence of VH of I100N antibody SEQ ID NO: 13: Amino acid sequence of VH of I100M antibody SEQ ID NO: 14: Amino acid sequence of VH of I100K antibody SEQ ID NO: 15: Amino acid sequence of VH of I100H antibody SEQ ID NO: 16: Amino acid sequence of VH of I100G antibody SEQ ID NO: 17: Amino acid sequence of VH of I100F antibody SEQ ID NO: 18: Amino acid sequence of VH of I100E antibody SEQ ID NO: 19: Amino acid sequence of VH of I100D antibody SEQ ID NO: 20: Amino acid sequence of VH of the D105A antibody SEQ ID NO: 21: Amino acid sequence of VH of the D105E antibody SEQ ID NO: 22: Amino acid sequence of VH of D105F antibody SEQ ID NO: 23: Amino acid sequence of VH of D105G antibody SEQ ID NO: 24: Amino acid sequence of VH of D105H antibody SEQ ID NO: 25: Amino acid sequence of VH of D105I antibody SEQ ID NO: 26: Amino acid sequence of VH of D105K antibody SEQ ID NO: 27: Amino acid sequence of VH of the D105L antibody SEQ ID NO: 28: Amino acid sequence of VH of D105M antibody SEQ ID NO: 29: Amino acid sequence of VH of D105P antibody SEQ ID NO: 30: Amino acid sequence of VH of the D105Q antibody SEQ ID NO: 31: Amino acid sequence of VH of D105R antibody SEQ ID NO: 32: Amino acid sequence of VH of D105V antibody SEQ ID NO: 33: Amino acid sequence of VH of D105W antibody SEQ ID NO: 34: Amino acid sequence of VH of D105Y antibody SEQ ID NO: 35: Amino acid sequence of VH of D105T antibody SEQ ID NO: 36: Amino acid sequence of VH of D105N antibody SEQ ID NO: 37: Amino acid sequence of VH of D105S antibody Amino acid sequence of Sequence ID No. 38: H2B11_A Amino acid sequence of SEQ ID NO: 39:J2H2B9_A Amino acid sequence of SEQ ID NO: 40: J2H2E9_A Amino acid sequence of SEQ ID NO: 41: 2H2E1_A Amino acid sequence of SEQ ID NO: 42: 2H2E5_A Amino acid sequence of SEQ ID NO: 43: 2H2E8_Y Amino acid sequence of SEQ ID NO: 44:L3G12 Amino acid sequence of SEQ ID NO: 45:L1H8 Amino acid sequence of SEQ ID NO: 46:2L3H7 Amino acid sequence of SEQ ID NO: 47: J2H2B9_Y Sequence ID 48: Amino acid sequence of CH in YTE SEQ ID NO: 49: Amino acid sequence of CH in G2_YTE SEQ ID NO: 50: Amino acid sequence of CH in G2_LS amino acid sequence of CH in SEQ ID NO: 51: G4PE_R409K_P Amino acid sequence of CH in SEQ ID NO: 52: G4PE_R409K_LS Sequence ID 53: Amino acid sequence of the G1 constant region Sequence ID 54: Amino acid sequence of the His-tag extracellular domain of human FcRn Sequence ID 55: Amino acid sequence of human β2-microglobulin Sequence ID 56: Amino acid sequence of the His-tag extracellular domain of the cynomolgus monkey FcRn. Sequence ID 57: Amino acid sequence of cynomolgus monkey β2 microglobulin SEQ ID NO: 58: Amino acid sequence of VL of A-9 antibody SEQ ID NO: 59: Amino acid sequence of VL of A-10 antibody< / super>

Claims

1. An antibody comprising a heavy chain variable region (hereinafter referred to as VH) containing the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region (hereinafter referred to as VL) containing the amino acid sequence represented by SEQ ID NO: 2, wherein at least the 100th or 105th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted with another amino acid residue, wherein the antibody or antibody fragment binds to FGF23.

2. The antibody or antibody fragment according to claim 1, wherein the 100th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in VH is substituted with one amino acid residue selected from alanine, asparagine, glycine, tyrosine, arginine, aspartic acid, histidine, tryptophan, and methionine residues.

3. The antibody or antibody fragment according to claim 1 or 2, wherein the 100th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in VH is substituted with an alanine residue or a tyrosine residue.

4. The antibody or antibody fragment according to any one of claims 1 to 3, wherein the 105th amino acid residue of the amino acid sequence represented by Sequence ID No. 1 in VH is substituted with one amino acid residue selected from alanine, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, valine, tryptophan, tyrosine, threonine, asparagine, and serine residues.

5. The antibody or antibody fragment according to any one of claims 1 to 4, wherein the antibody further comprises one substitution selected from (a1) to (a4) below. (a1) At least one substitution selected from the following: substitution of the 50th amino acid residue with a leucine residue in the amino acid sequence represented by Sequence ID No. 1 in VH, substitution of the 54th amino acid residue with a tryptophan residue, substitution of the 55th amino acid residue with a histidine residue, substitution of the 57th amino acid residue with a threonine residue, and substitution of the 58th amino acid residue with a phenylalanine residue. (a2) At least one substitution selected from the following: substitution of the 91st amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in VL with a methionine or leucine residue, substitution of the 92nd amino acid residue with a tyrosine residue, substitution of the 94th amino acid residue with an aspartic acid residue, and substitution of the 96th amino acid residue with an asparagine or aspartic acid residue. (a3) At least one substitution selected from the substitution of the 28th amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in VL with an aspartic acid residue, the 29th amino acid residue with a valine residue, the 31st amino acid residue with a threonine residue, and the 34th amino acid residue with a leucine residue, and (a4) At least one substitution selected from the substitution of the 92nd amino acid residue of the amino acid sequence represented by Sequence ID No. 2 in VL with a tyrosine or tryptophan residue, the 94th amino acid residue with an aspartic acid residue, and the 96th amino acid residue with an aspartic acid residue.

6. The antibody or antibody fragment according to any one of claims 1 to 5, wherein the antibody is selected from (c1) to (c10) below. (c1) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 39 and VL containing the amino acid sequence represented by SEQ ID NO: 2, (c2) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 47 and VL containing the amino acid sequence represented by SEQ ID NO: 2, (c3) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 44, (c4) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO: 44, (c5) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 45, (c6) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO: 45, (c7) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 46, (c8) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO: 46, (c9) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 3 and VL containing the amino acid sequence represented by SEQ ID NO: 58, and (c10) An antibody comprising VH containing the amino acid sequence represented by SEQ ID NO: 6 and VL containing the amino acid sequence represented by SEQ ID NO:

59.

7. The antibody or antibody fragment according to any one of claims 1 to 6, wherein the antibody subclass is IgG1, IgG2, IgG3, or IgG4.

8. The antibody or antibody fragment according to any one of claims 1 to 7, wherein the Fc region of the antibody is selected from (d1) to (d5) below. (d1) Fc region including substitution of the 252nd amino acid residue of the EU index with a tyrosine residue, substitution of the 254th amino acid residue with a threonine residue, and substitution of the 256th amino acid residue with a glutamic acid residue, (d2) Fc region including substitution of the 428th amino acid residue of the EU index with a leucine residue, and substitution of the 434th amino acid residue with a serine residue, (d3) Fc region including substitution of the 308th amino acid residue of the EU index with a proline residue, (d4) An Fc region including a substitution of the 250th amino acid residue of the EU index with a glutamine residue, and a substitution of the 428th amino acid residue with a leucine residue, (d5) An Fc region containing the substitution of the amino acid residue at EU index 434 with an alanine residue.

9. The antibody or antibody fragment according to any one of claims 1 to 8, wherein the heavy chain constant region of the antibody comprises an amino acid sequence represented by SEQ ID NO: 48, 49, 50, 51, or 52.

10. The antibody fragments are Fab, Fab', (Fab') ２ The antibody fragment according to any one of claims 1 to 9, which is one selected from a peptide comprising scFv, Diabody, dsFv, and CDR.

11. A nucleic acid having a base sequence encoding an antibody or antibody fragment according to any one of claims 1 to 10.

12. A vector containing nucleic acid as described in claim 11.

13. Transformed cells comprising the vector according to claim 12.

14. A method for producing an antibody or antibody fragment according to any one of claims 1 to 10, comprising culturing the transformed cells according to claim 13 in a culture medium and collecting an antibody or antibody fragment from the culture.

15. A composition comprising an antibody or an antibody fragment according to any one of claims 1 to 10.

16. A therapeutic agent for human FGF23-related disease comprising the antibody or antibody fragment described in any one of claims 1 to 10.

17. A method for treating human FGF23-related disease comprising the antibody or antibody fragment described in any one of claims 1 to 10.

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