Anti-FGF23 antibody or antibody fragment thereof

Abstract

The present invention pertains to: an antibody which binds to FGF23 and in which, with respect to an antibody that includes a heavy chain variable region (hereinafter, referred to as VH) having an amino acid sequence indicated in SEQ ID NO. 1 and a light chain variable region (hereinafter, referred to as VL) having an amino acid sequence indicated in SEQ ID NO. 2, at least the amino acid residue at positon 100 or the amino acid residue at position 105 in the amino acid sequence indicated in SEQ ID NO. 1 for VH is substituted with another amino acid residue; and a fragment of the antibody.

Description

[Technical Field]

[0001] The present invention relates to an anti-FGF23 antibody or an antibody fragment thereof. [Background technology]

[0002] Fibroblast growth factors (hereinafter referred to as FGFs) form a family of structurally similar polypeptides, and have been reported to have various functions, including not only fibroblast proliferation activity but also mesoderm and neuroectoderm proliferation, angiogenesis, and limb bud formation during development. In adults, they also function as homeostatic factors, such as tissue maintenance, repair, regeneration, and metabolism (Non-Patent Document 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 is composed of approximately 150 to 300 amino acids, with approximately 120 amino acids in the core sequence being identical at a rate of approximately 30 to 60%. The FGF family is classified into those expressed as secreted proteins that act on receptor tyrosine kinases and those expressed as intracellular proteins that act on voltage-dependent sodium channels and other molecules (Non-Patent Document 2).

[0004] FGF23 is a secretory protein that was identified in mice using a database search and PCR method based on its homology with FGF15, and then identified by a homology search. Human FGF23 is a polypeptide of 251 amino acid residues, and it is known that the N-terminal 24 residues function as a secretion signal and are cleaved during the protein maturation process (Non-Patent Document 3).

[0005] Hypophosphatemic diseases caused by excessive production of FGF23 are known, and are broadly classified into diseases for which the causative gene is known and acquired diseases (Non-Patent Document 4). Of the FGF23-related hypophosphatemic diseases for which the causative gene is known, the most common is X-linked hypophosphatemic rickets (hereafter referred to as XLH), which is caused by mutations in the phosphate-regulating endopeptidase homolog, X-linked (PHEX), and numerous PHEX gene mutations have been reported to date (Non-Patent Document 5).

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

[0007] Active vitamin D3 preparations and oral phosphate preparations have traditionally been used as symptomatic treatments for these FGF23-related hypophosphatemia, but long-term administration can lead to complications such as hypercalcemia and hypercalcemia, nephrocalcinosis, and persistent hyperparathyroidism (Non-Patent Documents 9 and 10).

[0008] Burosumab, an FGF23-neutralizing antibody, is known as a therapeutic agent for the above-mentioned diseases that are thought to be caused by the overproduction of FGF23.

[0009] 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 Summary of the Invention [Problem to be solved by the invention]

[0012] The present inventors have extensively studied the physical properties of the anti-human FGF23 antibody described in WO 2008 / 099969 and found that the antibody loses stability and undergoes degradation at low pH, and that this degradation is due to cleavage between D at position 99 and I at position 100 in the amino acid sequence of the heavy chain variable region of the antibody. It is generally known that antibodies are prone to aggregation when antibody formulations are concentrated, and that increasing the absolute value of the charge of the protein in the 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, one method of suppressing antibody aggregation is to lower the pH of the formulation. Therefore, when it is necessary to lower the pH of the antibody formulation, an antibody with a wide pH range is more desirable.

[0013] Therefore, an object of the present invention is to provide a novel anti-FGF23 antibody in which antibody degradation at a lower pH is suppressed compared to the anti-human FGF23 antibody described in WO 2008 / 099969. [Means for solving the problem]

[0014] As a result of intensive research into the above-mentioned problems, the present inventors found that the above-mentioned problems can be solved by using an anti-FGF23 antibody in which the 100th or 105th amino acid residue of the VH of the anti-FGF23 antibody described in WO 2008 / 099969 is substituted, and thus completed the present invention.

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

[0016] The anti-FGF23 antibody of the present invention is less susceptible to decomposition at low pH and exhibits superior stability compared to the anti-FGF23 antibody described in WO 2008 / 099969. The present invention can provide an anti-FGF23 antibody or an antibody fragment thereof, a nucleic acid having a nucleotide sequence encoding the antibody or the antibody fragment, a vector containing the nucleic acid, a transformed cell containing the vector, a method for producing the antibody or the antibody fragment, and a composition containing the antibody or the antibody fragment. DETAILED DESCRIPTION OF THE INVENTION

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

[0018] FGF23 is a type of fibroblast growth factor and a hormone derived from bone cells. Human FGF is a protein consisting of 251 amino acids, including a 24-amino acid secretion signal at the N-terminus, which is cleaved during the protein maturation process. The functions of FGF23 include inhibiting renal phosphorus reabsorption and vitamin D activation, primarily via the FGF receptor 1 (FGFR1) / α-Klotho complex on renal proximal tubule cells.

[0019] In the present invention, human FGF23 includes a polypeptide comprising the amino acid sequence of NCBI accession number NP_065689, a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of NCBI accession number NP_065689 and having the function of human FGF23, and a polypeptide consisting of an amino acid sequence 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] A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in NCBI Accession No. NP_065689 can be generated by 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 (1985)]. (1985)] or the like, can be obtained by introducing site-specific mutations into DNA encoding a polypeptide comprising the amino acid sequence shown in NCBI Accession No. NP_065689, for example.

[0021] The number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example, 1 to 20, more preferably 1 to several, for example, 1 to 5 amino acids.

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

[0023] DNA that hybridizes under stringent conditions refers to hybridizable DNA obtained by colony hybridization, plaque hybridization, Southern blot hybridization, DNA microarray, or the like, using DNA containing the nucleotide sequence of NM_020638 as a probe.

[0024] Specifically, the DNA can be identified by performing hybridization at 65°C in the presence of 0.7 to 1.0 mol / L sodium chloride using DNA derived from hybridized colonies or plaques, or a filter or slide onto which a PCR product or oligo DNA having the sequence has been immobilized [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University, (1995)], followed by washing the filter or slide at 65°C using 0.1 to 2x SSC solution (1x SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate).

[0025] Examples of hybridizable DNA include DNA having at least 60% or more homology with 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 found in the nucleotide sequences of genes encoding proteins in eukaryotes. Genes used in the present invention that have small mutations in their nucleotide sequences due to such polymorphisms are also included in the genes encoding human FGF23 of the present invention.

[0027] Unless otherwise specified, the homology values ​​in the present invention may be values ​​calculated using a homology search program known to those skilled in the art. For base sequences, the values ​​may be values ​​calculated using default parameters in BLAST [J. Mol. Biol., 215, 403 (1990)], and for amino acid sequences, the values ​​may be values ​​calculated using default parameters in BLAST2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649 (1997)].

[0028] The default parameters are: G (Cost to open gap) is 5 for nucleotide sequences and 11 for amino acid sequences; -E (Cost to extend gap) is 2 for nucleotide sequences and 1 for amino acid sequences; -q (Penalty for nucleotide mismatch) is -3; -r (reward for nucleotide match) is 1; -e (expect value) is 10; -W (wordsize) is 11 residues for nucleotide sequences and 3 residues for amino acid sequences; -y[Dropoff(X) for blast extensions in bits] is 20 for blastn and 7 for 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] A polypeptide containing a partial sequence of the amino acid sequence of NCBI Accession No. NP_065689 can be produced by methods known to those skilled in the art. Specifically, it can be produced 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 the deleted portion has been introduced. Furthermore, a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of NCBI Accession No. NP_065689 can be obtained by a method similar to that described above. Furthermore, a polypeptide consisting of the amino acid sequence of NCBI Accession No. NP_065689 or a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of NCBI Accession No. NP_065689 can also be produced by chemical synthesis methods such as the fluorenylmethyloxycarbonyl (Fmoc) method and the t-butyloxycarbonyl (tBoc) method.

[0030] In the present invention, deletion, substitution, addition, etc. of amino acids are also referred to as amino acid modification.

[0031] The antibodies of the present invention include polyclonal, monoclonal, and oligoclonal antibodies. A polyclonal antibody is a population of antibody molecules secreted by different clones of antibody-producing cells. A monoclonal antibody is an antibody secreted by a single clone of antibody-producing cells, recognizes only one epitope (also called an antigenic determinant), and has a uniform amino acid sequence (primary sequence) that constitutes the monoclonal antibody. An oligoclonal antibody is a population of antibody molecules that is a mixture of multiple different monoclonal antibodies.

[0032] The monoclonal antibody of the present invention may be an antibody produced by a hybridoma, or a recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.

[0033] Examples of epitopes include a single amino acid sequence, a three-dimensional structure consisting of an amino acid sequence, an amino acid sequence modified by post-translational modification, and a three-dimensional structure consisting of said amino acid sequence, which are recognized and bound by a monoclonal antibody.

[0034] Examples of post-translationally modified amino acid sequences include O-linked glycans in which glycans are bound to Tyr and Ser with OH substituents, N-linked glycans in which Gln and Asn with NH2 substituents are bound to, and sulfate groups in which sulfate molecules are bound to Tyr and Ser with OH substituents.

[0035] The binding of the antibody of the present invention to human FGF23 can be confirmed by measuring the binding affinity of the antibody of the present invention to human FGF23 using ELISA, surface plasmon resonance, etc. Alternatively, this can be confirmed by combining known immunological detection methods [Monoclonal Antibodies—Principles and Practice, Third Edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)], etc.

[0036] The amino acid residues or epitopes of human FGF23 to which the antibodies of the present invention bind can be determined by antibody binding experiments using deletions of some domains of human FGF23, mutants in which domains have been replaced with those from other proteins, and partial peptide fragments of human FGF23.

[0037] Alternatively, the amino acid residue or epitope 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 peptide fragments of human FGF23 digested with a protease and performing epitope mapping using known mass spectrometry.

[0038] Antibody molecules are also called immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into isotypes based on differences in molecular structure: IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM. IgG1, IgG2, IgG3, and IgG4, which share relatively high amino acid sequence homology, are collectively referred to as IgG.

[0039] An antibody molecule is composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (hereinafter referred to as L chains). Furthermore, the H chain is composed of VH and H chain constant region (also referred to as CH) from the N-terminus, while the L chain is composed of VL and L chain constant region (also referred to as CL) from the N-terminus. CH is known to have α, δ, ε, γ, and μ chains in each subclass. CH is further composed of 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. Furthermore, the CH2 domain and CH3 domain together are called the Fc region or simply Fc. CL is a region that contains the C λ Chain and C κ The chain is known.

[0040] The CH1 domain, hinge domain, CH2 domain, CH3 domain, and Fc region of the present invention can be identified by the amino acid residue numbering from the N-terminus according to the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services (1991)] (hereinafter simply referred to as EU index). Specifically, CH1 is identified as the amino acid sequence of positions 118 to 215 in the EU index, the hinge as the amino acid sequence of positions 216 to 230 in the EU index, CH2 as the amino acid sequence of positions 231 to 340 in the EU index, and CH3 as the amino acid sequence of positions 341 to 447 in the EU index.

[0041] The antibodies of the present invention particularly include recombinant antibodies such as recombinant mouse antibodies, recombinant rat antibodies, recombinant rabbit antibodies, human chimeric antibodies (hereinafter also simply referred to as chimeric antibodies), humanized antibodies (also referred to as human CDR-grafted antibodies), and human antibodies, which are produced by genetic engineering. The antibodies of the present invention also include recombinant antibodies produced by recombining the H chain (or VH) and L chain (or VL) derived from two different types of antibodies. The two different types of antibodies may be monoclonal antibodies derived from hybridomas, chimeric antibodies, humanized antibodies, or human antibodies. Furthermore, the antibodies of the present invention also include recombinant antibodies in which appropriate amino acid residues have been substituted when producing the above-mentioned recombinant antibodies.

[0042] A chimeric antibody is an antibody consisting of the VH and VL of an antibody from an animal other than a human (non-human animal) and the CH and CL of a human antibody. Any non-human animal can be used, such as a mouse, rat, hamster, or rabbit, as long as it is possible to produce a hybridoma from it.

[0043] Hybridomas are cells that produce monoclonal antibodies with desired antigen specificity, obtained by cell fusion of B cells obtained by immunizing a non-human animal with an antigen with myeloma cells derived from a mouse or other animal. Therefore, the variable regions of antibodies produced by hybridomas consist of the amino acid sequences of non-human animal antibodies.

[0044] Chimeric antibodies can be produced by obtaining cDNA encoding the VH and VL of a monoclonal antibody from a hybridoma derived from non-human animal cells that produces the monoclonal antibody, inserting the cDNA into an expression vector for animal cells containing DNA encoding the CH and CL of a human antibody to construct a human chimeric antibody expression vector, and introducing the vector into animal cells to express the antibody.

[0045] A humanized antibody is an antibody in which the amino acid sequences of the CDRs of the VH and VL of a non-human animal antibody have been grafted onto the corresponding CDRs of the VH and VL of a human antibody. The regions other than the CDRs of the VH and VL are called framework regions (hereinafter referred to as FR).

[0046] Humanized antibodies can be produced by constructing a cDNA encoding a VH amino acid sequence consisting of the amino acid sequence of the CDR of the VH of a non-human animal antibody and the amino acid sequence of the FR of the VH of any human antibody, and a cDNA encoding a VL amino acid sequence consisting of the amino acid sequence of the CDR of the VL of a non-human animal antibody and the amino acid sequence of the FR of the VL of any human antibody, and inserting each of these into an expression vector for animal cells containing DNA encoding the CH and CL of a human antibody to construct a humanized antibody expression vector, which can then be expressed and produced by introducing it into animal cells.

[0047] Human antibodies originally refer to antibodies that naturally occur in the human body, but also include human antibody phage libraries produced through recent advances in genetic engineering, cell engineering, and developmental engineering technologies, and antibodies obtained from human antibody-producing transgenic animals.

[0048] Human antibodies can be obtained by immunizing mice carrying human immunoglobulin genes with a desired antigen (Tomizuka K. et al., Proc Natl Acad Sci USA. 97, 722-7, 2000). Alternatively, human antibodies can be obtained without immunization by selecting human antibodies with the desired binding activity using a phage display library in which antibody genes have been amplified from human-derived B cells (Winter G. et al., Annu Rev Immunol. 12:433-55, 1994). Furthermore, human B cells can be immortalized using Epstein-Barr virus to generate cells that produce human antibodies with the desired binding activity, allowing human antibodies to be obtained (Rosen A. et al., Nature 267, 52-54, 1977).

[0049] Antibodies present in the human body can be obtained by, for example, immortalizing lymphocytes isolated from human peripheral blood by infecting them with EB virus or the like, and then cloning the lymphocytes to obtain lymphocytes that produce the antibodies.The antibodies can then be purified from the culture of the 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 the phage genome. Phages expressing antibody fragments with the desired antigen-binding activity can be recovered from the library using their binding activity toward an antigen-immobilized substrate as an indicator. These antibody fragments can also be converted into human antibody molecules consisting of two complete heavy chains and two complete light chains by genetic engineering techniques.

[0051] A human antibody-producing transgenic animal is an animal in which a human antibody gene has been integrated into the chromosome of the host animal. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into the early embryo of another mouse, and then allowing them to develop. Human antibodies can be produced from human antibody-producing transgenic animals by obtaining human antibody-producing hybridomas using hybridoma production methods typically used in non-human mammals, and culturing them to produce and accumulate human antibodies in the culture.

[0052] The amino acid sequences of VH and VL of the antibody of the present invention may be any of the amino acid sequences of VH and VL 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. κ or C λ is preferred.

[0054] The CH of the antibody of the present invention may be any immunoglobulin, but the 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 is linked to an antibody fragment, Fc fusion proteins (also called immunoadhesins) in which Fc is linked to a naturally occurring ligand or receptor, and Fc fusion proteins in which multiple Fc regions are fused together.

[0056] The antibodies or antibody fragments of the present invention also include antibodies or antibody fragments containing any post-translationally modified amino acid residues, such as deletion of a lysine residue at the C-terminus of the heavy chain (lysine clipping) or 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 refers to an antibody fragment that binds to human FGF23 and is less susceptible to degradation at low pH than an antibody comprising 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 antibodies (scFv), dimerized V regions (diabodies), disulfide-stabilized V regions (dsFv), and peptides containing multiple CDRs. Fab is a fragment obtained by treating an IgG antibody with the protease papain (cleaved at the 224th amino acid residue of the H chain). Fab is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is composed of approximately the N-terminal half of the H chain and the entire L chain linked by a disulfide bond (SS bond).

[0058] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000 that has antigen-binding activity and is slightly larger than Fab fragments obtained by treating IgG with the protease pepsin (cleaved at the 234th amino acid residue of the H chain). Fab' is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by cleaving the SS bond in the hinge region of the above-mentioned F(ab')2.

[0059] scFv is an antibody fragment with antigen-binding activity, which is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (P), such as a linker peptide consisting of any number of linkers (G4S) consisting of four Gly and one Ser residues.

[0060] Diabodies are antibody fragments formed by dimerization of scFvs with the same or different antigen-binding specificities, and have bivalent antigen-binding activity for the same antigen or specific antigen-binding activity for different antigens.

[0061] dsFv refers to polypeptides in which one amino acid residue in each of VH and VL is substituted with a cysteine ​​residue, and the cysteine ​​residues are linked via an S—S bond.

[0062] CDR-containing peptides comprise at least one region of the VH or VL CDR. In peptides containing multiple CDRs, the CDRs can be linked directly or via an appropriate peptide linker. The modified antibodies of the present invention can be produced by constructing DNA encoding the VH and VL CDRs, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism for expression. Alternatively, CDR-containing peptides can be produced by chemical synthesis, such as the Fmoc or tBoc method.

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

[0064] This antibody has higher stability at low pH and is less susceptible to antibody degradation than an antibody comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 1 and a VL comprising the amino acid sequence shown in SEQ ID NO: 2. Preferably, this antibody is an antibody that binds to FGF23 comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 1 and a VL comprising the amino acid sequence shown in SEQ ID NO: 2, in which at least the 100th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 in VH has been substituted with an alanine residue or a tyrosine residue.

[0065] In the present invention, low pH refers to weak acidity below pH 6 or acidity, and examples include pH 5, pH 4.5, pH 4, etc., but are not particularly limited thereto.

[0066] The antibodies of the present invention exhibit reduced degradation at low pH and superior stability compared to antibodies comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2. In the present invention, antibody degradation can be measured by size exclusion chromatography (hereinafter referred to as SEC) or SDS polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE), or the like.

[0067] The fact that the antibody of the present invention has reduced antibody degradation at low pH compared to an antibody comprising a VH containing the amino acid sequence shown in SEQ ID NO: 1 and a VL containing the amino acid sequence shown in SEQ ID NO: 2 can be confirmed, for example, by a method comprising the following steps (I) to (III), but is not limited to this method. (I) An antibody solution is prepared by replacing the solvent of an antibody solution containing an antibody of the present invention or an antibody comprising a VH having the amino acid sequence represented by SEQ ID NO: 1 and a VL having the amino acid sequence represented by SEQ ID NO: 2 with an appropriate solvent of pH 4, 4.5, or 5 using a column such as NAP™ 25 (GE Healthcare Life Sciences). (II) 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 the peaks (%) corresponding to antibody degradation products are detected by SEC. Alternatively, the antibody solution prepared in (I) above can be left standing 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 antibody degradation product. (III) If the peak (%) corresponding to the antibody degradation product detected by SEC in (II) above is reduced in the antibody of the present invention compared to an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2, or if the intensity of the band around 40 kDa in SDS-PAGE is reduced in the antibody of the present invention compared to an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2, it can be confirmed that the antibody degradation at low pH is suppressed in the antibody of the present invention compared to an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising 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 an Agilent Protein 230 Kit (Agilent Technologies Inc.) may be used. In this case, if the peak area percentage (%) of a band corresponding to an antibody degradation product detected at a size approximately 10 to 20 kDa smaller than the peak of the full-length H chain (e.g., 40 to 60 kDa) in the resulting electropherogram is lower for the antibody of the present invention than for an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2, it can be confirmed that antibody degradation at low pH is suppressed for the antibody of the present invention compared to an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2.

[0069] Specifically, examples of embodiments of the antibodies of the present invention that exhibit stability at low pH include the following. Compared to an antibody comprising a VH containing the amino acid sequence set forth in SEQ ID NO: 1 and a VL containing the amino acid sequence set forth in SEQ ID NO: 2, the intensity of the band corresponding to a molecular weight of approximately 40 kDa determined by SDS-PAGE is reduced after incubation at a low pH, such as pH 4, 4.5, or 5, and at 40°C for one month or two weeks, or at 25°C for three months. The intensity of the band can be assessed visually. Compared to an antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 1 and a VL having the amino acid sequence set forth in SEQ ID NO: 2, the peaks (%) corresponding to antibody degradation products detected by SEC after incubation at a low pH, such as pH 4, 4.5, or 5, and at 40°C for one month or two weeks, or at 25°C for three months, are reduced. The peaks (%) are preferably 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 an antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 1 and a VL having the amino acid sequence set forth in SEQ ID NO: 2. Compared to an antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2, the peak area percentage (%) of a band (e.g., 40-60 kDa) corresponding to an antibody degradation product detected at a size approximately 10-20 kDa smaller than the peak of the full-length H chain is reduced in an electropherogram using a Bioanalyzer Electrophoresis System (Agilent Technologies Inc.) and an Agilent Protein 230 Kit (Agilent Technologies Inc.) after incubation at a low pH (e.g., pH 4, 4.5, or 5) and 40°C for one month or two weeks, or at 25°C for three months, compared to an antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2. 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 an antibody comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0070] An embodiment of the antibody of the present invention includes the above-described antibody further comprising one substitution selected from the following (a1) to (a4): (a1) at least one substitution selected from the group consisting of substitution of the 50th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 in VH with a leucine residue, 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 substitution of the 91st amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL with a methionine residue or a 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 residue or an aspartic acid residue; (a3) at least one substitution selected from the group consisting of a substitution of an aspartic acid residue at position 28 of the amino acid sequence represented by SEQ ID NO: 2 in VL, a substitution of a valine residue at position 29, a substitution of a threonine residue at position 31, and a substitution of a leucine residue at position 34; and (a4) At least one substitution selected from the group consisting of a substitution of the 92nd amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL with a tyrosine residue or a tryptophan residue, a substitution of the 94th amino acid residue with an aspartic acid residue, and a substitution of the 96th amino acid residue with an aspartic acid residue.

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

[0072] The antibody of the present invention can improve its FGF23-binding activity and FGF23-neutralizing activity by substituting one amino acid residue selected from the above (a1) to (a4). FGF23-neutralizing activity refers to the activity of inhibiting the signal generated by binding of FGF23 to a receptor. An example of an FGF23 receptor is the complex of FGFR1 and αKlotho.

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

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

[0075] (b1) An antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2, in which the 50th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted 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 residue or a tyrosine residue;

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

[0077] (b3) An antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2, in which the 100th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted with an alanine residue or a tyrosine residue, and the 28th amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 in VL is substituted 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.

[0078] (b4) An antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2, in which the amino acid residue at position 100 in the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted with an alanine residue or a tyrosine residue, and the amino acid residue at position 91 in the amino acid sequence represented by SEQ ID NO: 2 in VL is substituted with a leucine residue, a tyros ...

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

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

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

[0082] Examples of Fc regions in which amino acid residues have been substituted to improve the FcRn-binding ability of antibodies include any one of the Fc regions (d1) to (d5) below, of which the Fc region (d1) is preferred. (d1) an Fc region comprising a substitution of the amino acid residue at position 252 in the EU index with a tyrosine residue, a substitution of the amino acid residue at position 254 with a threonine residue, and a substitution of the amino acid residue at position 256 with a glutamic acid residue; (d2) an Fc region comprising a substitution of the amino acid residue at position 428 of the EU index with a leucine residue and a substitution of the amino acid residue at position 434 with a serine residue; (d3) an Fc region comprising a substitution of the amino acid residue at position 308 of the EU index with a proline residue; (d4) an Fc region comprising a substitution of the amino acid residue at position 250 in the EU index with a glutamine residue and a substitution of the amino acid residue at position 428 with a leucine residue; and (d5) An Fc region containing a substitution of the amino acid residue at position 434 of the EU index with an alanine residue.

[0083] In the present invention, specific examples of amino acid sequences of heavy chain constant regions comprising Fc domains in which amino acid residues have been substituted to improve the FcRn-binding ability of antibodies include the amino acid sequences shown in SEQ ID NOs: 48, 49, 50, 51, and 52. Of these, the amino acid sequence shown in SEQ ID NO: 48 is preferred.

[0084] The monoclonal antibody or antibody fragment of the present invention includes derivatives of the antibody or antibody fragment in which a radioisotope, a low molecular weight drug, a high molecular weight drug, a protein, an antibody drug, or the like is chemically or genetically linked to the monoclonal antibody or antibody fragment of the present invention that binds to human FGF23.

[0085] Derivatives of antibodies or antibody fragments thereof can be produced by chemically binding radioisotopes, low molecular weight drugs, high molecular weight drugs, immunostimulants, proteins, antibody drugs, or nucleic acid drugs to the N-terminus, C-terminus, or appropriate substituents, side chains, or sugar chains of the H chain or L chain of the monoclonal antibody or antibody fragment of the present invention that binds to human FGF23.

[0086] Alternatively, the monoclonal antibody or antibody fragment thereof of the present invention that binds to human FGF23 can be produced by genetic engineering techniques, in which DNA encoding the monoclonal antibody or antibody fragment thereof is linked to DNA encoding the 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 radioisotopes include: 111 In, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 Radioisotopes can be directly bound to antibodies using the chloramine T method or the like. Alternatively, a substance that chelates radioisotopes can be bound to the antibody. Examples of chelating agents include 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).

[0088] Examples of low-molecular-weight drugs include anticancer drugs such as alkylating agents, nitrosoureas, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, platinum complex derivatives, M-phase inhibitors, and kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy, Inc. (1996)], steroid drugs such as hydrocortisone and prednisone, non-steroid drugs such as aspirin and indomethacin, immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, and antihistamines such as chlorpheniramine maleate and clemacitin [Inflammation and Anti-inflammatory Therapy, Ishiyaku Publishing Co., Ltd. (1982)].

[0089] Examples of anticancer drugs include amifostine (Ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), epirubicin, gemcitabine (Gemzar), daunorubicin, procarbazine, mitomatin, and the like. isin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol), docetaxel (Taxotere), 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, pegaspargase, pentostatin, pipobroman, tamoxifen, goserelin , leuprorenin, flutamide, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone, methylprednisolone, vindesine, nimustine, semustine, capecitabine, tomudex, 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 factor inhibitors such as Iressa or TarcevaReceptor (EGFR) inhibitors, radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine, all-trans retinoic acid, thalidomide, lenalidomide, anastrozole, fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (Targretin), tamoxifen, dexamethasone, progestins, estradiol Examples of such antihistamines include benzodiazepines, benzocaine, benzodiazepines (BPO), benzodiazepines (BPO), benzocaine ...

[0090] Examples of methods for binding a low molecular weight drug to an antibody include binding between the amino groups of the drug and the antibody via glutaraldehyde, or binding between the amino groups of the drug and the carboxyl groups 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, and hydroxypropyl methacrylamide. By conjugating these polymeric compounds to antibodies or antibody fragments thereof, 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 Pharmaceuticals, Hirokawa Shoten (1993)].

[0092] For example, a method for conjugating PEG to an antibody includes reacting it with a PEGylation modifying reagent [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)]. Examples of PEGylation modifying reagents include a modifying agent for the ε-amino group of lysine (Japanese Patent Laid-Open Publication No. 61-178926), a modifying agent for the carboxyl group of aspartic acid and glutamic acid (Japanese Patent Laid-Open Publication No. 56-23587), or a modifying agent for the guanidino group of arginine (Japanese Patent Laid-Open Publication No. 2-117920).

[0093] The immunostimulant may be a natural product known as an immunoadjuvant, and specific examples of immune-enhancing agents include β(1→3) glucan (e.g., lentinan or schizophyllan) or α-galactosylceramide (KRN7000).

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

[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), macrophage colony-stimulating factor (M-CSF), etc. Examples of toxin proteins include ricin, diphtheria toxin, ONTAK, etc., 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 the pathogenesis of tumors, antigens that regulate immune function, or antigens involved in angiogenesis at the lesion site.

[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, and epidermal growth factor receptor (EGFR).

[0098] Examples of antigens involved in the pathogenesis of tumors or antigens of antibodies that regulate immune function include 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. ligand (RANK), RANK ligand, CD25, folate receptor, cytokines (e.g., IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF) β, or TNFα) or receptors for these cytokines, or chemokines (e.g., SLC, ELC, I-309, TARC, MDC, or CTACK) or receptors for these chemokines.

[0099] Antigens for antibodies that inhibit angiogenesis at the lesion site 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] Examples of nucleic acid drugs include pharmaceuticals containing nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA, which act on living organisms by regulating gene function. For example, a conjugate with a nucleic acid drug that suppresses RORγt, the master transcription factor of Th17 cells, is conceivable.

[0101] When the antibody of the present invention or a derivative of the antibody fragment is used for detecting and measuring human FGF23 and diagnosing human FGF23-related diseases, the agent that binds to the antibody may be a label used in conventional immunological detection or measurement methods, such as an enzyme such as alkaline phosphatase, peroxidase, or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).

[0102] Another embodiment of the present invention is a composition comprising the antibody of the present invention or the antibody fragment thereof. Examples of the composition include a composition containing a monoclonal antibody that binds to human FGF23 or the antibody fragment thereof as an active ingredient. The composition comprising the antibody of the present invention or the antibody fragment thereof can be used to treat human FGF23-related diseases. One embodiment of the present invention provides a therapeutic agent for human FGF23-related diseases, comprising the antibody of the present invention.

[0103] The present invention also relates to a method for treating a human FGF23-associated disease, which comprises administering a monoclonal antibody or an antibody fragment thereof that binds to human FGF23.

[0104] Human FGF23-related diseases may be any disease involving human FGF23 or a human FGF23 receptor, and examples include tumor-induced osteomalacia (TIO), autosomal overt hypophosphatemic rickets (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 dialysis in renal failure, such as renal osteodystrophy, dialysis osteopathy, and renal tubular dysfunction.

[0105] The present invention also includes therapeutic agents for treating symptoms such as hypophosphatemia, impaired bone mineralization, bone pain, muscle weakness, skeletal deformities, growth disorders, and hypovitaminosis D, which are seen in diseases such as TIO, ADHR, XLH, fibrous dysplasia, McCune-Albright syndrome, and ARHR, which contain a monoclonal antibody that binds to human FGF23 or a fragment of the antibody as an active ingredient.

[0106] Therapeutic agents containing the antibodies or antibody fragments of the present invention may contain only the antibodies or antibody fragments as the active ingredient, but are usually preferably provided as pharmaceutical formulations prepared by mixing them with one or more pharmacologically acceptable carriers and using any method known in the technical field of pharmaceuticals.

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

[0108] The dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., 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 detecting or measuring FGF23, which comprises a monoclonal antibody that binds to human FGF23 or a fragment of the antibody. The present invention also relates to a method for detecting or measuring FGF23 using a monoclonal antibody that binds to human FGF23 or a fragment of the antibody. In the present invention, methods for detecting or measuring human FGF23 include any known methods, such as immunological detection or measurement methods.

[0110] Immunological detection or measurement methods are methods that use labeled antigens or antibodies to detect or measure the amount of antibodies or antigens, including, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western blotting, and physicochemical techniques.

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

[0112] In the present invention, biological samples to be used for detecting or measuring human FGF23 include, but are not limited to, tissues, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium, as long as they may contain human FGF23.

[0113] A diagnostic agent containing the monoclonal antibody or its antibody fragment of the present invention may contain a reagent for carrying out an antigen-antibody reaction and a reagent for detecting the reaction, depending on the intended diagnostic method. Reagents for carrying out the antigen-antibody reaction include buffers, salts, etc. Detection reagents include reagents used in conventional immunological detection or measurement methods, such as a labeled secondary antibody that recognizes the monoclonal antibody or its antibody fragment, or a substrate corresponding to the label.

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

[0115] The method for producing the antibody, the method for treating a disease, and the method for diagnosing a disease of the present invention will be specifically explained below.

[0116] 1. Antibody production method (1) Antigen preparation Human FGF23, which can be used as an antigen, can be obtained by introducing an expression vector containing a cDNA encoding full-length or partial human FGF23 into E. coli, yeast, insect cells, or animal cells. Human FGF23 can also be obtained by purifying human FGF23 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 as antigens directly. Furthermore, synthetic peptides containing partial sequences of human FGF23 can be prepared by chemical synthesis methods such as the Fmoc or 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 the C- or N-terminus.

[0117] The human FGF23 used in the present invention can be produced by expressing DNA encoding the human FGF23 in host cells using methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or 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 a portion encoding human FGF23 downstream of the promoter of an appropriate expression vector. Instead of the full-length cDNA, a DNA fragment of an appropriate length containing a portion encoding a polypeptide prepared based on the full-length cDNA may be used. Next, the resulting recombinant vector is introduced into a host cell compatible with the expression vector to obtain a transformant that produces the polypeptide.

[0119] Any expression vector can be used as long as it is capable of autonomous replication in the host cell or integration into the chromosome and contains a suitable promoter at a position where the DNA encoding the polypeptide can be transcribed. Any host cell can be used as long as it can express the target gene, such as a microorganism belonging to the genus Escherichia, 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 a vector that can autonomously replicate in prokaryotes and contains a promoter, a ribosome binding sequence, a DNA containing a portion encoding human FGF23, and a transcription termination sequence. Although the recombinant vector does not necessarily require a transcription termination sequence, it is preferable to place the transcription termination sequence directly downstream of 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 Shine-Dalgarno sequence (also called SD sequence), which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

[0122] Furthermore, bases in the base sequence of the DNA encoding the human FGF23 can be substituted to provide codons optimal for expression in the host, thereby improving the production rate of the desired human FGF23.

[0123] Any expression vector can be used as long as it can function in the host cell to be used. Examples of such vectors include pBTrp2, pBTac1, and pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Patent Application Laid-Open 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)], and pBluescript II. SK(-) (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 Application Laid-Open No. 60-221091], pGKA2 [prepared from E. coli IGKA2 (FERM BP-6798), Japanese Patent Application Laid-Open No. 60-221091], pTerm2 (U.S. Pat. Nos. 4,686,191, 4,939,094, and 160,735), pSupex, pUB110, pTP5, pC194, and pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), and pME18SFL3 are examples.

[0124] Any promoter may be used as long as it is functional in the host cell used. Examples include promoters derived from E. coli or phages, such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. Other examples include artificially designed and modified promoters, such as a tandem promoter with two Ptrp promoters in tandem, the tac promoter, the lacT7 promoter, and the letI 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, and E. coli DH5α.

[0126] Any method for introducing a recombinant vector into a host cell can be used as long as it is a method for introducing DNA into the host cell to be used, such as a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

[0127] When animal cells are used as hosts, any expression vector can be used as long as it can function in animal cells. Examples of such vectors include pcDNAI, pCDM8 (Funakoshi), pAGE107 [JP Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (JP Patent Publication No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), and pAGE103 [J. Biochemistry, 101, 1307]. (1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97 / 10354), N5KG1val (U.S. Pat. No. 6,001,358), INPEP4 (Biogen-IDEC), and transposon vectors (WO 2010 / 143698).

[0128] Any promoter that can function in animal cells can be used, including, for example, the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the SV40 early promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, an SRα promoter, or a promoter or enhancer of Moloney murine leukemia virus. The enhancer of the IE gene of human CMV may also be used together 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, II (pp. 883-900)], CHO cells (CHO / DG44 cells) lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) [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 referred to as YB2 / 0), mouse myeloma cells NSO, mouse myeloma cells SP2 / 0-Ag14, and Syrian hamster cells BHK or HBT5637 (Japanese Patent Laid-Open Publication No. 63-000299).

[0130] Any method for introducing DNA into animal cells can be used to introduce a recombinant vector into a host cell, including, for example, electroporation [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open Publication No. 2-227075), or lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

[0131] Human FGF23 can be produced by culturing the thus obtained transformant derived from a microorganism or animal cell harboring a recombinant vector incorporating DNA encoding human FGF23 in a medium, allowing the human FGF23 to be produced and accumulated in the culture medium, and then collecting it from the culture medium. The method for culturing the transformant in a medium can be performed according to a conventional method used for culturing hosts.

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

[0133] When culturing a microorganism transformed with a recombinant vector using an inducible promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside may be added to the medium when culturing a microorganism transformed with a recombinant vector using the lac promoter, and indoleacrylic acid may be added to the medium when culturing a microorganism transformed with a recombinant vector using the trp promoter.

[0134] Examples of media for culturing transformants obtained using animal cells as hosts include commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's Modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, and media supplemented with fetal bovine serum (FBS) or other ingredients. Culture is typically performed for 1 to 7 days under conditions such as pH 6-8, 30-40°C, and 5% CO2. Antibiotics such as kanamycin or penicillin may be added to the medium during culture, if necessary.

[0135] Methods for expressing the gene encoding human FGF23 include, for example, direct expression, as well as secretory production and 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 within host cells, secreting it outside the host cells, or producing it on the outer membrane of the host cells. An appropriate method can be selected by changing the structure of the host cells used or the human FGF23 to be produced.

[0137] When human FGF23 is produced inside a host cell or on the outer membrane of the host cell, human FGF23 can be actively secreted outside the host cell by using the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the methods described in Japanese Patent Application Laid-Open No. 05-336963 or International Publication No. 94 / 23021. Furthermore, the amount of human FGF23 produced can also be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Application Laid-Open No. 2-227075).

[0138] The resulting human FGF23 can be isolated and purified, for example, as follows: When human FGF23 is expressed in a dissolved state within the cells, the cells are collected by centrifugation after the culture is completed, suspended in an aqueous buffer solution, and then disrupted using an ultrasonic homogenizer, French press, Manton-Gaulin homogenizer, Dynomill, or the like to obtain a cell-free extract. The supernatant obtained by centrifuging the cell-free extract can be used to obtain purified samples by conventional protein isolation and purification methods, such as solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with organic solvents, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Chemical), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing, either alone or in combination.

[0139] When human FGF23 is expressed as an insoluble form within cells, the cells are recovered and disrupted 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. The solubilized solution is diluted or dialyzed to restore the human FGF23 to its normal three-dimensional structure, and a purified polypeptide preparation can be obtained by the same isolation and purification method as described above.

[0140] When human FGF23 or its derivatives such as glycosylated forms are secreted extracellularly, the human FGF23 or its derivatives such as glycosylated forms can be recovered from the culture supernatant. The culture can be treated by a method such as centrifugation as described above to obtain a soluble fraction, and a purified preparation can be obtained from the soluble fraction by the same isolation and purification method as described above.

[0141] Human FGF23 used in the present invention can also be produced by chemical synthesis methods such as the Fmoc or tBoc method, or by using a peptide synthesizer manufactured by Advanced Chemtech, Perkin-Elmer, Pharmacia, Protein Technology Instruments, Synthecel-Vega, Perceptive, Shimadzu, or other companies.

[0142] (2) Immunization of animals and preparation of antibody-producing cells for fusion Animals such as 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 also be used as immunized animals.

[0143] Immunization is carried out by administering the antigen subcutaneously, intravenously, or intraperitoneally to animals together with an appropriate adjuvant, such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine. When the antigen is a partial peptide, it is conjugated with a carrier protein such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin) and used as an immunogen.

[0144] After the first administration, antigen is administered 5 to 10 times every 1 to 2 weeks. Blood is collected from the retinal venous plexus 3 to 7 days after each administration, and the serum antibody titer is measured using an enzyme immunoassay [Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] or similar. Animals whose serum shows 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 removed from the immunized animal, and the antibody-producing cells are collected. When using spleen cells, the spleen is minced and disaggregated, then centrifuged, and red blood cells are removed to obtain antibody-producing cells for fusion.

[0146] (3) Preparation of myeloma cells Myeloma cells include established mouse cell lines, 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)], and 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), subcultured in normal medium 3 to 4 days before cell fusion, and then cultured at 2 × 10 7 Ensure that there are at least 100 cells.

[0148] (4) Cell fusion and preparation of monoclonal antibody-producing hybridomas The antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) are thoroughly washed with Minimum Essential Medium (MEM) or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter of distilled water, pH 7.2), mixed at a cell ratio of 5–10:1, and centrifuged. The supernatant is then discarded. After thoroughly loosening the precipitated cells, a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium, and dimethyl sulfoxide is added to the mixture while stirring at 37°C. 1–2 mL of MEM medium is added several times every 1–2 minutes, followed by the addition of MEM medium until the total volume reaches 50 mL. After centrifugation, the supernatant is discarded. After gently loosening the precipitated cells, the antibody-producing cells for fusion are gently suspended in HAT medium (normal medium supplemented with hypoxanthine, thymidine, and aminopterin). This suspension is cultured 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 cell population that reacts with an antigen containing human FGF23 but does not react with an antigen that does not contain human FGF23 is selected by a hybridoma selection method such as the binding assay described below. Next, cloning is performed by limiting dilution, and those that show stable and strong antibody titers are selected as monoclonal antibody-producing hybridomas.

[0150] (5) Preparation of purified monoclonal antibodies The monoclonal antibody-producing hybridoma obtained in (4) is injected intraperitoneally into 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 then raised for two weeks). The hybridoma develops into ascites tumors within 10-21 days. Ascites fluid is collected from the mice and centrifuged to remove solids. The fluid is then salted out with 40-50% ammonium sulfate, and purified using caprylic acid precipitation, DEAE-Sepharose column, protein A column, or gel filtration column. The IgG or IgM fraction is collected and used as purified monoclonal antibody.

[0151] Alternatively, the monoclonal antibody-producing hybridomas obtained in (4) can be cultured in RPMI 1640 medium or similar medium supplemented with 10% FBS, then centrifuged to remove the supernatant. The cells are then suspended in Hybridoma SFM medium and cultured for 3 to 7 days. The resulting cell suspension is then centrifuged, and the supernatant is purified using a Protein A or Protein G column to collect the IgG fraction, yielding purified monoclonal antibodies. Hybridoma SFM medium can also be supplemented with 5% Daigo GF21.

[0152] The antibody subclass is determined by enzyme immunoassay using a subclass typing kit. Protein amount is quantified by the Lowry method or by calculation based on absorbance at 280 nm.

[0153] (6) Selection of monoclonal antibodies Monoclonal antibodies are selected by measuring the binding ability of the antibody to human FGF23 using ELISA as described below.

[0154] Human FGF23 is dispensed into a plate such as a 96-well plate, and a test substance such as serum, hybridoma culture supernatant, or purified monoclonal antibody is dispensed as the first antibody and allowed to react. Next, the plate is thoroughly washed with PBS or the like, and an anti-immunoglobulin antibody labeled with an enzyme reagent or the like is dispensed as the second antibody and allowed to react. After that, the plate is thoroughly washed with PBS or the like, and a substrate is added. The extinction coefficient of each well is measured using a plate reader to select monoclonal antibodies that specifically react with human FGF23.

[0155] 2. Production of recombinant antibodies As examples of recombinant antibodies, the methods for producing human chimeric antibodies and humanized antibodies are described below. Recombinant mouse, rat, and rabbit antibodies can also be produced using similar methods.

[0156] (1) Construction of recombinant antibody expression vectors A recombinant antibody expression vector is an expression vector for animal cells that incorporates DNA encoding the CH and CL of a human antibody, and can be constructed by cloning the DNA encoding the CH and CL of a human antibody into an expression vector for animal cells.

[0157] The C region of a human antibody can be any human antibody CH and CL. For example, the CH of the γ1 subclass and the CL of the κ class of human antibodies can be used. Although cDNA is used to encode the CH and CL of a human antibody, chromosomal DNA consisting of exons and introns can also be used. Any expression vector for animal cells can be used as long as it can incorporate and express genes encoding the C region of a human antibody. 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 for animal cell expression vectors include the SV40 early promoter [J. Biochem., 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)], and immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)].

[0158] For recombinant antibody expression vectors, tandem recombinant antibody expression vectors in which the antibody H and L chains are present on the same vector are used [J. Immunol. Methods, 167, 271 (1994)] because of the ease of constructing the recombinant antibody expression vector, the ease of introducing it into animal cells, and the balance of antibody H and L chain expression levels in animal cells. However, recombinant antibody expression vectors in which the antibody H and L chains are present on separate vectors can also be used. Examples of tandem recombinant antibody expression vectors include pKANTEX93 (WO 97 / 10354) and pEE18 [Hybridoma, 17, 559 (1998)].

[0159] (2) Identification of cDNA encoding the V region of non-human animal antibodies and analysis of their amino acid sequences The cDNA encoding the VH and VL of a non-human antibody and the amino acid sequence can be obtained as follows.

[0160] mRNA is extracted from hybridoma cells producing a non-human antibody, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or plasmid to create a cDNA library. From the library, recombinant phages or recombinant plasmids carrying cDNA encoding VH or VL are isolated using DNA encoding the C or V region of a mouse antibody as a probe. The entire nucleotide sequence of the VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of the VH or VL is deduced from the nucleotide sequence.

[0161] Non-human animals used to produce hybridoma cells that produce non-human antibodies include mice, rats, hamsters, and rabbits, but any animal can be used as long as it is possible to produce hybridoma cells.

[0162] Total RNA from hybridoma cells can be prepared by the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] or by using a kit such as the RNA Easy Kit (Qiagen).

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

[0164] For cDNA synthesis and cDNA library construction, 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 SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Invitrogen) or ZAP-cDNA Synthesis (registered trademark) Kit (Stratagene) are used.

[0165] When constructing a cDNA library, any vector capable of incorporating cDNA synthesized using mRNA extracted from hybridoma cells as a template can be used, such as ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPII (Stratagene), λgt10, λgt11 [DNA Cloning: A Practical Approach, 1, 49 (1985)], Lambda BlueMid (Clontech), λExCell, pT7T3-18U (Pharmacia), pCD2 [Mol. Cell. Biol., 3, 280 (1983)], or pUC18 [Gene, 33, 103 (1985)].

[0166] Any E. coli strain capable of introducing, expressing, and maintaining a cDNA library constructed using a phage or plasmid vector can be used for the introduction of the cDNA library, such as 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)].

[0167] To select cDNA clones encoding the VH or VL of non-human antibodies from a cDNA library, methods such as colony hybridization using isotope- 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 or a cDNA library synthesized from mRNA as a template, followed by the polymerase chain reaction method (hereinafter referred to as PCR; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)).

[0169] The selected cDNA is digested with appropriate restriction enzymes and cloned into a plasmid such as pBluescript SK(-) (Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method, such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], followed by an automated nucleotide sequence analyzer such as the ABI PRISM3700 (PE Biosystems) or ALF DNA Sequencer (Pharmacia).

[0170] The complete amino acid sequences of VH and VL are deduced from the determined nucleotide sequences 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 the antibody, including the secretory signal sequence. The complete amino acid sequences of VH and VL of the antibody, including the secretory signal sequence, can be deduced 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)], thereby determining the length and N-terminal amino acid sequence of the secretory signal sequence and further determining the subgroup to which they belong. The amino acid sequences of each CDR of VH and VL can also be determined 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 obtained complete amino acid sequences of VH and VL can be used to perform a homology search using, for example, the BLAST method [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 a human chimeric antibody expression vector or a human chimeric antibody variant expression vector A human chimeric antibody expression vector can be constructed by cloning cDNA encoding the VH or VL of a non-human antibody upstream of the gene encoding the CH or CL of a human antibody into the recombinant antibody expression vector obtained in (1).

[0173] To link the 3'-end of the cDNA encoding the VH or VL of a non-human antibody to the 5'-end of the CH or CL of a human antibody, VH and VL cDNAs are prepared with the nucleotide sequence of the linker designed to encode the appropriate amino acids and to have an appropriate restriction enzyme recognition sequence. The VH and VL cDNAs thus prepared are cloned upstream of the respective genes encoding the CH or CL of the human antibody into the recombinant antibody expression vector obtained in (1) so that they are expressed in the appropriate form, thereby constructing a human chimeric antibody expression vector.

[0174] Alternatively, the cDNA encoding the non-human antibody VH or VL can be amplified by PCR using synthetic DNA containing 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 A cDNA encoding the VH or VL of a humanized antibody can be constructed as follows.

[0176] The amino acid sequence of the FR of the VH or VL of a human antibody is selected for grafting the amino acid sequence of the CDR of the VH or VL of a non-human antibody. Any amino acid sequence derived from a human antibody can be used for the selected FR. For example, the amino acid sequences of the FR of human antibodies registered in databases such as the Protein Data Bank, or the consensus amino acid sequences of each subgroup of FRs of human antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], etc., can be used. To minimize loss of antibody binding activity, the amino acid sequence of the FR should be selected to have as high a homology as possible (at least 60% or more) with the amino acid sequence of the FR of the VH or VL of the original antibody.

[0177] Next, the amino acid sequences of the CDRs of the original antibody are grafted onto the amino acid sequences of the FRs of the VH or VL of the selected human antibody, respectively, to design the amino acid sequences of the VH or VL of the humanized antibody. The designed amino acid sequences are converted into DNA sequences taking into account the codon usage frequency found in the nucleotide sequence of the antibody gene [Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services (1991)], and DNA sequences encoding the amino acid sequences of the VH or VL of the humanized antibody are designed.

[0178] Based on the designed DNA sequence, several synthetic DNAs each about 100 bases long are synthesized and used in a PCR reaction. In this case, taking into account the reaction efficiency of the PCR reaction and the length of DNA that can be synthesized, preferably six synthetic DNAs each are designed for VH and VL. Furthermore, by introducing appropriate restriction enzyme recognition sequences into the 5' or 3' end of the synthetic DNA 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 a plasmid such as pBluescript SK(-) (Stratagene), and the nucleotide sequence is determined using a method similar to that described in (2) to obtain a plasmid containing a DNA sequence encoding the amino acid sequence of VH or VL of the desired humanized antibody.

[0180] Alternatively, full-length VH and full-length VL can be synthesized as single long DNA strands based on the designed DNA sequences and used instead of the PCR amplification products. Furthermore, by incorporating appropriate restriction enzyme recognition sequences into both ends of the synthetic long DNA strands, 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 a humanized antibody When only the CDRs of the VH and VL of a non-human antibody are grafted onto the FRs of the VH and VL of a human antibody, the antigen-binding activity of the resulting humanized antibody is reduced compared to that of the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)]. In humanized antibodies, the reduced antigen-binding activity can be increased by identifying, within the amino acid sequences of the FRs of the VH and VL of a human antibody, amino acid residues directly involved in antigen-binding, amino acid residues that interact with amino acid residues in the CDRs, and amino acid residues that maintain the three-dimensional structure of the antibody.

[0182] To identify the amino acid residues in FRs involved in antigen-binding activity, the three-dimensional structure of an antibody can be constructed and analyzed using X-ray crystallography [J. Mol. Biol., 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)]. Alternatively, several modified antibodies can be produced for each antibody, and the correlation with each antibody's antigen-binding activity can be examined repeatedly through trial and error to obtain a humanized antibody with the desired antigen-binding activity.

[0183] The amino acid residues in the FRs of the VH and VL of a human antibody can be modified by carrying out the PCR reaction described in (4) using synthetic DNA for modification. The nucleotide 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 vector A humanized antibody expression vector can be constructed by cloning the cDNA encoding the VH or VL of the constructed recombinant antibody upstream of the gene encoding the CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1).

[0185] For example, by introducing an appropriate restriction enzyme recognition sequence into the 5' or 3' end of the synthetic DNAs located at both ends of the synthetic DNAs used to construct the VH or VL of the humanized antibody obtained in (4) and (5), they can be cloned upstream of the respective genes encoding the CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1) so that they can be expressed in an appropriate form.

[0186] Furthermore, when producing genetically engineered antibodies such as the above-mentioned chimeric antibodies and humanized antibodies, a vector for expressing a VL-substituted chimeric antibody can be constructed by preparing an antibody expression vector that combines the H chain (or VH) and L chain (or VL) derived from two different types of antibodies.

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

[0188] Any host cells capable of expressing a recombinant antibody can be used to introduce the expression vector; 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 an expression vector into COS-7 cells, the DEAE-dextran method [Methods in Nucleic Acids Res., CRC Press (1991)] or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] can be used.

[0190] After introduction of the expression vector, the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant are measured using enzyme-linked immunosorbent assays (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 transformants 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 appropriate host cells, transformants that stably express the recombinant antibody can be obtained. The expression vector can be introduced into the host cell by electroporation (Japanese Patent Application Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)).

[0192] Any host cells can be used to introduce a recombinant antibody expression vector, as long as they are capable of expressing a recombinant antibody. For example, CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat. #11619), rat myeloma cell YB2 / 3HL.P2.G11.16Ag.20 (ATCC No.: CRL1662, also referred to as YB2 / 0), mouse myeloma cell NS0, mouse myeloma cell SP2 / 0-Ag14 (ATCC No.: CRL1581), mouse P3X63-Ag8.653 cell (ATCC No.: CRL1580), and CHO cell (CHO / DG44 cell) lacking the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] etc.

[0193] Alternatively, host cells can be used that have reduced or absent activity of proteins such as enzymes involved in the synthesis of the intracellular sugar nucleotide GDP-fucose, proteins such as enzymes involved in glycosylation in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of N-glycosidically linked complex glycans, or proteins involved in the transport of the intracellular sugar nucleotide GDP-fucose to the Golgi apparatus, such as CHO cells lacking the α1,6-fucosyltransferase gene (WO 2005 / 035586, WO 02 / 31140), or Lec13 cells that have acquired lectin resistance [Somatic Cell and Molecular Genetics, 12, 55 (1986)].

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

[0195] Animal cell culture media include RPMI1640 medium (Invitrogen), GIT medium (Nihon Pharmaceuticals), EX-CELL301 medium (JRH), IMDM medium (Invitrogen), Hybridoma SFM medium (Invitrogen), and media containing various additives such as FBS. The resulting transformed strain is cultured in the medium, resulting in the expression and accumulation of the recombinant antibody in the culture supernatant. The expression level and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or other methods. The expression level of the recombinant antibody produced by the transformant can also be increased by 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)]. Protein purification methods such as gel filtration, ion exchange chromatography, and ultrafiltration can also be combined.

[0197] The molecular weight of the H chain, L chain, or entire antibody molecule of the purified recombinant antibody 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 of the present invention or its antibody fragment 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 ELISA, surface plasmon resonance, or the like.

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

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

[0202] Therapeutic agents containing the monoclonal antibodies or antibody fragments thereof of the present invention may contain only the antibody or antibody fragment as the active ingredient, but are usually provided as pharmaceutical formulations prepared by mixing the antibody or antibody fragment with one or more pharmacologically acceptable carriers and using methods known in the technical field of pharmaceuticals.

[0203] Examples of administration routes include oral administration and parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, or intravenous administration. Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, and tapes.

[0204] Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.

[0205] Liquid preparations such as emulsions or syrups are produced using additives such as water, sugars such as sucrose, sorbitol, or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil, or soybean oil, preservatives such as p-hydroxybenzoic acid esters, or flavors such as strawberry flavor or peppermint.

[0206] Capsules, tablets, powders, granules, etc. are produced using additives such as excipients such as lactose, glucose, sucrose, or mannitol; disintegrating agents 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.

[0207] Formulations suitable for parenteral administration include injections, suppositories, and sprays. Injections are prepared using carriers such as saline solutions, glucose solutions, or mixtures of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats, or carboxylic acids.

[0208] Sprays are prepared using carriers that do not irritate the recipient's oral and respiratory mucosa, disperse the monoclonal antibody or antibody fragment of the present invention as fine particles, and facilitate absorption. Examples of carriers include lactose and glycerin. Sprays can also be prepared as aerosols or dry powders. Furthermore, the above-mentioned parenteral preparations can also contain the additives listed above for formulations suitable for oral administration.

[0209] 5. Method for diagnosing diseases using the anti-human FGF23 monoclonal antibody or antibody fragment thereof 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] Human FGF23-related diseases can be diagnosed, for example, by detecting or measuring human FGF23 present in the patient's body by immunological techniques.

[0211] Immunological techniques are methods for detecting or measuring the amount of antibody or antigen using labeled antigens or antibodies, such as radioactive-labeled immunosorbent assays, enzyme immunoassays, fluorescent immunoassays, luminescent immunoassays, Western blotting, or physicochemical techniques.

[0212] In the radioactive substance-labeled immunoassay, for example, an antibody of the present invention or an antibody fragment thereof is reacted with an antigen or cells expressing the antigen, and then a radiolabeled anti-immunoglobulin antibody or an antibody fragment thereof is reacted, followed by measurement using a scintillation counter or the like.

[0213] In enzyme immunoassays, for example, an antibody or antibody fragment of the present invention is reacted with an antigen or cells expressing the antigen, followed by an anti-immunoglobulin antibody or binding fragment labeled with an enzyme or the like, followed by addition of a substrate and measurement of the absorbance of the reaction solution using an absorptiometer. For example, a sandwich ELISA method or the like is used. The label used in enzyme immunoassays may be a publicly known enzyme label [Enzyme Immunoassay, Igaku-Shoin (1987)].

[0214] For example, alkaline phosphatase labeling, peroxidase labeling, luciferase labeling, or biotin labeling may be used. Sandwich ELISA 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, 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 pre-adsorbed onto a plate (e.g., a 96-well plate), and the second antibody or antibody fragment is then labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin. Cells or their lysates, tissues or their lysates, cell culture supernatant, serum, pleural fluid, ascites, or ocular fluid isolated from a living body are then reacted with a labeled monoclonal antibody or antibody fragment, and a detection reaction appropriate for the labeling substance is carried out. The antigen concentration in the test sample is calculated from a calibration curve prepared by serially diluting a known concentration of antigen. The antibodies used in sandwich ELISA may be either polyclonal or monoclonal antibodies, or antibody fragments such as Fab, Fab', or F(ab)2. The combination of two antibodies used in sandwich ELISA 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] Fluorescence immunoassay is performed according to the methods described in literature such as "Monoclonal Antibodies - Principles and Practice, Third Edition, Academic Press (1996) and "Monoclonal Antibody Experiment Manual," Kodansha Scientific (1987)." Labels used in fluorescence immunoassay include known fluorescent labels such as those used in "Fluorescent Antibody Method," Soft Sciences (1983). For example, FITC or RITC can be used.

[0216] Luminescence immunoassay is performed according to the method described in Bioluminescence and Chemiluminescence, Clinical Tests 42, Hirokawa Shoten (1998). Labels used in luminescence immunoassay include known luminescent labels, such as acridinium esters or lophine.

[0217] In Western blotting, antigens or cells expressing the antigen are fractionated using SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel) [Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)], the gel is then blotted onto a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose membrane, and the membrane is reacted with an antibody or antibody fragment that recognizes the antigen, followed by reaction with an anti-mouse IgG antibody or binding fragment labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or a biotin label, and the label is visualized for measurement.

[0218] Physicochemical techniques include, for example, forming aggregates by binding the antigen, human FGF23, with the monoclonal antibody or its antibody fragment of the present invention, and then detecting these aggregates. Other physicochemical techniques include capillary tube analysis, one-dimensional immunodiffusion, immunoturbidimetry, and latex immunoturbidimetry [Clinical Test Methods Summary, Kanehara Publishing (1998)]. Latex immunoturbidimetry uses a carrier such as polystyrene latex with a particle size of approximately 0.1 to 1 μm sensitized with an antibody or antigen. When an antigen-antibody reaction occurs with the corresponding antigen or antibody, scattered light in the reaction solution increases and transmitted light decreases. This change is detected as absorbance or integrating sphere turbidity, and the antigen concentration in the test sample is measured.

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

[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 WO 2008 / 099969 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2. Hereinafter, a human IgG1 antibody comprising a VH containing the amino acid sequence shown in SEQ ID NO: 1 and a VL containing the amino acid sequence shown in 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 one month (hereinafter referred to as the 40°C 1M sample) or at -80°C for one month and then thawed (hereinafter referred to as the initial sample).

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

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

[0224] [Example 2] Preparation of modified I100 antibody Antibodies were produced by the method described below in which the 100th amino acid residue, I, in the amino acid sequence of SEQ ID NO: 1 in VH of antibody A was substituted with an amino acid residue listed in Table 1. Hereinafter, all or part of the antibodies listed in Table 1 will also be referred to as I100 modified antibodies.

[0225] [Table 1]

[0226] The necessary plasmids were prepared by introducing gene fragments corresponding to the nucleotide sequences encoding the amino acid sequences of the VH of each antibody listed in Table 1 into expression vectors using the seamless cloning method (contracted to FASMAC). The H-chain expression vector used was the pCI-OtCAG_hG1 vector, which contains a signal sequence and a human γ-chain constant region sequence. For all clones, human IgG1 was used as the H-chain constant region.

[0227] The amino acid sequence of the VL of the I100 modified antibody was the same as that of antibody A (SEQ ID NO: 2). The L chain expression vector used was the pCI-OtCMV_hK vector, which contains a signal sequence and a human κ chain constant region sequence. The completed plasmid was mass-produced using a 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 attached instructions. The L-chain expression vector and H-chain expression vector were mixed at a 1:2 ratio and introduced. After plasmid introduction, the cells were cultured for 3 days under conditions of 37°C, 5% CO2, and 125 rpm. The cell culture suspension was then 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 in the column was equilibrated with PBS, and then the culture supernatant was added to the column. The column was washed twice with PBS, once with Wash buffer 1 (PBS with 1 M NaCl) and once with Wash buffer 2 (20 mM citric acid, 50 mM NaCl, pH 5.0), and the antibody was eluted using elution buffer (20 mM citric acid, 50 mM NaCl, pH 3.4).

[0230] The resulting antibody solution was neutralized by adding 1 / 10 volume of neutralization buffer (1 M phosphate-NaOH, pH 7.0), and the solvent of the antibody solution was replaced with PBS using a NAP25 (GE Healthcare). After buffer replacement, the antibody solution was concentrated by ultrafiltration using Amicon Ultra-4 Centrifugal Filter Units (Millipore). The absorbance A280 was measured using a Nanodrop (Thermo Scientific) to determine and adjust the antibody solution concentration. 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 antibodies The I100 modified antibody and antibody A obtained in Example 2 were assayed for binding activity to recombinant human FGF-23 (R&D Systems, Cat No. 2604-FG-025 / CF) as follows.

[0232] Anti-human IgG antibodies were immobilized on a CM5 sensor chip (Global Life Science Technologies Japan, Cat. No. BR100530) using a Human Antibody Capture Kit (Global Life Science Technologies Japan, Cat. No. BR-1008-39) according to the attached protocol.

[0233] An antibody prepared at 1 μg / mL was added to the flow cell on which the anti-human IgG antibody had been immobilized at a flow rate of 10 μL / min for 30 seconds.

[0234] Next, recombinant human FGF-23 was diluted two-fold starting from 130.5 ng / mL to five concentrations, and the binding reaction was monitored for 2 minutes and the dissociation reaction for 10 minutes at a flow rate of 30 μL / min. The measurement was performed using a single-cycle method. The acquired sensorgrams were analyzed using Bia Evaluation Software (Global Life Science Technologies Japan, Inc.), 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 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 confirm that almost all I100 modified antibodies, including I100A antibody and I100Y antibody, have reduced antigen binding activity compared to antibody A.

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

[0239] [Example 4] Confirmation of the degradation inhibitory effect of I100 modified antibodies For the I100 modified antibody and antibody A 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 solution was left standing at 40°C for one month or two weeks, and then subjected to SDS-PAGE under reducing conditions. Table 4 shows the results of a comparison of the intensity of the band around 40 kDa corresponding to the antibody degradation product between each antibody and antibody A. If the intensity of this band was lower with the modified antibody compared to antibody A, it was determined that degradation was suppressed with the modified antibody compared to antibody A.

[0240] The values ​​entered in the column for degradation inhibitory effect in Table 4 were set as follows: 1 was entered for antibodies whose degradation was more inhibited than that of antibody A, 2 was entered for antibodies whose degradation was similar to that of antibody A, and 3 was entered for antibodies whose degradation was more enhanced than that of antibody A.

[0241] [Table 4]

[0242] As shown in Table 4, I100A, I100Y, I100W, I100R, I100N, I100M, I100H, I100G, and I100D showed reduced degradation compared to antibody A. I100L, I100V, I100T, I100S, I100K, I100F, and I100E showed degradation to the same extent as antibody A. Furthermore, I100Q showed enhanced degradation compared to antibody A.

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

[0244] The measurement sample was prepared in D-PBS buffer to a concentration of 0.5 mg / mL. Measurements were performed using a Micro Cal VP-Capillary DSC system (Spectris Corporation). Measurements were performed using a program that increased the temperature from 25°C to 100°C at 1°C per minute. The results are shown in Tables 5 and 6.

[0245] [Table 5]

[0246] [Table 6]

[0247] As can be seen from Tables 5 and 6, for I100A, I100G, I100N, and I100R, the Fab peak and CH2 peak were detected overlapping, and the thermal stability of Fab was improved by 3 to 8 degrees compared to antibody A. From the above, it was confirmed that I100A, I100G, I100N, and I100R have higher structural stability than antibody A.

[0248] [Example 6] Preparation of D105 modified antibody Using a method similar to that used in Example 2, an antibody was prepared in which the 105th D in the amino acid sequence of VH containing the amino acid sequence shown in SEQ ID NO: 1 of antibody A was replaced with the amino acid residue shown in Table 7 (hereinafter, part or all of this antibody will also be referred to as the D105 modified antibody).

[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 in the same manner as in Example 3. The results are shown in Tables 8 and 9. A_1 and A_2 in Table 8 are both measurement results for antibody A.

[0251] [Table 8]

[0252] [Table 9]

[0253] Tables 8 and 9 confirm that the D105 modified antibody had reduced antigen-binding activity compared to antibody A.

[0254] [Example 8] Confirmation of degradation inhibitory effect of D105 modified antibody The D105 modified antibody obtained in Example 6 was examined for its inhibitory effect on antibody degradation in the same manner as in Example 4. The antibody solution was left to stand at 40°C for 2 weeks.

[0255] The results are shown in Table 10. The numerical value shown in the column for degradation inhibitory effect in Table 10 is 1 for antibodies whose degradation was more inhibited than that of antibody A, 2 for antibodies whose degradation was similar to that of antibody A, and 3 for antibodies whose degradation was more enhanced than that of antibody A.

[0256] [Table 10]

[0257] Table 10 confirms that degradation of the 17 D105 modified antibodies, excluding D105E, was suppressed more than that of antibody A. For all antibodies, the intensity of the approximately 40 kDa band corresponding to the antibody degradation product on SDS-PAGE was significantly lower than that of antibody A, confirming that antibody degradation was significantly suppressed.

[0258] [Example 9] Measurement of the neutralizing activity of I100 modified antibody and D105 modified antibody against human FGF23 The neutralizing activity against human FGF23 of 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), which showed less antibody degradation than antibody A in Examples 4 and 8, was measured using the method described below.

[0259] To measure neutralizing activity, we used a promoter assay using HEK293 cells stably expressing αKlotho that had been transformed with a luciferase expression vector containing a promoter derived from the mouse Egr1 gene (hereafter referred to as mEgr1 / αKL / HEK293). mEgr1 / αKL / HEK293 cells were generated using a method similar to that described in Nature 2006 Dec 7;444(7120).

[0260] In this promoter assay, when FGF23 binds to αKlotho on mEgr1 / αKL / HEK293, a signal flows within the cell, causing luciferase gene expression, and the fluorescence of the luciferase can be detected. When FGF23 is neutralized by an FGF23-neutralizing antibody, the fluorescence intensity decreases.

[0261] The antibody was diluted to 100 μg / ml in a buffer containing 10 mM sodium L-glutamate and 262 mM D-sorbitol to prepare a stock solution. The standard culture medium for mEgr1 / αKL / HEK293 cells was DMEM (Thermofisher) supplemented with 10% (vol%) Fetal Bovine Serum (Thermofisher) and 1% (vol) penicillin / streptomycin.

[0262] The antibody was diluted 1000-fold with the standard culture medium (100 ng / ml) to the highest concentration, and eight √10-fold dilutions were prepared and used for evaluation. Evaluation was performed using a 384-well plate. Cells were added at 2000 cells / well, and the FGF23 concentration was 4 ng / ml. FGF23 was the same as in Example 3, diluted with standard culture medium.

[0263] After adding each antibody to the cells, they were cultured for 24 hours, and then FGF23 was added and cultured for 4 hours. Luciferase fluorescence intensity was measured using the Bright-Glo™ Luciferase Assay System (Promega) with the Envision multiplate reader (PerkinElmer).

[0264] For the results obtained, the fluorescence intensity when FGF23 was added was set to 100% and the fluorescence intensity when no FGF23 was added was set to 0%, and the percentage of fluorescence intensity when each antibody was added was calculated, and the IC50 values ​​(ng / ml) are shown in Table 11.

[0265] [Table 11]

[0266] As shown in Table 11, all of the modified antibodies had reduced FGF23 neutralizing activity compared to antibody A.

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

[0268] The CH-modified antibody was prepared by substituting the CH (human IgG1) of antibody A with human IgG2, IgG2AAAS (a human IgG2 modified antibody in which valine at position 234 in the EU index is substituted with alanine, glycine at position 237 is substituted with alanine, and proline at position 331 is substituted with serine in the Fc of human IgG2) (Michael, S., et al., J. Immunol., 1997, 159: 3613; J. Immunol. 2000, 164: 4178-4184), or IgG4PE_R409K (human A total of 23 types of antibodies were produced by adding the following Fc amino acid modifications (10-1) to (10-5) to the Fc region of three types of IgG4 isotype antibodies (human IgG4 modified antibody in which serine at position 228 in the EU index was substituted with proline, leucine at position 235 with glutamic acid, and arginine at position 409 with lysine) (WO 2006 / 033386) to CH, or antibody A or antibody A isotype antibodies, which are known to improve the binding activity to human and monkey FcRn.

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

[0270] Hereinafter, the CH-modified antibody in which the CH of antibody A was replaced with the CH of human IgG2 will be referred to as antibody A_G2. CH-modified antibodies in which the CH of other subclasses was replaced will also be referred to in the same manner.

[0271] Hereinafter, the CH-modified antibodies obtained by adding the amino acid alterations (10-1) to (10-5) above 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, the CH-modified antibodies obtained by adding the amino acid alterations (10-1) to (10-5) above to antibody A_G2, A_G2AAS, and A_G4PE_R409K will be similarly referred to.

[0272] A plasmid containing a nucleotide sequence encoding the amino acid sequences of the H chain variable and constant regions of antibody A prepared in Example 2 was digested with restriction enzymes NheI and BamHI to prepare a vector fragment from which the constant region portion had been removed. Using this plasmid fragment, FASMAC synthesized gene fragments containing nucleotide sequences encoding the amino acid sequences of the above 23 types of CH-modified antibodies and introduced them into appropriate expression vectors to prepare the required plasmids. Antibodies were prepared using the obtained plasmids in the same manner as in Example 2.

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

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

[0275] DNA sequences encoding human FcRn (only amino acids 1 to 297 of the full-length amino acid sequence of human FcRn were used to express it 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. Transient expression was carried out using this plasmid with the Expi293 Expression System Kit (Life Technologies) in the same manner as in Example 2.

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

[0277] After obtaining the culture supernatants, human and monkey FcRn were purified using Ni-NTA Agarose (QIAGEN, Cat. No. 30210) according to the standard method 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, Cat. No. BR100530). Human FcRn and monkey FcRn solutions prepared at 10 μg / mL were added to the flow cell on which Tetra His Antibody BSA Free was immobilized at a flow rate of 10 μL / min for 120 seconds. Next, five concentrations of each Fc-modified antibody, diluted three-fold starting from 450 μg / mL, were added at a flow rate of 30 μL / min. The binding reaction was monitored for 2 minutes, and the dissociation reaction was monitored 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, Inc.), and the dissociation constant [kd1 / ka1 = KD] of each antibody was calculated. The results are shown in Tables 12 and 13.

[0280] [Table 12]

[0281] [Table 13]

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

[0283] [Example 12] Preparation of CH-modified antibodies of I100 and D105 modified antibodies Of the I100 and D105 modified antibodies, which were confirmed in Examples 4 and 8 to have less antibody degradation than antibody A, CH modified antibodies were produced by adding the amino acid modifications (12-1) to (12-5) below to each of the D105N, I100A, I100H, and I100Y antibodies using the method described below.

[0284] (12-1) Amino acid modification in which M at position 252 in the EU index is replaced with Y, S at position 254 with T, and T at position 256 with E

[0285] (12-2) Amino acid modifications in which CH is substituted from human IgG1 to human IgG2, and M at position 252 in the EU index is substituted with Y, S at position 254 with T, and T at position 256 with E

[0286] (12-3) Amino acid alterations: CH is replaced from human IgG1 to human IgG2, and M at position 428 in the EU index is replaced with L, and N at position 434 is replaced with S

[0287] (12-4) Amino acid alteration: CH is replaced from human IgG1 to human IgG4PE_R409K, and V at position 308 in the EU index is replaced with P

[0288] (12-5) Amino acid alterations in which CH is replaced from human IgG1 to human IgG4PE_R409K, and M at position 428 in the EU index is replaced with L, and N at position 434 is replaced with S

[0289] Hereinafter, the CH-modified antibodies obtained by modifying the D105N antibody with the amino acids (12-1) to (12-5) above will be referred to as the D105N_YTE, D105N_G2_YTE, D105N_G2_LS, D105N_G4PE_R409K_P, and D105N_G4PE_R409K_LS antibodies, respectively. Similar descriptions will be used for other antibodies. The amino acid sequences of the CHs of the CH-modified antibodies in which the amino acid modifications (12-1) to (12-5) above were performed are set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively.

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

[0291] [Example 13] Measurement of FGF23-neutralizing activity of heavy chain constant region-modified antibodies The FGF23-neutralizing activity of the 20 types of CH modified antibodies prepared in Example 12 was measured in the same manner 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 changed. From the above, it was confirmed that the amino acid alterations in the heavy chain constant region of each antibody did not affect the FGF23 neutralizing activity of the antibody.

[0294] [Example 14] Preparation of modified antibodies with improved antigen-binding activity In order to improve the antigen-binding activity of the I100A and I100Y antibodies as shown in Tables 2 and 3, Abwiz Bio performed affinity maturation using the phage display method to obtain the amino acid sequences (14-1) to (14-9) below.

[0295] (14-1) A VH comprising the amino acid sequence of SEQ ID NO: 1, in which the amino acid at position 51 is substituted with V, the amino acid at position 54 with F, the amino acid at position 55 with W, the amino acid at position 57 with R, the amino acid at position 58 with W, and the amino acid at position 100 with A in the amino acid sequence of SEQ ID NO: 1 (H2B11_A, amino acid sequence: SEQ ID NO: 38).

[0296] (14-2) A VH comprising the amino acid sequence of SEQ ID NO: 1, in which the amino acid at position 50 is substituted with L, the amino acid at position 54 with W, the amino acid at position 55 with H, the amino acid at position 57 with T, the amino acid at position 58 with F, and the amino acid at position 100 with A in the amino acid sequence of SEQ ID NO: 1 (J2H2B9_A, amino acid sequence: SEQ ID NO: 39).

[0297] (14-3) A VH comprising the amino acid sequence of SEQ ID NO: 1, in which the amino acid at position 50 is substituted with V, the amino acid at position 54 with F, the amino acid at position 55 with C, the amino acid at position 57 with F, the amino acid at position 58 with V, and the amino acid at position 100 with A in the amino acid sequence of SEQ ID NO: 1 (J2H2E9_A, amino acid sequence: SEQ ID NO: 40).

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

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

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

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

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

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

[0304] [Example 15] Preparation of modified antibodies The modified antibodies A-1 to A-8 listed in Table 15 were prepared using the amino acid sequence information obtained in Example 14 and the VH amino acid sequence listed in (15-1).

[0305] (15-1) A VH comprising the amino acid sequence represented by SEQ ID NO: 1, in which the amino acid at position 50 is substituted with L, the amino acid at position 54 with W, the amino acid at position 55 with H, the amino acid at position 57 with T, the amino acid at position 58 with F, and the amino acid at position 100 with Y in the amino acid sequence represented by SEQ ID NO: 1 (J2H2B9_Y, amino acid sequence: SEQ ID NO: 47).

[0306] [Table 15]

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

[0308] [Example 16] Evaluation of modified antibodies The eight types of modified antibodies prepared in Example 15 and antibody A as a control were used to evaluate the antigen-binding activity, FGF23-neutralizing activity, and thermal stability. For each modified antibody, various measurements were performed by diluting samples adjusted to a concentration of 0.5 mg / mL in D-PBS (Nacalai Tesque, Inc., Code 14249-24) as the buffer, as needed.

[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. However, instead of immobilizing an anti-human IgG antibody on a CM5 sensor chip, a Protein A sensor chip (Global Life Science Technologies Japan, Cat. No. 29127555) was used. Table 16 shows the binding rate constant (ka) and dissociation rate constant (kd) of each antibody, as well as the dissociation constant calculated from the binding rate constant (ka) and dissociation rate constant (kd) [kd1 / ka1 = KD].

[0310] [Table 16]

[0311] Table 16 confirms that all of the antibodies A-1 to A-8 have 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 antibody A. Furthermore, in all of the antibodies A-1 to A-8, the 100th amino acid residue of VH is A or Y.

[0313] From the above, it was confirmed that substituting the 100th amino acid residue of VH of antibody A with A or Y reduces the antigen-binding activity of the antibody, but substituting additional amino acid residues contained in antibodies A-1 to A-8 improves the antigen-binding activity of the antibody to the same level as 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. Table 16 confirms that all modified antibodies have 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 antibody and the I100Y antibody was lower than that of antibody A. In addition, in all of the antibodies A-1 to A-8, the 100th amino acid residue of VH is A or Y.

[0316] From the above, it was confirmed that, in the case of antibody A, substituting the 100th amino acid residue of VH with A or Y reduces the antibody's FGF23 neutralizing activity, but by substituting additional amino acid residues contained in antibodies A-1 to A-8, the neutralizing activity of the antibody is improved to the same level as that of antibody A.

[0317] 16-3) Confirmation of isoelectric point The isoelectric point of each antibody was 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, and pI Marker 5.12 (Protein Simple, Cat. No. 102224) and pI Marker 9.77 (Protein Simple, Cat. No. 102219) were used as the acidic marker and basic marker, respectively. Measurements were performed according to standard protocols.

[0318] The results are shown in Table 16. Table 16 confirms that all modified antibodies had a lower pI than antibody A.

[0319] 16-4) Confirmation of antibody degradation inhibition rate The degradation inhibition rate was confirmed for antibodies A-1 to A-8 using the same method as in Example 4. In this example, each antibody was left standing at 40°C for 2 weeks, and antibody degradation was confirmed using a Bioanalyzer Electrophoresis System (Agilent Technologies, Inc.) and an Agilent Protein 230 Kit (Agilent Technologies, Inc., Cat. 5067-1517). Sample preparation and electrophoresis conditions were performed according to the protocol provided with the kit. In the resulting electropherogram, the antibody H chain band was detected at 63 kDa, and a band corresponding to the antibody degradation product was detected at approximately 55 kDa. The peak area ratio (%) of the band corresponding to the antibody degradation product and the ratio (%) of the degradation band peak area ratio 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 of the antibodies 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 difference in the thermal stability of each antibody site between antibody A and antibody A_YTE. On the other hand, it was confirmed that the thermal stability of the Fab of antibody A-1 and antibody A-3, which have the same heavy chain constant region amino acid sequence as antibody A_YTE, was improved by approximately 3 degrees 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 shown in Table 20 were produced in the same manner as in Example 2 (hereinafter, the A-9 antibody and A-10 antibody may also be referred to as modified antibodies).

[0327] The L chain expression vector for the A-9 antibody was the pcDNA3.4 vector (Invitrogen) instead of the pCI vector used in Example 2. For both the A-9 and A-10 antibodies, the L chain expression vector and the H chain expression vector were mixed at a 1:1 ratio and transfected into cells.

[0328] [Table 20]

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

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

[0331] The VL of the A-10 antibody was designed and used as follows:

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

[0333] [Example 18] Evaluation of modified antibodies The two types of modified antibodies prepared in Example 17 and antibody A as a control were used to evaluate the antigen-binding activity, FGF23-neutralizing activity, and thermal stability. For each modified antibody, various measurements were performed by diluting samples adjusted to a concentration of 1 mg / mL in D-PBS (Nacalai Tesque, Inc., Code 14249-24) as needed.

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

[0335] [Table 21]

[0336] Table 21 confirms that the A-9 antibody has twice the antigen-binding activity of antibody A, and the A-10 antibody has the same level of antigen-binding activity as antibody A. In Example 3, the I100A antibody and I100Y antibody had lower antigen-binding activity than antibody A. Furthermore, the A-9 antibody and A-10 antibody have A and Y as the 100th amino acid residues in VH, respectively. From the above, it was confirmed that substituting the 100th amino acid residue of VH of antibody A with A or Y reduces the antigen-binding activity of the antibody, but substituting additional amino acid residues in antibody A-9 and antibody I100Y improves the binding activity of the antibody to the same level as 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. Table 21 confirms that the FGF23 neutralizing activity of antibody A-9 is three times stronger than that of antibody A, and that antibody A-10 is two times stronger than that of antibody A.

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

[0339] From the above, it was confirmed that substituting the 100th amino acid residue of VH of antibody A with A or Y reduces the antibody's FGF23 neutralizing activity, whereas substituting additional amino acid residues present in antibodies A-9 and A-10 improves the antibody's FGF23 neutralizing activity more than that of antibody A.

[0340] [Example 19] Confirmation of degradation inhibition rate of modified antibodies The degradation inhibition rate was confirmed using four types of modified antibodies, A-1 antibody, A-3 antibody, A-5 antibody, and A-8 antibody, as well as antibody A as a control.

[0341] The antibody used in this example was purified by inserting the target gene sequence into a mammalian expression vector, introducing it into CHO cells, and then affinity purifying the supernatant of the mammalian cell culture medium using MabSelect SuRe (Global Life Science Technologies Japan, Inc.) and cation exchange chromatography. The solvent in the antibody solution was replaced with a pH 4 or pH 4.5 solvent containing 10 mM sodium L-glutamate, 262 mM D-sorbitol, and 0.05 mg / mL Polysorbase 80 using NAP25 (Global Life Science Technologies Japan, Inc.), resulting in a protein concentration of 1 mg / mL.

[0342] The resulting antibody solution was incubated at 40°C for one month and at 25°C for three months, then frozen at -80°C. After thawing, the antibody solution was analyzed for antibody degradation using a PA800Plus biopharmaceutical analysis system (SCIEX). Sample preparation and analysis were performed according to the method of Oscar Salas-Solano et al. (2006, Anal. Chem.: 6583-6594), with appropriate modifications.

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

[0344] [Table 22]

[0345] [Table 23]

[0346] As shown in Tables 22 and 23, as in Example 16, even under 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 degradation of all of the A-1 antibody, A-3 antibody, A-5 antibody, and A-8 antibody was suppressed compared to antibody A.

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

[0348] The antibody A and antibody A-1 used in this example were purified by inserting the target gene sequence into a mammalian expression vector, introducing the vector into CHO cells, and then affinity purifying the supernatant of the mammalian cell culture medium using MabSelect SuRe (Global Life Science Technologies Japan, Inc.) and cation exchange chromatography. The solvent in the antibody solution was replaced with a solvent containing 10 mM sodium L-glutamate, 262 mM D-sorbitol, and 0.05 mg / mL polysorbate at pH 4, pH 4.5, or pH 5 using NAP25 (Global Life Science Technologies Japan, Inc.), resulting in a protein concentration of 1 mg / mL.

[0349] The resulting antibody solution was left standing at 40°C for one month, after which antibody degradation was confirmed using a microchip electrophoresis system, LabChip (Perkin-Elmer), and a Protein Clear Reagent Kit (Perkin-Elmer, Cat. CLS960014). Sample preparation and electrophoresis conditions were performed according to the protocol provided with the kit.

[0350] The peak area ratios (%) of the bands corresponding to the antibody degradation products measured under reducing conditions are shown in Table 24 at pH 4.0, Table 25 at pH 4.5, and Table 26 at 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 the A-9 antibody and the A-10 antibody was also suppressed compared to the A antibody when stored at 40°C for one month under buffer conditions of pH 4.0, pH 4.5, or pH 5.0.

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

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

[0357] [Table 27]

[0358] On the other hand, after a single subcutaneous administration of 3 mg / kg of burosumab (KRN23, Crysvita) to cynomolgus monkeys, serum inorganic phosphorus concentrations increased from the third day after administration, and by the 42nd day after administration, inorganic phosphorus concentrations had decreased to the same level as the vehicle-treated group and baseline (day 0 of administration) (Crysvita Subcutaneous Injection Pharmaceutical Interview Form, revised December 2021 (5th edition), p. 44).

[0359] Therefore, it was demonstrated that the A-8 antibody prolonged the duration of serum inorganic phosphorus concentrations in cynomolgus monkeys compared to burosumab.

[0360] In addition, A-1 antibody and A-5 antibody were subcutaneously administered at 3 mg / kg to cynomolgus monkeys, and serum inorganic phosphorus concentrations were measured in the same manner as above. As a result, these antibodies, like burosumab, increased serum inorganic phosphorus concentrations from day 3 after administration, and reduced inorganic phosphorus concentrations to baseline 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, and the amino acid sequences of the heavy chain constant regions of the A-1 antibody, A-5 antibody, and A-8 antibody are represented by SEQ ID NO: 48. Of the A-1 antibody, A-5 antibody, and A-8 antibody, which have the same heavy chain constant region amino acid sequence, only the A-8 antibody maintained serum inorganic phosphorus concentrations longer than burosumab in cynomolgus monkeys.

[0362] Therefore, it was suggested that the longer duration of serum inorganic phosphorus concentration in cynomolgus monkeys with the A-8 antibody compared with burosumab, A-1 antibody, and A-5 antibody is due to differences in the amino acid sequence of the variable region, not differences in the amino acid sequence of the heavy chain constant region.

[0363] [Example 22] The serum inorganic phosphorus concentration (mg / dL) was measured in the same manner as in Example 21. Test antibodies, A-9 antibody and A-10 antibody (3.0 mg / kg), were subcutaneously administered. Blood samples were collected over a 57-day period after administration, and the serum inorganic phosphorus concentration 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 the A-10 antibody, the serum inorganic phosphorus concentration increased from the third day after administration, and even on the 57th day after administration, it remained higher than the baseline inorganic phosphorus concentration (phosphorus concentration on Day 0 in Table 28).

[0367] The amino acid sequence of the heavy chain constant region of the A-9 antibody and the A-10 antibody is the same as that of the A-1 antibody, the A-5 antibody, and the A-8 antibody, and is the amino acid sequence shown in SEQ ID NO: 48.

[0368] Therefore, it was suggested that the longer duration of serum inorganic phosphorus concentrations in cynomolgus monkeys for the A-9 and A-10 antibodies compared to burosumab, A-1, and A-5 antibodies is due to differences in the amino acid sequences of the variable regions, as with the A-8 antibody, rather than differences in the amino acid sequences of the heavy chain constant regions.

[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 present invention. This application is based on Japanese patent applications filed on August 10, 2022 (Patent Application No. 2022-128011) and October 12, 2022 (Patent Application No. 2022-164256), which are incorporated by reference in their entireties. All references cited herein are incorporated by reference in their entireties. [Sequence List 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 D105A antibody SEQ ID NO: 21: Amino acid sequence of VH of 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 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 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 SEQ ID NO: 38: Amino acid sequence of H2B11_A SEQ ID NO: 39: Amino acid sequence of J2H2B9_A SEQ ID NO: 40: Amino acid sequence of J2H2E9_A SEQ ID NO: 41: Amino acid sequence of 2H2E1_A SEQ ID NO: 42: Amino acid sequence of 2H2E5_A SEQ ID NO: 43: Amino acid sequence of 2H2E8_Y SEQ ID NO: 44: Amino acid sequence of L3G12 SEQ ID NO: 45: Amino acid sequence of L1H8 SEQ ID NO: 46: Amino acid sequence of 2L3H7 SEQ ID NO: 47: Amino acid sequence of J2H2B9_Y SEQ ID NO: 48: Amino acid sequence of YTE CH SEQ ID NO: 49: Amino acid sequence of CH of G2_YTE SEQ ID NO: 50: Amino acid sequence of CH of G2_LS SEQ ID NO: 51: Amino acid sequence of CH of G4PE_R409K_P SEQ ID NO: 52: Amino acid sequence of CH of G4PE_R409K_LS SEQ ID NO: 53: Amino acid sequence of the G1 constant region SEQ ID NO: 54: Amino acid sequence of human FcRn extracellular domain His-tag SEQ ID NO: 55: Amino acid sequence of human β2 microglobulin SEQ ID NO: 56: Amino acid sequence of the His-tagged extracellular domain of cynomolgus monkey FcRn SEQ ID NO: 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 or antibody fragment thereof selected from the following (c1) to (c10) that binds to FGF23. (c1) an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 39 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2; (c2) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 47 and VL comprising the amino acid sequence represented by SEQ ID NO: 2; (c3) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 3 and VL comprising the amino acid sequence represented by SEQ ID NO: 44; (c4) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 6 and VL comprising the amino acid sequence represented by SEQ ID NO: 44; (c5) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 3 and VL comprising the amino acid sequence represented by SEQ ID NO: 45; (c6) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 6 and VL comprising the amino acid sequence represented by SEQ ID NO: 45; (c7) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 3 and VL comprising the amino acid sequence represented by SEQ ID NO: 46; (c8) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 6 and VL comprising the amino acid sequence represented by SEQ ID NO: 46; (c9) an antibody comprising VH comprising the amino acid sequence represented by SEQ ID NO: 3 and VL comprising the amino acid sequence represented by SEQ ID NO: 58; and (c10) An antibody comprising a VH having an amino acid sequence represented by SEQ ID NO: 6 and a VL having an amino acid sequence represented by SEQ ID NO:

59.

2. The antibody or antibody fragment thereof according to claim 1, wherein the subclass of the antibody is IgG1, IgG2, IgG3 or IgG4.

3. The antibody or antibody fragment thereof according to claim 1, wherein the Fc region of the antibody is one selected from the following (d1) to (d5): (d1) an Fc region comprising a substitution of the amino acid residue at position 252 of the EU index with a tyrosine residue, a substitution of the amino acid residue at position 254 with a threonine residue, and a substitution of the amino acid residue at position 256 with a glutamic acid residue; (d2) an Fc region comprising a substitution of the amino acid residue at position 428 of the EU index with a leucine residue and a substitution of the amino acid residue at position 434 with a serine residue; (d3) an Fc region comprising a substitution of the amino acid residue at EU index 308 with a proline residue; (d4) an Fc region comprising a substitution of the amino acid residue at position 250 of the EU index with a glutamine residue and a substitution of the amino acid residue at position 428 with a leucine residue; and (d5) An Fc region comprising a substitution of the amino acid residue at EU index position 434 with an alanine residue.

4. The antibody or antibody fragment thereof according to claim 1, wherein the heavy chain constant region of the antibody comprises the amino acid sequence represented by SEQ ID NO: 48, 49, 50, 51, or 52.

5. The antibody fragment is Fab, Fab', (Fab') ２ 2. The antibody fragment of claim 1, which is selected from the group consisting of scFv, diabody, and dsFv.

6. A nucleic acid having a base sequence encoding the antibody or antibody fragment thereof according to any one of claims 1 to 5.

7. A vector containing the nucleic acid of claim 6.

8. A transformed cell comprising the vector of claim 7.

9. A method for producing an antibody or antibody fragment described in any one of claims 1 to 5, comprising culturing in a culture medium a transformed cell containing a vector containing a nucleic acid having a base sequence encoding the antibody or antibody fragment described in any one of claims 1 to 5, and collecting the antibody or antibody fragment from the culture.

10. A composition comprising the antibody or antibody fragment thereof according to any one of claims 1 to 5.

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