HK1172917B - Anti fgf23 antibody and a pharmaceutical composition comprising the same - Google Patents

Description

anti-FGF 23 antibody and pharmaceutical composition containing same

The present application is a divisional application of an invention patent application having a national application number of 200880004814.5(PCT/JP2008/052918) entitled "anti-FGF 23 antibody and pharmaceutical composition containing the same", filed on 14/2/2008.

Technical Field

The invention relates to an anti-FGF 23 antibody capable of specifically binding with FGF23 antigen. Furthermore, the present invention relates to a medicament useful for preventing or treating mineral metabolism disorder caused by excess FGF23 or other causes, which comprises an anti-FGF 23 antibody as an active ingredient. In particular, the present invention relates to a medicament for the treatment of hypophosphatemic rickets (hypophosphatemic rickets) and osteomalacia (osteomalachi).

Background

Fibroblast growth factor was first isolated and purified from bovine pituitary as a substance that stimulates the growth of the fibroblast cell line NIH3T 3. Then, a number of similar proteins are isolated from different tissues, and the protein products constitute a polypeptide family (FGF family). To date, 22 proteins have been isolated and purified from members of the FGF family in vertebrates. As for the biological activity of these proteins, it is known that they have not only fibroblast growth activity but also various other functions such as mesodermal and neuroectodermal growth activity, angiogenesis activity, limb bud formation activity at a specific developmental stage, and the like. FGF also has positional and temporal variability in gene expression. It is usually expressed only at certain developmental stages or at certain sites in adults. At least four genes encoding FGF receptors are currently known, FGFR1, FGFR2, FGFR3, and FGFR 4. In addition, with regard to FGFR1, FGFR2, FGFR3, it is known that the receptor proteins produced have different extracellular domains for each receptor due to different cleavage patterns. In addition, it is currently known that heparin and heparan sulfate proteoglycans control their activity by interacting with FGF and FGF receptors. In addition, many of them have similar structures to FGF and belong to the FGF family, but their biological activities, receptor binding properties, and the like are hardly known. There is a review (see for details Ornitz, D. et al, Genome biology, 2: 3005.1-3005.12, 2001) summarizing the properties of these FGF family members.

The first clone of FGF23 (also sometimes referred to as FGF-23 in general) was obtained from mice using database search and PCR methods for homology of FGF 15. Subsequently, human FGF23 was also cloned using the mouse FGF23 homologous sequence. Human FGF23 is a polypeptide consisting of 251 amino acid residues. In addition, it is expected that, as a secretion signal sequence, an amino acid sequence consisting of up to 24 amino acids is cleaved at the N-terminus of the polypeptide at the time of protein secretion (see, for details, Yamashita, T. et al, biochem. Biophy. Res. Commun., 277: 494-498, 2000). Then, in the case of an autosomal dominant type of rickets/osteomalacia (hereinafter referred to as ADHR), it was found that the region of gene mutation in ADHR patients was narrowed (narrow down), and with the progress of the identification of the relevant gene, it was found that a specific missense mutation causing this was made in FGF23 gene in ADHR patients (for details, see White, K.E., et al, Nature Genet., 26: 345-348, 2000). These findings strongly suggest that FGF23 has important physiological significance in vivo. On the other hand, it is what determines the biological activity of FGF23, which has been studied by neoplasmic osteomalacia (neoplasmic osteomalacia), a type of hypophosphatemic rickets and osteomalacia. Among these diseases, malignant tumors produce and secrete a liquid disease-initiating factor, and as expected, diseases such as hypophosphatemic rickets and osteomalacia are caused by this disease-initiating factor.

In the study of the disease-initiating factor produced by malignant tumors, FGF23 was cloned as a gene overexpressed in tumors. Furthermore, by administration of this factor, hypophosphatemic rickets/osteomalacia were reproduced (see Shimada, T.et al, Proc. Natl. Acad. Sci., 98: 6500-6505, 2001 and International Publication Number WO02/14504 pampthlet for details). Based on the above studies, FGF23 has been shown to exhibit biological functions related to the metabolic control associated with the phosphorus and calcium elements in vivo. In addition, it was thus shown that FGF23 fulfills its function by circulating in vivo as a systemic factor. Furthermore, recent studies have shown higher concentrations of FGF23 in the blood vessels of patients with neoplastic osteomalacia compared to healthy persons (see for details Yamazaki, Y. et al, J.Clin.Endocrinol.Metab., 87: 4957-doped 4960, 2002 and Jonsson, K.B., et al, N.Engl.J.Med., 348: 1656-doped 1663, 2003).

In addition, X chromosome-linked inherited rickets with hypophosphatemia (hereinafter referred to as XLH) is known to be a disease clinically having similar manifestations to ADHR and patients with neoplastic cartilage. Patients with this disease also contain high levels of FGF23 in their blood (see for details Yamazaki, y. et al, j. clin. endocrinol. meta. pab., 87: 4957-.

In other words, the causes of vitamin D-resistant rickets and osteomalacia found in neoplastic osteomalacia and XLH, etc. have not been previously clarified, but it has been suggested that this secreted pathogenic factor is FGF 23. In addition, in the study of other types of mineral metabolic diseases such as fibroblastic dysplasia, Ma-Aus syndrome, autosomal recessive hypophosphatemic rickets and the like, there have been reports on the correlation between FGF23 at a high concentration in blood and hypophosphatemia (hypophosphatemia), rickets and osteomalacia (see the documents Riminucci, M. et al, J.Clin. invest., 112: 683-692, 2003; Yamamoto, T. et al, J.bone Miner. sub. Metab., 23: 231-237, 2005; Lorenz-Depiereux, B. et al, Nat. Genet., 38: 1248-1250, 2006).

The reports show that the excessive expression of FGF23 in vivo can cause diseases such as hypophosphatemia, hypophosphatemia accompanied rickets, osteomalacia and the like. In addition, abnormally high levels of FGF23 have been reported in the serum of patients with chronic renal insufficiency, hyperphosphatemia. There is evidence that overexpression of FGF23 may be associated with partial mineral metabolism disorders that occur in chronic renal insufficiency (see Gupta, A. et al, J.Clin. Endocrinol. Metab., 89: 4489-4492, 2004, Larsson, T. et al, Kidney Int., 64: 2272-2279, 2003 for details).

Since the cause of these diseases is the overexpression of FGF23, inhibition of the activity of FGF23 or elimination of FGF23 may become a treatment for these diseases. To date, there have been reported studies of anti-FGF 23 mouse monoclonal antibodies for inhibiting FGF23 activity (see, for details, Yamashita, T. et al, biochem. Biophy. Res. Commun., 277: 494-498, 2000). In this report, when anti-FGF 23 murine monoclonal antibodies 2C3B and 3C1E were administered to normal mice, the mouse endogenous FGF23 function was inhibited and phosphorus excretion via the kidneys was also inhibited. By interfering with the expression of vitamin D metabolizing enzymes in the kidney, the interference results indicate an increase in serum phosphorus and 1 α, 25-dihydroxyvitamin D (hereinafter referred to as 1, 25D). In addition, a monoclonal antibody against FGF23 mouse was repeatedly administered to Hyp mice, which are model mice of XLH disease with high FGF23 serum, hypophosphatemia, bone lengthening dysfunction and calcification dysfunction, and as a result, it was observed that the mice had an increased phosphorus concentration in blood and the symptoms of bone lengthening dysfunction and calcification dysfunction were improved. From these results, it can be seen that an antibody inhibiting the activity of FGF23 is available as a therapeutic drug for FGF23 overexpression type diseases. However, the 2C3B and 3C1E antibodies used in literature reports are murine antibodies. Murine antibodies can be used as foreign by human hosts and cause the human host to produce a so-called "human anti-murine antibody" (i.e., HAMA) response, which can lead to serious side effects (see, for details, Van Kronenbergh, M.J., et al, Nucl. Med. Commun.9: 919-.

To avoid this problem, one solution has been to develop chimeric antibodies (see, for details, specification of European patent application No. 120694 and specification of European patent application No. 125023). Chimeric antibodies comprise antibody fragments derived from two or more species (e.g., murine antibody variable regions and human antibody constant regions, etc.). The advantage of this type of chimeric antibody is that the binding properties of the original murine antibody to the antigen are retained. However, on the other hand, chimeric antibodies still induce a "human anti-chimeric antibody" (i.e., "HACA") response in a human host (see Bruggemann, M. et al, J.exp.Med., 170: 2153-2157, 1989 for details).

In addition, recombinant antibodies have been developed in which only a portion of the substituted antibody is a Complementary Determining Region (CDR) (see GB21886 2188638A and us 5585089). An antibody (i.e., a humanized antibody) composed of murine CDRs, human variable framework regions and constant regions was prepared using CDR grafting techniques (see, for details, Riechmann, L. et al, Nature, 332: 323-327, 1988). It is known that, using the above-described method, an anti-FGF 23 murine antibody (e.g., 2C3B antibody, etc.) can be humanized by substituting a human antibody sequence for the murine antibody. However, when humanization occurs, there is a possibility that the affinity for an antigen is reduced.

In addition, the main current treatment for rickets with hypoxemia caused by XLH and the like is oral administration of vitamin D preparation and phosphoric acid at regular intervals. However, this treatment means causes a problem in that it causes a considerable burden to the patient and his family, which is determined by the number of times and dosage of administration per day. Therefore, in order to alleviate such a burden on patients and their families, a (sustained diagnosis) hypophosphatemia therapeutic drug having a long-lasting effect on serum phosphate concentration and serum 1,25D concentration is required, so that the interval between administrations is prolonged.

Disclosure of Invention

An object of the present invention is to provide a human anti-FGF 23 antibody and a pharmaceutical composition thereof, such as a drug for prevention or treatment or the like with little side effect, which inhibits FGF23 action by using the antibody and thereby prevents and treats diseases.

Further, it is an object of the present invention to provide an antibody, which is an anti-FGF 23 antibody useful as a therapeutic agent for hypophosphatemia and which can produce a sustained increase in serum phosphate (phosphate) concentration and serum 1,25D concentration for a longer period of time with a single dose as compared with the existing anti-FGF 23 antibody. It is another object of the present invention to provide a pharmaceutical composition, for example, a medicament for preventing and treating FGF 23-related diseases using the antibody.

At present, the main treatment of rickets with hypophosphatemia is the administration of vitamin D and phosphate by oral administration several times a day. However, oral administration of a large dose of a drug several times a day causes a problem that the patient is forced to bear a large burden. The anti-FGF 23 human monoclonal antibody, C10 antibody, obtained in the present invention was confirmed to have a longer lasting effect on serum phosphate concentration and serum 1,25D concentration. These indicate that the C10 antibody is expected to have significant therapeutic advantages over currently available drugs for treating hypophosphatemia.

The invention is as follows:

[1] an anti-human FGF23 antibody or a functional fragment of the antibody, comprising the heavy chain variable region and/or the light chain variable region of an antibody produced by hybridoma cell C10 (accession number: FERM BP-10772).

[2] An anti-human FGF23 antibody or a functional fragment of such antibody, comprising the amino acid sequence of SEQ ID NO: 12 from position 20Q to position 136S and/or seq id NO: 14 from position 23 a to position 128K.

[3] An anti-human FGF23 antibody or a functional fragment of said antibody, wherein said anti-human FGF23 antibody or said functional fragment of said antibody comprises a heavy chain variable region and/or a light chain variable region amino acid sequence; and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 12 from position 20Q to position 136S, the light chain variable region amino acid sequence is as set forth in SEQ ID NO: 14 from position 23 a to position 128K.

[4] An anti-human FGF23 antibody produced by hybridoma cell C10 (claim number FERM BP-10772) or a functional fragment of such an antibody.

[5] An antibody, or a functional fragment of such an antibody, which binds to all or part of an epitope on human FGF23 that binds to an antibody produced by hybridoma cell C10 (claim number FERM BP-10772), or to all or part of an epitope on human FGF 23.

[6] An anti-human FGF23 antibody or a functional fragment of the antibody, which comprises the heavy chain variable region as described in [3] above, having any one or all of the following Complementarity Determining Regions (CDRs): consisting of SEQ ID NO:40, and a Complementarity Determining Region (CDR)1 represented by the amino acid sequence of SEQ ID NO:41, and a CDR2 represented by the amino acid sequence of SEQ ID NO:42, CDR3 as shown in the amino acid sequence of seq id no.

[7] An anti-human FGF23 antibody or a functional fragment of the antibody, comprising the light chain variable region as described in [3] above, having any one or all of the following Complementarity Determining Regions (CDRs): consisting of SEQ ID NO:43, and a Complementarity Determining Region (CDR)1 represented by the amino acid sequence of SEQ ID NO:44, and a CDR2 represented by the amino acid sequence of SEQ ID NO:45, CDR3 shown in the amino acid sequence of seq id no.

[8] An anti-human FGF23 antibody or a functional fragment of the antibody, said anti-human FGF23 antibody or functional fragment of the antibody comprising a heavy chain variable region having any one or all of the following Complementarity Determining Regions (CDRs): consisting of SEQ ID NO:40, and a Complementarity Determining Region (CDR)1 represented by the amino acid sequence of SEQ ID NO:41, and a CDR2 represented by the amino acid sequence of seq id NO:42, CDR3 as set forth in the amino acid sequence of seq id no; the anti-human FGF23 antibody or functional fragment of the antibody further comprises a light chain variable region having any one or all of the following Complementarity Determining Regions (CDRs): consisting of SEQ ID NO:43, and a Complementarity Determining Region (CDR)1 represented by the amino acid sequence of SEQ ID NO:44, and a CDR2 represented by the amino acid sequence of SEQ ID NO:45, CDR3 shown in the amino acid sequence of seq id no.

[9]Such as [1]]-[8]The anti-human FGF23 antibody of any one of claims, or a functional fragment of such an antibody, wherein: said functional fragment is selected from Fab, Fab', F(ab′)₂Disulfide-stabilized fv (dsfv), dimerized V region (diabody), single chain fv (scFv), and peptide fragments of CDRs.

[10] The anti-human FGF23 antibody according to any one of [1] to [8], or a functional fragment of the antibody, which comprises a heavy chain and/or a light chain having an amino acid sequence in which one or more amino acid residues are deleted, substituted or added.

[11] The anti-human FGF23 antibody according to any one of [1] to [10], wherein the antibody is of the IgG, IgA, IgE or IgM class.

[12] The anti-human FGF23 antibody according to [11], wherein the subclass of the antibody is IgG1, IgG2, IgG3, or IgG 4.

[13] A pharmaceutical composition comprising as an active ingredient an anti-human FGF23 antibody according to any one of [1] to [12], or a functional fragment of the antibody.

[14] A pharmaceutical composition which can control phosphorus metabolism and/or vitamin D metabolism by FGF23 and which comprises as an active ingredient an anti-human FGF23 antibody as described in any one of [1] to [12] or a functional fragment of such an antibody.

[15] A pharmaceutical composition for the prophylaxis or treatment of a disease associated with a disorder of mineral metabolism, which comprises as an active ingredient an anti-human FGF23 antibody or a functional fragment of the antibody as described in any one of [1] to [12 ].

[16] The pharmaceutical composition according to [15], wherein the disease associated with metabolic disorders of minerals is selected from neoplastic osteomalacia, ADHR, XLH, dysplasia of bone fibroid, Mao-Aoshi syndrome and autosomal recessive hypophosphatemia.

[17] A pharmaceutical composition for preventing or treating a disease selected from the group consisting of osteoporosis, rickets, hypercalcemia, hypocalcemia, ectopic calcification, bone sclerosis, Pagey's disease, hyperparathyroidism, hypoparathyroidism and pruritus, which comprises as an active ingredient the anti-human FGF23 antibody or a functional fragment of the antibody as described in any one of [1] to [12],

[18] hybridoma cell C10 (Soxhlet number FERM BP-10772).

[19] A nucleic acid encoding a heavy chain variable region amino acid sequence consisting of SEQ ID NO: 11 from the 58 th C to the 408 th A nucleotide sequence shown in FIG. 11.

[20] A nucleic acid encoding a light chain variable region amino acid sequence consisting of SEQ ID NO: 13 from the 67 th G to the 384 th A nucleotide sequence.

[21] A vector comprising the nucleic acid as described in [19] or [20 ].

[22] A host cell comprising the vector as described in [21 ].

[23] A method for producing an anti-human FGF23 antibody or a functional fragment of the antibody, comprising the steps of: culturing the host cell as described in [22] to express an anti-human FGF23 antibody or a functional fragment of the antibody.

Drawings

FIG. 1 is a schematic diagram of the construction process of C10 expression vector

FIG. 2 shows the nucleotide sequence (SEQ ID NO: 30) and amino acid sequence (SEQ ID NO: 31) of the antibody heavy chain gene in N5KG 1-C10-LH. The amino acid sequence marked with a rectangular box in the figure is a secretion signal sequence (leader sequence).

FIG. 3 shows the nucleotide sequence (SEQ ID NO: 32) and amino acid sequence (SEQ ID NO: 33) of the antibody light chain gene in N5KG 1-C10-LH. The amino acid sequence marked with a rectangular box in the figure is a secretion signal sequence (leader sequence).

FIG. 4 shows the structure of the C10 expression vector.

FIG. 5A shows the results of measurement of the purified full-length human FGF23 protein by sandwich enzyme-linked immunosorbent assay (ELISA), in which 2C3B antibody or C10 antibody was used as the immobilized antibody and 3C1E antibody was used as the test antibody.

FIG. 5B shows the results of assays performed on supernatants from cultures of FGF 23-expressing cynomolgus monkey (cynomolgus monkey) cells using sandwich enzyme-linked immunosorbent assay (ELISA) using 2C3B antibody or C10 antibody as immobilized antibody and 3C1E antibody as detection antibody.

The graph in fig. 6 shows the results of measuring the serum phosphorus concentration of cynomolgus monkeys at various time points over time when the cynomolgus monkeys were administered either 2C3B antibody or C10 antibody. The measurements are expressed as mean +/-standard error. In addition, the significant difference from the solvent-administered group was examined by Student-test, and the value measured with the significant difference (p < 0.05) was marked with an x number on the graph.

The graph in fig. 7 shows: after 5 days of solvent administration to the macaque and the serum phosphorus concentration of the macaque as a reference, the soluble 2C3B antibody or C10 antibody of the macaque is administered for 5 days, and the serum phosphorus concentration of the macaque is increased.

The graph in fig. 8 shows: the results of the test of the concentration of 1,25D in cynomolgus monkey serum at different time points over time when cynomolgus monkey is dosed with solvent, 2C3B antibody or C10 antibody. The measurements are expressed as mean +/-standard error. In addition, when the test for the difference in significance between the test group and the solvent administration group was performed on the same day by the student's T test method, the measurement values having a significant difference (p < 0.05) were marked with an index on the graph.

FIG. 9 is a graph showing the detection of non-forced expression of cell culture supernatant (as control), FGF 23-expressing human cell culture supernatant, and FGF 23-expressing cynomolgus monkey cell culture supernatant using the C15 antibody and protein hybridization (Western blotting) technique.

FIG. 10 shows the structure of the vector pPSs FGF 23.

FIG. 11 shows the structure of the pUS FGF23KI vector.

FIG. 12 shows the structure of 3 alleles: a drug resistance gene (loxp-peo) having an allelic structure^r) Is directed to an allelic structure of human FGF23(-SP) + drug resistance gene (loxp-peo) among others^r) The KI vector was oriented by pUS hFGF23, one of the allelic structures was the drug resistance gene (loxp-peo)^r、loxpv-puro^r) Knocked out, and the position of the probe for southern blot analysis. The detailed explanation of the proper nouns appearing in the figures is as follows:

hFGF23 (-SP): a human FGF23 gene lacking a specific signal peptide coding region. C_κ: mouse Ig_κA gene constant region. loxpv-puro: a puromycin resistance gene having loxpv sequence at both ends thereof, the loxpv sequence being a loxp sequence having a partial mutation. loxp-neo^r: a neomycin phosphotransferase resistance gene with loxp sequences at both ends.

Ck 3' probe: southern blotting (Southern blotting) analysis probes for screening hFGF23(-SP) + loxpv-puro^rGene transfer and loxpv-puro^rKnock-out cloning of the gene. 3' KO-Probe: southern blot analysis probes for screening loxp-neo^rGene transfer and knockout cloning. E: EcoRI restriction sites.

FIG. 13 is a graph showing the results of measurement of the serum FGF23 concentration 7 days before the treatment with the control antibody or C10 antibody. The measurements are expressed as mean +/-standard error. In addition, the test group was tested for significant differences from the wild type mouse group by Student's T-test, with the significant differences (p < 0.001) indicated by the x-signs on the graph.

FIG. 14 is a graph showing the results of measurements of serum phosphate concentration 7 days before the control or C10 antibody treatment and 3 days after the control or C10 antibody treatment was first administered. The measurements are expressed as mean +/-standard error. In addition, the test group was tested for significant differences from the wild type mouse group by the student's T test method, and the group with significant differences (p < 0.001) was marked with a mark on the graph. In addition, when a significant difference between the group to which the hFGF23 KI-type mouse control antibody is administered and the test group is examined in one day, the group to which the hFGF23 KI-type mouse C10 antibody is administered, which has a significant difference (p < 0.001), is marked with # # in the graph.

FIG. 15 is a graph showing the results of serum phosphorus concentration measurements 1 day after the fifth control antibody or C10 antibody administration. The measurements are expressed as mean +/-standard error. In addition, the test group was tested for significant differences from the wild type mouse group by the student's T test method, and the group with significant differences (p < 0.001) was marked with a mark on the graph. In addition, when significant differences between the group to which the hFGF23KI type mouse control antibody is administered and the test group are examined, the group to which the hFGF23KI type mouse C10 antibody is administered, which has significant differences (p < 0.001), is marked with # # in the graph.

FIG. 16 is a graph showing the results of the grip strength test of animals 1 day after the fourth control antibody or C10 antibody administration. The measurements are expressed as mean +/-standard error. In addition, the test group was tested for significant differences from the wild type mouse group by the student's T test method, and the group with significant differences (p < 0.001) was marked with a mark on the graph. In addition, when significant differences between the group to which the hFGF23KI type mouse control antibody is administered and the test group are examined, the group to which the hFGF23KI type mouse C10 antibody is administered, which has significant differences (p < 0.001), is marked with # in the graph.

FIG. 17 shows a stained image of mouse femoral tissues obtained from mice treated for 1 day with the fifth control antibody or C10 antibody, using a Villanueva-Goldner staining procedure.

FIG. 18 is a graph showing the measurement of the weight of tibial ash in mice as a function of the dry weight of the tibia, wherein the femur used was derived from the mice treated for 1 day after the fifth administration of the control antibody or C10 antibody. The measurements are expressed as mean +/-standard error. In addition, the test group was tested for significant differences from the wild type mouse group by the student's T test method, and the group with significant differences (p < 0.001) was marked with a mark on the graph. In addition, when significant differences between the group to which the hFGF23KI type mouse control antibody is administered and the test group are examined, the group to which the hFGF23KI type mouse C10 antibody is administered, which has significant differences (p < 0.001), is marked with # # in the graph.

[ plain text of sequence Listing ]

SEQ ID NO: 1-3, 5-27, 30-33, 44-50 synthesis

Detailed Description

The present invention will be described in detail below with reference to the definitions of terms used in the present invention.

I. Antibodies of the invention

1. anti-FGF 23 antibodies and functional fragments thereof

The antibodies of the invention are anti-FGF 23 antibodies, which are members of the Fibroblast Growth Factor (FGF) family.

In the present invention, the anti-FGF 23 antibody binds to FGF23 or a portion thereof, is reactive with FGF23 or a portion thereof, or recognizes FGF23 or a portion thereof. The anti-FGF 23 antibody is also referred to as an anti-FGF 23 antibody. In the present invention, the antibody is an immunoglobulin, wherein all structural regions constituting the heavy chain variable region, the heavy chain constant region, the light chain variable region and the light chain constant region of the immunoglobulin are derived from a gene encoding the immunoglobulin. The antibody is preferably a monoclonal antibody. Here, the portion of FGF23 denotes SEQ ID NO:4, and the part of the amino acid sequence of the full-length amino acid sequence of the FGF23 is an FGF23 peptide segment comprising a continuous amino acid sequence. Preferably, the antibody comprises SEQ ID NO: 12 from Q at position 20 to S at position 136 and/or SEQ id no: 14 from position 23 a to position 128K. More preferably, the antibody is produced by hybridoma cell C10. SEQ ID NO: 12 is the amino acid sequence of the heavy chain variable region of the anti-FGF 23 antibody, including the leader sequence, SEQ ID NO: 12 from position 20Q to position 136S is the mature amino acid sequence fragment formed after the leader sequence is cut out of the amino acid sequence. In addition, SEQ ID NO: 14 is the amino acid sequence of the light chain variable region of the anti-FGF 23 antibody, including the leader sequence, SEQ ID NO: the amino acid sequence from the 23 rd position A to the 128 th position K in 14 is a mature amino acid sequence fragment formed after the leader sequence of the amino acid sequence is cut off. With respect to the antibody types, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin E (IgE), and immunoglobulin M (IgM) may all be used. Immunoglobulin g (IgG) is preferred, and further, for the subclass of immunoglobulin g (IgG), IgG1, IgG2, IgG3, and IgG4 may be used, and IgG1, IgG2, and IgG4 are preferred. More preferably IgG 1.

The antibodies of the invention also include anti-FGF 23 antibodies comprising novel Complementarity Determining Region (CDR) amino acid sequences.

CDRs are present in the antibody variable region, and this part of the structure provides antigen recognition specificity. The portion of the variable region other than the complementarity determining region functions to maintain the structure of the complementarity determining region and is called a Framework Region (FR). The constant regions are present at the C-terminus of the heavy and light chains and are referred to as the heavy chain constant region (CH) and the light chain constant region (CL), respectively.

The complementarity determining regions present in the heavy chain variable region are complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR 3). These 3 complementarity determining regions in the heavy chain variable region are collectively referred to as heavy chain complementarity determining regions. Similarly, the 3 complementarity determining regions present in the light chain variable region are complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR 3). These 3 complementarity determining regions in the light chain variable region are collectively referred to as light chain complementarity determining regions. The Sequences of these complementarity determining regions can be obtained by the method described in "protein Sequences of Immunological importance" of the United states department of public health and Human Services (1991), and the like (Sequences of Proteins of Immunological Interest, US Dept. health and Human Services (1991)).

The antibody of the invention preferably has the amino acid sequence of SEQ ID NO:40, CDR1 shown in SEQ ID NO:41 and CDR2 shown in SEQ ID NO:42 as the heavy chain complementarity determining region of an antibody, at least any one or all of the complementarity determining regions of CDR3 shown in fig. 42. In addition, the antibody of the present invention preferably has the amino acid sequence of SEQ ID NO:43, CDR1 shown in SEQ ID NO:44 and CDR2 shown in seq id NO:45 as the light chain complementarity determining region of an antibody, at least any one or all of the complementarity determining regions of CDR3 shown in seq id no. The antibody of the invention is preferably an antibody that binds to FGF23 and has the amino acid sequence of seq id NO:40, CDR1 shown in SEQ ID NO:41 and CDR2 shown in SEQ ID NO:42 as the heavy chain complementarity determining region of an antibody and having the amino acid sequence shown in SEQ ID NO:43, CDR1 shown in SEQ ID NO:44 and CDR2 shown in SEQ ID NO: CDR3 shown in 45 serves as the light chain complementarity determining region of the antibody.

The CDR sequences of the antibody of the present invention are not particularly limited. However, the antibody of the invention preferably comprises SEQ ID NO: 40-45, more preferably comprises 3 heavy chain complementarity determining regions, and even more preferably comprises 6 complementarity determining regions as shown. The amino acid sequence other than the complementarity determining region is not particularly limited. The antibody of the present invention includes a so-called CDR-grafted antibody in which the amino acid sequence other than the complementarity determining region is derived from another antibody, particularly an antibody derived from another species. In these CDR-grafted antibodies, the amino acid sequences other than the CDRs are preferably derived from a humanized antibody or a human antibody. If necessary, the FR may be subjected to addition, deletion, substitution and/or insertion of one or more amino acid residues. The method for producing a humanized antibody or a human antibody can be a known method.

"functional fragment" refers to a portion (partial fragment) of an antibody having one or more activities (actions) of the antibody against an antigen. In other words, it refers to a fragment that retains the binding ability to an antigen, reactivity to an antigen, or recognition ability to an antigen. For example, Fv, disulfide-stabilized Fv (dsfv), single-chain Fv (scFv), and polymers thereof. More specific examples include those containing Fab, Fab ', F (ab')₂scFv, diabody: (diabody), dsFv and CDR peptides [ D.J.King, Applications and engineering of Monoclonal antibodies, 1998T.J.International Ltd]。

Among these fragments, the Fab fragment, which is an antibody fragment having an antigen-binding activity and a molecular weight of about 50,000, was obtained by treating an antibody that binds to FGF23 with papain. The Fab fragment contains nearly half of the amino-terminal side of a single heavy chain (H chain) and a single intact light chain (L chain) linked together by a disulfide bond.

The Fab fragments of the present invention can be prepared by papain treatment of antibodies that bind FGF 23. Or the antibody Fab fragment is coded by inserting the DNA sequence into an expression vector for prokaryote or an expression vector for eukaryote, and then the vector is transferred into the prokaryote and eukaryote to express the vector.

Among the fragments obtained by treating IgG with pepsin, F (ab')₂The fragment is an antibody fragment having an activity of binding to an antigen and having a molecular weight of about 100,000. F (ab')₂The fragments are larger than the Fab fragments which are disulfide bonded together through the hinge region.

F (ab')₂Fragments may be prepared by pepsin treatment of antibodies that bind FGF23, or by linking Fab's together using disulfide or thioether bonds as described below.

Fab' is an antibody fragment with a molecular weight of about 50,000 that has antigen binding activity. Wherein, the F (ab')₂The disulfide bonds of the hinge region are cleaved.

Fab 'of the present invention can be prepared by treating F (ab') of the present invention capable of binding to FGF23 with a reducing agent dithiothreitol₂And obtaining the product. Or the antibody Fab' fragment is coded by inserting the DNA sequence into an expression vector for prokaryote or an expression vector for eukaryote, and then the vector is transferred into the prokaryote and eukaryote to express the vector.

scFv is an antibody fragment having antigen-binding activity, which is a VH-P-VL or VL-P-VH type polypeptide comprising a single heavy chain variable region (hereinafter referred to as VH) and a single light chain variable region (hereinafter referred to as VL) linked by a suitable peptide linker (hereinafter referred to as P).

The scFv fragment can be obtained by obtaining cDNA which is combined with FGF23 and codes VH and VL in the antibody, constructing a DNA sequence for coding scFv, inserting the DNA sequence for coding the antibody fragment into an expression vector for prokaryotes or an expression vector for eukaryotes, transferring the vector into prokaryotes and eukaryotes, and expressing the vector.

A diabody is an antibody fragment formed by scFv dimerization, which has bivalent antigen binding activity. The binding activity of the bivalent antigen may be the same or different.

The diabody of the present invention can be prepared by obtaining cDNA encoding VH and VL in the antibody conjugated to FGF23 of the present invention, constructing a DNA sequence encoding scFV in such a manner that the length of the amino acid sequence of the peptide fragment linker is 8 residues or less, inserting the DNA sequence into an expression vector for prokaryote or an expression vector for eukaryote, transferring the vector into prokaryote or eukaryote, and expressing the vector.

In dsFv, one amino acid residue in each of the VH and VL fragments that make up sFv is replaced with a cysteine residue, and the two polypeptide fragments are linked together by a disulfide bond formed between these cysteine residues. The selection of amino acid residues substituted with cysteine residues can be made based on the prediction of the three-dimensional structure of the antibody according to the method used by Reiter et al (protein engineering, 7: 697-704, 1994).

The dsFv fragment of the invention can be prepared by obtaining cDNA encoding VH and VL in the antibody of the invention combined with FGF23, constructing a DNA sequence encoding dsFv, inserting the DNA sequence encoding the antibody fragment into an expression vector for prokaryote or an expression vector for eukaryote, transferring the vector into prokaryote or eukaryote, and expressing the vector.

The constructed peptide segment containing the complementarity determining region at least contains more than one VH or VL complementarity determining region. Peptide fragments containing multiple complementarity determining regions may be joined directly or by suitable peptide fragment linkers.

The peptide fragment containing the complementarity determining region can be prepared by constructing a DNA sequence encoding the VH and VL Complementarity Determining Regions (CDRs) of the antibody of the present invention bound to FGF23, inserting the DNA sequence into an expression vector for prokaryote or an expression vector for eukaryote, transferring the vector into prokaryote or eukaryote, and expressing the vector.

Furthermore, the peptide fragment containing the complementarity determining region can be prepared by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method).

Furthermore, a "functional fragment" is a fragment of an antibody that binds to an antigen (FGF 23). Such "functional fragment" is preferably a fragment that binds FGF23 and comprises the amino acid sequence of SEQ ID NO: 12 from position 20Q to position 136S, and/or SEQ ID NO: 14 from position 23 a to position 128K. Such "functional fragment" is preferably a fragment comprising SEQ ID NO: 40-45, and which binds to FGF 23. Such "functional fragments" are more preferably derived from the variable regions of antibodies produced by the C10 hybridoma and bind FGF 23.

The antibody of the present invention includes antibody derivatives, wherein radioisotopes, low molecular weight drugs, macromolecular drugs, proteins, and the like are chemically or genetically engineered to bind to the anti-FGF 23 antibody of the present invention or a functional fragment of the antibody.

The antibody derivative of the present invention can be prepared by chemically binding a radioisotope, a low molecular weight drug, a macromolecular drug, a protein or the like to the N-terminus or C-terminus of the heavy chain or light chain of the anti-FGF 23 antibody or "functional fragment" of the antibody of the present invention, a suitable substituent or side chain of the anti-FGF 23 antibody or "functional fragment" of the antibody, a sugar chain of the anti-FGF 23 antibody or "functional fragment" of the antibody of the present invention, or the like (Koutai Kogaku Nyuumon, Osamu Kanamitsu, Chijin shokan, 1994).

Alternatively, protein-bound antibody derivatives can be prepared by linking a DNA sequence encoding the anti-FGF 23 antibody of the present invention or a "functional fragment" of the antibody to a DNA sequence encoding a protein to be bound, inserting the DNA sequence into an expression vector, transferring the expression vector into an appropriate host cell, and expressing the vector.

For the radioactive isotope, examples thereof include¹³¹I、¹²⁵I, and the like. For example, the radioisotope can be conjugated to the antibody by a method such as T-chloramine labeling.

Low molecular weight drugs include: alkylating agents (alkylating agents) including nitrogen mustards (nitrogen mustards), cyclophosphamide (cyclophosphamide); antimetabolites such as 5-fluorouracil (5-fluorouricil), methotrexate (methotrexate), and the like; antibiotics (antibiotics) such as daunomycin (daunomycin), bleomycin (bleomycin), mitomycin C (mitomycin C), daunorubicin (daunorubicin), and doxorubicin (doxorubicin), and the like; plant alkaloids such as vincristine (vincristine), vinblastine (vinblastine) and vindesine (vindesine); anticancer drugs such as hormonal drugs tamoxifen (tamoxifen nd) and dexamethasone (dexamethasone) (Clinical Oncology; Japanese Clinical Oncology Research Meeting, Japanese Journal of Cancer and chemotherapy Co., 1996); steroids (steroids) such as cortisol (hydrocortisone) and prednisone (prednisone), etc.; non-steroids including aspirin (aspirin) and indomethacin (indomethacin); immunomodulators such as gold thiomalate (gold thiomalate) and penicillamine (penicillamine), etc.; immunosuppressants (immunosupressors) such as cyclophosphamide (cyclophosphamide) and azathioprine (azathioprine), etc.; anti-inflammatory drugs (anti-antibiotics) such as antihistamines (anti-histamines) drugs chlorpheniramine maleate and clemastine (clemastine) and the like (Inflammation and anti-Inflammation treatment method, Ishiyaku publishing corp. ltd., 1982) can be bound by a known method, for example, examples of a method for binding daunomycin and an antibody include a method of binding between amino groups of daunomycin and an antibody by glutaraldehyde, and a method of binding together an amino group of daunomycin and a carboxyl group of an antibody by water-soluble carbodiimide. By combining a low-molecular-weight drug and an antibody, an antibody derivative having the function of the low-molecular-weight drug can be produced.

For the macromolecular drugs, examples thereof include polyethylene glycol (hereinafter, referred to as PEG), albumin, dextran, polyethylene oxide, styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, hydroxypropyl methacrylamide and the like. By binding these macromolecular compounds to antibodies or "functional fragments" of antibodies, several effects are expected: (1) improving the stability of the product to various chemical, physical and biological factors. (2) Remarkably prolong the half-life in blood, (3) the immunogenicity disappears, the antibody production is inhibited, etc. (Bioconjugate Pharmaceutical, Hirokawa Shoten, 1993). An example of one method for combining PEG and an antibody is a method of reacting with a PEG-modifying reagent. Examples of PEG modifying agents include epsilon-amino group modifier lysine (Laid-Open Patent publication Number S61-178926), carboxyl group modifier aspartic acid and glutamic acid (Laid-Open Patent publication Number S56-23587), guanidino group modifier arginine (Laid-Open Patent publication Number H2-117920), and the like.

The antibody bound to the protein can also be prepared as a fusion antibody. In other words, by linking cDNA encoding the antibody or a functional fragment of the antibody to cDNA encoding a specific protein, DNA encoding a fusion protein composed of the antibody and the specific protein is constructed. This DNA is inserted into an expression vector for prokaryotes or eukaryotes. After the expression vector is transferred into prokaryotes and eukaryotes, the fusion antibody combined with the specific protein can be produced after the vector is expressed.

As for the anti-FGF 23 antibody or a functional fragment of the antibody described in the present invention, the evaluation of its binding activity to human FGF23 or its functional inhibitory activity against human FGF23 can be carried out by Immunological assays such as ELISA (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1988; Monoclonal Antibodies: Princles and Practice, academic Press Limited, 1996) or by measuring its binding dissociation constant by a biosensing technique such as a Biacore biosensor (Journal of Immunological Methods, 145: 229. quadrature. 240, 1991) and by detecting the inhibition of the early growth reactive gene-1 promoter activity by the stimulation of human FGF23 by cells expressed with klothoc (Nature, 444: 774, 2006), etc.

In the present invention, a "human antibody" is defined as an antibody derived from a gene expression product of a human antibody gene. As described below, the antigen can be obtained by administering the antigen to a transgenic animal having the ability to produce the human antibody by transferring the human antibody gene site to the animal. Examples of such transgenic animals include mice. Established methods for producing human antibody-producing mice are described, for example, in International patent publication No. WO 02/43478.

Examples of the antibody according to the present invention include an antibody produced by a C10 hybridoma cell (C10 antibody) described in the following examples. On 2.2007, C10 hybridoma cells have undergone international deposition under the Budapest treaty, deposited at the independent institute of Advanced Industrial Science and Technology, national institute of Technology, Central 6, 1-1Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan, with the accession number FERM ABP-10772 (recognition: C10).

The antibody or functional fragment thereof of the present invention also includes a monoclonal antibody or functional fragment thereof comprising a heavy chain and/or a light chain, each of the amino acid sequences constituting the heavy chain and/or the light chain having one or several amino acid deletions, substitutions and additions. Here, "1 or several", "several" means 9 or less, preferably 5 or less, more preferably 3 or less, and particularly preferably 2. Partial modifications (deletions, substitutions, insertions, additions) of an amino acid sequence as described previously may be introduced into the amino acid sequence constituting an antibody or functional fragment according to the invention by partial modifications of the nucleotide sequence encoding this amino acid sequence. Partial modifications of these nucleotide sequences can be introduced by conventional methods of known site-specific mutagenesis methods [ Proc Natl Acad Sci usa., 81: 5662-5666, 1984]. The antibodies of the invention include antibodies of all immunoglobulin types and isotypes.

The anti-FGF 23 antibody of the present invention can be produced by the following production method. For example, a conjugate (conjugate) of FGF23, or a portion of FGF23, or a portion of FGF23, and a suitable pharmaceutical carrier (e.g., bovine serum albumin, etc.) capable of causing antigenic growth of the antigen thereof, together with a corresponding adjuvant (freund's complete or incomplete adjuvant, etc.) as necessary, is used to immunize a desired non-human mammal, such as a human antibody-producing transgenic mouse. For FGF23, either native or recombinant FGF23 can be used. Alternatively, immunosensitization can be generated by transferring a gene encoding FGF23 into an expression vector and expressing the FGF23 protein in the animal. Monoclonal antibodies are produced by culturing hybridomas (formed by fusing antibody-producing cells derived from an immunized animal with myeloma cells having no antibody-producing ability) to produce monoclonal antibodies having specific affinity for an antigen used for immunization, and selecting clones producing the monoclonal antibodies.

The antibodies of the present invention include those that are converted into different subclasses by using genetic engineering modification techniques well known to those skilled in the art (see, for example, European patent application EP 314161). That is, a subclass of antibody different from the original subclass can be produced by using DNA encoding the variable region of the antibody of the present invention by genetic engineering techniques.

2. Production of the antibodies of the invention

The production of a monoclonal antibody involves the following steps. Namely:

(1) preparation of an antigenic protein or an expression vector for the antigenic protein to be used as an immunogen, (2) extraction of blood sample from an animal and analysis of antibody titer in the blood sample by antigen injection or antigen expression in the animal in vivo after immunization of the animal, and after determination of spleen separating time, preparation of antibody-producing cells (3) preparation of myeloma cells (4) fusion of antibody-producing cells and myeloma cells (5) screening of hybridoma cell masses capable of producing an antibody of interest (6) hybridoma cell masses broken into individual cell clones (7) optionally culturing the hybridomas or feeding of the animals into which the hybridomas have been transplanted for production of large amounts of monoclonal antibodies (8) measurement of the biological activity and recognition specificity of the monoclonal antibodies produced in this manner or measurement of the characteristics as a labeling agent.

The method for producing the anti-FGF 23 monoclonal antibody according to the above procedure will be described in detail below. However, the production method of such an antibody is not limited to this method. For example, antibody-producing cells and myeloma cells other than spleen cells (production of such antibodies) may be used.

(1) Purification of antigens

As antigens, there may be used: by using gene recombination technology, a DNA sequence coding for FGF23 is integrated into a suitable expression plasmid, and after FGF23 is produced in a host such as Escherichia coli or animal cells, etc., the FGF23 protein is purified. Since the primary structure of the human FGF23 protein is well known [ GenBank accession No.: no. aag09917, SEQ ID NO: 4], an incomplete peptide fragment chemically synthesized by a method well known to those skilled in the art and composed of an amino acid sequence derived from FGF23 can also be used as the antigen.

(2) Preparation procedure of antibody-producing cells

The antigen mentioned in the above (1) which has been prepared is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or aluminum potassium, etc., and the mixture is used as an immunogen to immunize an experimental animal. For the experimental animals, transgenic mice having the ability to produce human-derived antibodies are most suitably used. Such mice are described in references [ tomizuka et al, Proc Natl Acad Sci usa, 97: 722-727, 2000] is described by Tomizuka et al.

When mice are immunized, the immunogen can be administered by any one of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, toe injection, and the like. Intraperitoneal injection, toe injection or intravenous injection is preferred.

One immunization may be performed or multiple immunizations may be performed at appropriate intervals. Subsequently, the antibody titer against the antigen present in the serum of the immunized animal is measured. When an animal having a very high antibody titer is used as a production raw material for antibody-producing cells, the efficiency of the subsequent operation steps is improved. Generally, it is preferred to use antibody producing cells derived from animals 3-5 days after final immunization for subsequent cell fusion.

Here, examples of the method for detecting an antibody titer include various known techniques such as a radioactive isotope immunoassay (hereinafter referred to as "RIA method"), a fixed enzyme immunoassay (hereinafter referred to as "ELISA method"), a fluorescent antibody method, a passive hemagglutination method, and the like. The RIA method or ELISA method is suitable from the viewpoint of detection sensitivity, rapidity, accuracy and feasibility of automated operation.

The detection of the titer of the antibody of the present invention, for example, by using an ELISA assay, can be carried out by the following procedure. First, an anti-human antibody antigen is adsorbed to a fixed surface of an ELISA96 well plate or the like. This immobilized surface, which has not adsorbed the antigen, is then blocked with a protein unrelated to the antigen, such as Bovine Serum Albumin (BSA). After rinsing the surface, the surface is contacted with a reagent (e.g., serum from a human antibody-producing transgenic mouse) diluted in a continuous concentration gradient as a primary antibody to bind the antigen to the anti-FGF 23 antibody in the sample. Then, an enzyme-labeled anti-human antibody is added as a secondary antibody that binds to the human antibody. After rinsing, a substrate for the enzyme is added. Then, the change in light absorption due to the color change caused by the degradation of the substrate was detected to calculate the antibody titer.

(3) Myeloma preparation step

As the myeloma, a cell derived from a mammal such as a mouse, rat, guinea pig, hamster, rabbit, or human, which does not have an antibody-producing ability by itself, can be used. Generally, it is preferred to use a mouse-derived cell line, such as the 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1(P3-U1) [ Yelton, D.E., et al, Current Topics in Microbiology and Immunology, 81: 1-7, 1978]P3/NSI/1-Ag4-1(NS-1) [ Kohler, G. et al, European J.immunology, 6: 511-519, 1976]Sp2/O-Ag14(SP2/O) [ Shulman, M. et al, Nature, 276: 269-270, 1978]P3x63ag8.653(653) [ Kearney, j.f. et al, j.immunology, 123: 1548-1550, 1979]P3X63Ag8(X63) [ Horibata, k. and Harris, a.w., Nature, 256: 495-497, 1975]And the like. The subculture of these cell lines is carried out using a suitable medium, for example 8-azaguanine medium [ a medium RPMI-1640 supplemented with glutamine, 2-mercaptoethanol, gentamicin and Foetal Calf Serum (FCS), also 8-azaguanine]Iscove's Modified Dulbecco's Medium (IMDM) or Dulbecco's Modified Eagle Medium (DMEM medium). However, 3-4 days before cell fusion, the cell line is subcultured in normal medium (e.g., DMEM medium containing 10% FCS) and the number of cells prepared for cell fusion is not less than 2X10⁷。

(4) Cell fusion

Antibody-producing cells are lymphocytes of plasma cells and their precursors. These cells can be obtained from any location in the individual organism. Generally, spleen, lymph node, bone marrow, tonsil, peripheral blood, or suitable combinations thereof can be fused. Generally, spleen cells are most commonly used. .

After the last immunization of the animal, a site where the antibody-producing cells exist, such as the spleen, is isolated from a mouse which can obtain a prescribed antibody titer, thereby producing spleen cells as antibody-producing cells. Next, spleen cells and myeloma cells are fused. For the fusion method of spleen cells and myeloma cells obtained in step (3), the most commonly used method is to use polyethylene glycol. The method has relatively low cytotoxicity, and the fusion operation is simple and easy. This method has the following steps, for example:

spleen cells and myeloma cells are washed with serum-free medium (e.g., DMEM) or phosphate-buffered saline (PBS), mixed at a ratio of 5: 1 to 10: 1, and centrifuged. The supernatant was removed, the pellet was broken up and 1mL of serum-free medium containing 50% (w/v) polyethylene glycol (MW 1000-4000) was added with stirring. Then, 10mL of serum-free medium was slowly added, followed by centrifugation. The supernatant was discarded, and the precipitated cells were suspended in a normal medium (hereinafter, HAT medium) containing appropriate amounts of hypoxanthine/aminopterin/thymidine (hereinafter, HAT) lysate and human interleukin-6 (hereinafter, IL-6). These cells were equally divided equally to each well of the plate (hereinafter referred to as plate) and incubated at 37 □ in the presence of 5% CO₂Cultured under the conditions of (1) for about two weeks. During the culture, HAT medium was supplemented as necessary.

(5) Screening of hybridoma cell masses:

when the above-mentioned myeloma cell is an 8-azaguanine-resistant strain, that is, if it is a Hypoxanthine Guanine Phosphoribosyltransferase (HGPRT) -deficient cell strain, the myeloma cell which is not fused or the fused cell formed by fusing only the myeloma cells cannot survive in a medium containing HAT. On the other hand, a fused cell formed only from an antibody-producing cell or a fused cell hybridoma formed from an antibody-producing cell and a myeloma cell is viable. However, the life cycle of fused cells formed by fusion of antibody-producing cells alone is limited. Therefore, only hybridoma cells of a fused type formed from antibody-producing cells and myeloma cells can survive by continuous culture in a medium containing HAT. Thus, hybridoma cells can be selected.

For hybridomas that are growing in clones, the medium is changed to a medium that does not contain aminopterin (hereinafter referred to as HT medium). Then, a part of the culture supernatant was collected and the anti-FGF 23 antibody titer was measured by, for example, ELISA.

The above is an example of a method using an 8-azaguanine resistant cell line. However, other cell lines may be used in the screening method for hybridoma cells. In these cases, the composition of the medium used may also vary.

(6) Cloning procedure

Hybridoma cells determined to produce the specific antibody are transferred to another culture plate and cloned by detecting antibody titer using the same antibody titer detection method as used in (2). Examples of cloning methods include: a limiting dilution method in which hybridoma cells are diluted so that each culture well in a culture plate contains only one hybridoma cell and are cultured; a soft agar culture method in which hybridoma cells are cultured in a soft agar medium and clones are collected; a method in which a micromanipulator is used to transfer and culture one cell at a time; sorter sorting cloning, in which single cells and the like are separated using a cell sorter. Limiting dilution is relatively simple and common.

With respect to the culture wells, antibody titers were observed in the culture wells, and, for example, when cloning was repeated 2 to 4 times by the limiting dilution method, cell lines producing stable antibody titers were selected for use as anti-FGF 23 monoclonal antibody-producing hybridoma cell lines.

(7) Preparation of monoclonal antibodies by hybridoma cell culture

The HT medium was changed to the normal medium to culture the cloned hybridoma cells. For large-scale culture, there are circulation culture using a large-volume culture flask, spinner culture, culture using a hollow fiber system, and the like. The anti-FGF 23 monoclonal antibody is obtained by purification of the large-scale culture supernatant by methods well known to those skilled in the art, such as gel filtration and the like. Furthermore, peritoneal fluid containing a large amount of anti-FGF 23 monoclonal antibody was obtained by growing the hybridoma in the peritoneum of the same strain of mouse (e.g., BALB/c) or nu/nu mouse, rat, guinea pig, hamster, rabbit, etc. A simple antibody purification method is to use a commercially available monoclonal antibody purification kit (e.g., MAbTrap GII kit; GEHealthcare Bioscience Co.), etc.

The monoclonal antibodies obtained by these methods have high antigen specificity against FGF 23.

In addition, recombinant Antibodies can be prepared by cloning the gene encoding the ANTIBODY from ANTIBODY-producing cells such as myeloma using gene recombination techniques (Delves, P.J., ANTIBODY PRODUCTION ESTINAL TECHNIQUES, 1997 WILEY, Shepherd, P. and Dean C., Monoclonal Antibodies, 2000 OXORDUNIVERTY PRESS, Goding, J.W., Monoclonal Antibodies: principles and cloning, 1993 ACADEMIC PRESS), integrating the gene into a suitable vector and transferring into a host (e.g., mammalian cell lines, E.coli, yeast cells, insect cells, plant cells, etc.).

The present invention includes nucleic acids containing the gene sequences of antibodies produced by hybridomas producing the antibodies of the present invention, and particularly includes the following nucleic acids containing the heavy chain variable regions and the light chain variable regions of antibodies produced by hybridomas of the present invention. Herein, nucleic acids include DNA and RNA. In addition, the present invention also includes nucleic acids of the mature fragments of the heavy chain variable region and the light chain variable region as described in the present invention, wherein the mature fragments refer to the heavy chain variable region and the light chain variable region peptide fragments remaining after the removal of the signal peptide sequence. Further, the nucleic acid of the present invention includes, in addition to the above-mentioned nucleic acids, nucleic acids having codons corresponding to amino acids of the amino acid sequence of the antibody of the present invention and amino acids of the heavy chain variable region and/or the light chain variable region of the antibody.

For the preparation of a gene encoding a monoclonal antibody derived from the hybridoma, a method is employed according to which DNA encoding each of the light chain variable region, light chain constant region, heavy chain variable region, heavy chain constant region of the monoclonal antibody can be prepared by PCR or the like. In this preparation method, oligo DNA designed based on the anti-FGF 23 antibody gene or its amino acid sequence was used as a primer. As the template, DNA prepared from the hybridoma can be used as the template. The DNAs may be expressed after being integrated into a suitable vector and transferred into a host cell, or may be co-expressed after being integrated together into a suitable vector.

As the vector, a phage or plasmid capable of autonomous growth in a microbial host can be used. As the plasmid DNA, examples thereof include plasmids derived from Escherichia coli, Bacillus subtilis, yeast or the like. For the phage DNA, examples thereof include lambda phage.

All hosts capable of expressing the gene of interest can be used as transformation hosts. Examples thereof include bacteria (E.coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.), insect cells, and the like.

Methods for transferring a gene into a host are well known, and many methods (e.g., calcium ion method, electroporation method, protoplast method, lithium acetate method, calcium phosphate method, lipofection method, etc.) can be exemplified. Further, examples of the method for transferring a gene into the following animals include the following methods: microinjection, a method of transferring a gene into an embryonic cell by using an electroporation technique or a liposome transfer method, and a nuclear transfer method.

In the present invention, the anti-FGF 23 antibody can be obtained by culturing the transformant and collecting the culture product. Here, "culture product" means any one of the following: (a) a culture supernatant, (b) cultured cells or a homogenate thereof, and (c) a secretion of the transformant. For culturing the transformant, a medium suitable for the host used is used, and a culture method such as a static culture method, a roller bottle culture method or the like is used.

When the target antibody is produced in bacterial cells or cells after the completion of the culture, the antibody is collected by homogenizing the bacterial cells or cells. When the target antibody is produced outside the bacterial cells, the culture solution is used as it is, or the bacterial cells or cells are removed by centrifugation or the like. Subsequently, the target antibody is separated and purified from the culture product by a conventional biochemical method using various chromatographic techniques for protein separation and purification, either alone or in appropriate combination

In addition, by using a transgenic animal construction technology, an animal host such as a transgenic cow, a transgenic goat, a transgenic sheep or a transgenic pig, in which the target antibody gene is integrated as an endogenous gene, is constructed. A large amount of monoclonal antibodies derived from the target antibody gene can be obtained from the milk secreted from these transgenic animals (Wright, G., et al, Bio/Technology 9: 830-834, 1991). When a hybridoma is cultured in vitro, the hybridoma is grown, maintained, and stored according to various conditions such as the characteristics of the cultured cells, the purpose of experimental study, and the culture method. Known nutrient media or nutrient media of various origins and prepared in known basal media can be used for producing the monoclonal antibody in the culture supernatant.

(8) Analysis of the monoclonal antibody

The analysis of the isotype and subclass of the monoclonal antibodies produced in these methods can be performed by the following methods. First, examples of the identification method include a plate double diffusion (Ouchterlony) method, an ELISA method, an RIA method, or the like. The plate double diffusion method is simple, but when the concentration of the monoclonal antibody is low, a concentration operation is necessary. On the other hand, when the ELISA method or the RIA method is used, the culture supernatant can be directly reacted with the antigen adsorbed on the immobilization surface, and by using an antibody reacting with the isotype and subclass of each immunoglobulin as a secondary antibody, the isotype and subclass of the monoclonal antibody can be identified.

In addition, protein quantification can be performed by the Folin/Lowry method and by a method of calculating the light absorption at 280nm [1.4(OD280) ═ immunoglobulin 1mg/mL ].

The identification of the epitopes recognized by the monoclonal antibodies (epitope mapping) was performed as follows. First, the respective partial structures of the molecules recognized by the monoclonal antibodies were constructed. For the construction of the partial structure, the method is as follows: one method is to prepare peptide fragments of each part of the molecule by adopting the well-known oligopeptide synthesis technology; one method is to incorporate a DNA sequence encoding the peptide fragment of the target moiety into a suitable expression plasmid using genetic recombination techniques, the peptide fragment being produced in vivo or in vitro in a host such as E.coli. However, in order to achieve the above object, the above two methods are generally used in combination. For example, a series of antigenic protein polypeptides that are successively shortened in random length from the carboxy terminus or the amino terminus is constructed using genetic recombination techniques well known to those skilled in the art. The reactivity of the monoclonal antibodies with these polypeptides was then studied and their recognition sites roughly determined.

Hereinafter, as described in more detail, oligopeptides or variants of the corresponding portions of these peptides are synthesized using oligopeptide synthesis techniques well known to those skilled in the art. In order to determine the epitope, the binding ability of monoclonal antibodies containing the monoclonal antibody as an active ingredient in the prophylactic or therapeutic agent of the present invention or competitive inhibitory activity of these polypeptides on the binding ability of the monoclonal antibody to the corresponding antigen was investigated. As a simple method for obtaining various oligopeptides, commercially available kits (e.g., SPOTs kit (Genosis Biotechnologies), polypeptide series synthesis kit (Chiron Co) utilizing polypeptide synthesis technology, etc.) can be used

(9) Production of the antibody fragment

The antibody fragment is produced by using genetic engineering or protein chemistry methods based on the antibody described in (7) above.

For the genetic engineering technique, examples thereof are: constructing a gene encoding an antibody fragment of interest, and expressing the gene using a suitable host such as animal cells, plant cells, insect cells, E.coli, etc., and purifying the antibody fragment.

For the protein chemistry method, examples thereof are: proteases (e.g., papain, pepsin, etc.) are used for specific site cleavage and purification.

As the antibody fragment, there may be mentioned, for example, a fragment containing Fab, F (ab')₂Fab', scFv, diabodies, dsFv, peptide fragments of CDRs, etc. The production method of each antibody fragment will be described in detail below.

(i) Production of Fab

Fab can be prepared by protein chemistry by treating IgG with papain. After papain treatment, if the primary antibody is of the IgG subclass having protein A binding ability, the IgG molecules and Fc fragment are separated by passing through a protein A column, and homogeneous Fab is recovered (monoclonal antibodies: Principles and Practice, third edition, 1995). If the antibody is of IgG subclass without protein A binding ability, Fab is recovered from the fraction eluted in the low salt concentrate by ion exchange chromatography (Monoclonal Antibodies: Principles and Practice, third edition, 1995). In addition, for obtaining Fab by genetic engineering techniques, Escherichia coli is used in most cases, or insect cells and animal cells and the like are used to produce Fab. The DNA encoding the antibody variable region as described in the above 2(7) was cloned into a Fab expression vector to construct a Fab expression vector. For the Fab expression vector, any plasmid that allows integration and expression of Fab DNA can be used. An example is pIT106(Science, 240: 1041-. Fab expression vectors are transformed into appropriate E.coli and can be generated and aggregated in inclusion bodies or periplasm. For Fab from inclusion bodies, it can be activated by methods commonly used for refolding of proteins. In addition, when Fab is expressed in the periplasm, Fab with activity is released into the culture supernatant. Homogeneous fabs were obtained by refolding or Fab from culture supernatants purified using antigen-bound columns (Antibody Engineering, a Practical Guide, w.h. freeman and company, 1992).

(ii)F(ab′)₂Production of

F(ab′)₂Can be prepared by treating IgG with pepsin using protein chemistry. After pepsin treatment, homogeneous F (ab') can be recovered by the same purification procedure as for Fab₂(MonoclonaLantibodies: Principles and Practice, third edition, 1995). F (ab ') can also be obtained by treating Fab ' described in the following (iii) with a maleimide (e.g., o-PDM, bismaleimide hexane, etc.) to form a thioether bond, or treating with DTNB (5, 5 ' -dithio-bis (nitrobenzoic acid)) to form an S-S bond₂(Antibody Engineering，A Practical Approach，IRL PRESS，1996)。

(iii) Production of Fab

Fab 'can be prepared by treating F (ab') as described in (ii) above with a reducing agent such as dithiothreitol and the like₂And (4) preparing. In addition, Fab' can be produced by genetic engineering techniques, in most cases using E.coli, or using insect cells and animal cells, etc. For example, the DNA encoding the antibody variable region in the above 2(7) is cloned into a Fab 'expression vector to construct a Fab' expression vector. For the Fab 'expression vector, any plasmid that allows integration and expression of the DNA of Fab' can be used. An example is pAK19(BIO/TECHNOLOGY, 10: 163-167, 1992) and the like. The Fab' expression vector was transformed into the appropriate E.coli. Fab' can be generated and accumulated in inclusion bodies or in the periplasm. For Fab' from inclusion bodies, it can be activated by methods commonly used for refolding of proteins. Furthermore, when Fab' is expressed in the periplasm, it is partially digested and osmosized by lysozymeBacteria are homogenized by means of osmotic shock, ultrasonic lysis, etc., and these (Fab') can be recovered from the outside of the cell. Homogeneous Fab's were purified either by refolding treatment or from bacterial homogenates using protein G chromatography columns etc. (Antibody Engineering, A Practical Approach, IRL PRESS, 1996).

(iv) Production of scFv

The scFv can be produced by using a phage, Escherichia coli, insect cells, animal cells, or the like, by using a genetic engineering technique. For example, the DNA encoding the antibody variable region in the above-mentioned 2(7) is cloned into an scFv expression vector to construct an scFv expression vector. For the scFv expression vector, any plasmid that allows DNA of scFv to be integrated and expressed can be used. Examples include pCANTAB5E (GEHealthcare Bioscience Co.), pHFA (Human Antibodies & hybrids, 5: 48-56, 1994), and the like. The scFv expression vector is transferred into an appropriate E.coli, and the helper phage is infected to obtain a phage in which scFv is expressed on the phage surface in a form fused with a phage surface protein. In addition, scFv can be generated and aggregated in inclusion bodies or periplasm of E.coli transferred with the scFv expression vector. For scFv from inclusion bodies, activation can be achieved by methods commonly used for refolding of proteins. In addition, when scFv is expressed in the periplasm, bacteria are homogenized by partial digestion with lysozyme, osmotic shock, ultrasonic lysis, etc., and these (scFv) are recovered extracellularly. Homogeneous scFv were obtained by refolding treatment or purification of scFv from bacterial homogenate using cation exchange chromatography or the like (Antibody Engineering, A Practical Approach, IRL PRESS, 1996).

(v) Production of doublets

When the gene engineering technology is used to produce the binary bodies, Escherichia coli is mainly used, and insect cells and animal cells can also be used. For example, a binary expression vector is constructed by preparing DNA in which VH and VL of the antibody described in the above 2(7) are linked so that the amino acid residue encoded by the linker is 8 residues or less, and cloning it into a binary expression vector. Any plasmid that allows integration and expression of the binary DNA can be used as a binary expression vector. Examples thereof include pCANTAB5E (GEHealthcare Bioscience), pHFA (Human Antibodies hybrids, 5, 48, 1994), etc., and duplexes can be produced and aggregated in inclusion bodies or periplasm of E.coli transferred into a binary expression vector. The diploids obtained from inclusion bodies can be activated by the refolding methods commonly used for proteins. When the (double body) is expressed in the periplasm, the bacteria are homogenized by partial digestion with lysozyme, osmotic shock, ultrasonic lysis or the like, and the (double body) is recovered from the outside of the cell. The homogeneous duplexes are obtained by treatment with cation exchange chromatography or the like (Antibody Engineering, A Practical Approach, IRL PRESS, 1996), refolding or purification of the duplexes from a bacterial homogenate.

(vi) Production of dsFv

In the production of dsFv by genetic engineering techniques, Escherichia coli is mainly used, and insect cells and animal cells are also used. First, mutations were introduced into suitable sites of the DNAs encoding the antibody VH and VL in the above-mentioned (ii), (iv) and (v), thereby obtaining DNAs in which the encoded amino acid residues were substituted with cysteine. Each of the DNAs prepared as described can be cloned into dsFv expression vectors to construct VH and VL expression vectors. Any plasmid that allows the integration and expression of the DNA of dsFv can be used as the dsFv expression vector. Such as pUL19(Protein Engineering, 7: 697-704, 1944), etc. The VH and VL expression vectors can be transformed into suitable E.coli and can be produced and aggregated in inclusion bodies or in periplasm. VH and VL from inclusion bodies or periplasm are obtained and mixed, and dsFvs can be activated by the refolding method commonly used for proteins. After refolding treatment, further purification can be achieved by ion exchange chromatography and gel filtration (Protein Engineering, 7: 697-704, 1994).

(vii) Production of CDR peptides

The peptide containing CDR can be prepared by chemical synthesis method (such as Fmoc method or Boc method). In addition, the CDR peptide expression vector can be obtained by preparing a DNA encoding a peptide containing CDR and cloning the DNA in a suitable expression vector. Any plasmid that can integrate and express the DNA encoding the CDR peptide can be used as the expression vector. Such as pLEX (Invitrogen), and pAX4a + (Invitrogen). The expression vector can be transformed into a suitable E.coli, and it can be produced and aggregated in inclusion bodies or in periplasm. CDR peptides are obtained from inclusion bodies or periplasm and purified by ion exchange chromatography and gel filtration (Protein Engineering, 7: 697-.

3. Characterization of the antibodies of the invention and functional fragments of such antibodies

The antibodies of the invention and functional fragments of such antibodies have any of the following characteristics:

(a) FGF23 binding assay; and a polypeptide having the sequence of SEQ ID NO:4 from amino acid residue 25 to amino acid residue 251.

(b) In vitro testing; FGF23 activity is inhibited in an assay whereby FGF23 activity can be detected. An example of a method for detecting FGF23 activity in vitro is stimulation with FGF23 causing promoter activation of early growth response gene-1 (Nature, 444: 770-774, 2006).

(c) In vivo experiments; when administered to humans, inhibit endogenous FGF23 activity and increase serum phosphorus and serum 1,25D concentrations. For example, when administered to cynomolgus monkeys, they cause an increase in serum phosphorus concentration for about 3 times or more, preferably about 5 times, as compared to conventional antibodies (2C3B antibodies (mouse monoclonal antibodies against FGF23 protein, disclosed in WO03/057733, anti-FGF 23 antibodies produced by hybridoma cells having the accession number FERM BP-7838), and the duration of the increase in serum phosphorus concentration and serum 1,25D concentration is long, e.g., they cause an increase in serum phosphorus concentration for about 3 times or more, preferably about 5 times, as long as 2C3B antibodies, and they cause an increase in serum 1,25D concentration for about 1.5 times or more, preferably about 2.5 times, as long as 2C3B antibodies.

The invention also includes nucleic acids encoding the amino acid sequences of the anti-FGF 23 antibodies of the invention. The nucleic acid may be DNA or RNA. The nucleic acid of the present invention is preferably a nucleic acid encoding the amino acid sequence of an antibody produced by C10 hybridoma. One example is a nucleic acid encoding the amino acid sequence of the heavy chain variable region (the heavy chain nucleic acid sequence of the C10 antibody) consisting of the amino acid sequence of SEQ ID NO: 11 from position 58C to position 408 a. In addition, another example is a nucleic acid encoding an amino acid sequence of a light chain variable region consisting of SEQ ID NO: 13 from G at position 67 to a at position 384.

Pharmaceutical compositions

Formulations, i.e., pharmaceutical compositions containing the human anti-FGF 23 antibody of the invention or a functional fragment of such an antibody, are included within the scope of the invention. Such a formulation, in addition to the antibody or functional fragment of the antibody, preferably comprises a physiologically acceptable diluent or pharmaceutical carrier, or may be a mixture with other drugs, such as other antibodies or antibiotics. Suitable pharmaceutical carriers include: physiological saline, phosphate buffered physiological saline glucose solution, buffered physiological saline, but not limited to these carriers. In addition, the antibody may be lyophilized and reconstituted after adding the above buffer if necessary. The administration method comprises the following steps: oral administration, or parenteral administration such as oral administration, intrabronchial administration, intrarectal administration, subcutaneous injection, intramuscular injection, intravenous injection, etc., preferably administration by intravenous injection. Administration can be by various dosage forms including: aerosols, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments and adhesive preparations (tapes).

When formulated into liquid preparations such as emulsions and syrups, additives such as: water; sugars such as sucrose, sorbitol, fructose, and the like; alcohols such as polyethylene glycol, propylene glycol, and the like; oils such as sesame oil, olive oil, soybean oil, etc.; preservatives such as parabens and the like; flavoring agents such as strawberry flavoring agent and peppermint.

When prepared into capsules, tablets, powders, granules, etc., additives such as: excipients such as lactose, glucose, sucrose, mannitol, and the like; disintegrating agents such as starch, sodium oxalate and the like; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin, etc.; surfactants such as fatty acid esters and the like; plasticizers such as glycerol and the like.

Additives that can be used in the formulation for injection include: water; saccharides such as sucrose, sorbitol, xylose, trehalose, fructose, etc.; sugar alcohols such as mannitol, xylitol, and sorbitol, etc.; buffers such as phosphate buffer, citrate buffer, glutamate buffer, and the like; surfactants such as fatty acid esters and the like.

Suitable formulations for parenteral administration include: injection, suppository, aerosol, etc. In the case of injections, they are usually provided in the form of single-dose ampoules or in multidose containers. It may be a suitable powder which is reconstituted at the time of use with a suitable pharmaceutical carrier, such as sterile water, which does not contain a pyrogen. These preparations contain additives such as emulsifying agents, suspending agents and the like which are conventionally used for formulation of preparations. The injection can be administered by, for example, intravenous infusion, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intradermal injection, etc. In addition, the dose to be administered varies depending on the age of the subject, the mode of administration, and the frequency of administration, and can be adjusted within a wide range.

Suppositories can be formulated with pharmaceutical carriers (e.g., cocoa butter, hydrogenated fats, carboxylic acids, etc.). An aerosol can be formulated with a carrier that does not cause irritation to the oral and respiratory mucosa of the subject (patient) and that allows the antibody or functional fragment of the antibody to diffuse into microparticles for easy absorption, either by itself or by itself.

Specific examples of the carrier include lactose, glycerin, and the like. Depending on the nature of the antibody or functional fragment of the antibody and the nature of the carrier used, agents such as aerosols, dry powders and the like may be used. In addition, additive components used in oral preparations may also be added to these parenteral preparations.

The dose used generally varies depending on the symptoms, age, body weight, etc., but generally, it is about 0.01mg to 1000mg per day for oral administration to adults. The dose may be administered once or in several divided doses. In parenteral administration, the amount administered can be 0.01mg to 1000mg per time by subcutaneous injection, intramuscular injection or intravenous injection.

The invention includes a method for preventing or treating the following diseases by using the antibody or the functional fragment of the antibody or the pharmaceutical composition containing the antibody or the functional fragment of the antibody, and in addition, the invention also includes the application of the antibody or the functional fragment of the antibody in the preparation of medicines for preventing or treating the following diseases.

Diseases that can be prevented and treated by the antibody of the present invention, or a functional fragment of the antibody, include: diseases with excessive FGF23 activity, such as tumor-induced osteomalacia (tumor-induced osteomalacia), ADHR, XLH, fibrofic dysplasia, McCune + Albright syndrome, and diseases accompanied by abnormal mineral metabolism, such as autosomal recessive hypophosphatemia. In addition, the present invention is expected to improve the therapeutic effect on syndromes associated with diseases such as hypophosphatemia, bone salt deposition failure, bone pain, muscle weakness, bone malformation, growth disorder, and hypo-1, 25D. Since FGF23 plays an important role in physiological conditions, FGF23 calcium metabolism control activity mediated by phosphorus metabolism and vitamin D metabolism control can be regulated by the antibody of the present invention, or a functional fragment of the antibody, and thus, they (the antibody of the present invention, or the functional fragment of the antibody) can be used for the prevention and treatment of diseases caused by abnormalities in mineral metabolism and vitamin D metabolism, such as osteoporosis, rickets (including hypophosphatemic rickets and vitamin D-resistant rickets), hypercalcemia, hypocalcemia, ectopic calcification (ectogenic calcium), bone sclerosis, Paget's disease, hyperparathyroidism, hypoparathyroidism, pruritus, and the like. In addition, the antibody of the present invention or a functional fragment of the antibody can be used for the prevention and treatment of diseases caused by renal failure dialysis and renal failure complications, such as renal osteodystrophy, dialysis bone disease, and renal tubular dysfunction. On the other hand, it has been reported that 1,25D has not only the above-mentioned activity on mineral metabolism (e.g., calcium metabolism, etc.), but also a cell growth inhibitory effect, a cell differentiation activity, and the like. Therefore, the antibody of the present invention or a functional fragment of the antibody can also be used for the prevention and treatment of diseases caused by cells whose growth and differentiation are regulated by 1, 25D.

Furthermore, it is well known that overexpression of FGF23 by tumors can lead to lesions in tumor-induced chondropathy. Therefore, it is thought that by using the antibody of the present invention linked to a radioactive substance (e.g., a radioisotope, etc.), or to various toxin therapeutic agents (e.g., a low molecular weight drug, etc.), and the aggregation of the antibody in tumors that overproduce FGF23, tumor regression will result.

Formulation examples

The preparation comprising the antibody of the present invention or a functional fragment of the antibody is used in the form of an ampoule of a sterile solution or suspension dissolved in water or a pharmacologically acceptable solution other than water. In addition, sterile powders, preferably lyophilized antibody molecules of the invention, may be filled into ampoules and diluted with a pharmacologically acceptable solution for use.

Examples

The present invention is described in more detail below by way of examples, but it is not intended that the present invention be limited only by these descriptions of the examples.

Example 1

Preparation of recombinant human FGF23 expression vector.

(1) Construction of expression vector for human FGF23H protein.

A cDNA encoding the human FGF23 gene was amplified using a human cDNA library of tumors causing tumor-induced osteomalacia (template), by performing 35 PCR cycles using F1EcoRI primer (SEQ ID NO: 1) and LHissNot primer (SEQ ID NO: 2) and LA-Taq DNA polymerase, each PCR cycle consisting of: incubation was carried out at 96 ℃ for 1min, then at 96 ℃ for 30 seconds, at 55 ℃ for 30 seconds and at 72 ℃ for 30 seconds. The F1EcoRI primer, after annealing, binds to a sequence present at the 5 '-upstream distal end of the nucleotide fragment encoding the human FGF23 gene, and introduces an EcoRI restriction site at the 5' -end of the nucleotide fragment encoding the human FGF23 gene obtained by amplification. The LHisNot primer contains: a sequence that anneals to the 5' -end of the stop codon in the nucleotide sequence encoding human FGF23 gene, a sequence encoding a terminal (terminal) codon that follows the sequence encoding the His6 tag sequence (His-His) and the NotI restriction site. As a result, the amplified fragment encoded human FGF23 protein to which a His 6-tagged sequence was added at the carboxy terminus of the FGF23 protein, and a NotI restriction enzyme site was added downstream thereof. The amplified fragment was digested with EcoRI and NotI and ligated with an animal cell expression vector pcDNA3.IZeo (Invitrogen) which was also digested with EcoRI and NotI. The expression vector constructed in this way was cloned and its nucleotide sequence was determined, thereby confirming that the expression vector encodes a target protein, i.e., human FGF23 protein, to which a His6 tag sequence was added. The vector was designated pcDNA/hFGF 23H.

F1EcoRI：CCGGAATTCAGCCACTCAGAGCAGGGCACG(SEQ IDNO：1)

LHisNot：

ATAAGAATGCGGCCGCTCAATGGTGATGGTGATGATGGATGAACTTGGCGAA(SEQ ID NO：2)

(2) Construction of human FGF23 protein expression vector

Fragments were amplified using pcDNA/hFGF23H as template, with 25 PCR cycles using F1EcoRI and LNot primers (SEQ ID NO: 3) and LA-Taq DNA polymerase, each PCR cycle consisting of: the temperature was maintained at 94 ℃ for 1min, then at 94 ℃ for 30 sec, at 55 ℃ for 30 sec and at 72 ℃ for 1 min. After completion of the reaction, the fragment encoding human FGF23 was digested with EcoRI and NotI, and then purified. These purified fragments were cloned by inserting them into EcoRI and NotI restriction enzyme sites of pEAK8/IRES/EGFP vector, which is an animal cell expression vector obtained by ligating ribosome entry sequence (IRES) and Enhanced Green Fluorescent Protein (EGFP) within pEAKS (edge biosystem) molecules. The nucleotide sequence of the thus obtained plasmid was determined, and it was confirmed that it encodes the human FGF23 protein. The vector was designated as pEAK8/IRES/EGFP/hFGF 23.

LNot：ATAAGAATGCGGCCGCTCAGATGAACTTGGCGAA(SEQID NO：3)

(example 2)

(1) Expression of recombinant human FGF23 protein and recombinant mutant human FGF23H protein.

pcDNA/hFGF23H was linearized by cleavage of the FspI restriction enzyme site on the ampicillin resistance Gene in the vector and purified, then mixed with murine CHO clone-1 cells (Shirahata, S., et al, Biosci Biotech Biochem, 59: 345-347, 1995) and transfected into these cells using electroporation using a Gene Pulser II (Bio Rad) punch. After these cells were cultured with MEM alpha medium (Gibco BRL) containing 10% FCS for 24 hours, Zecocin (Invitrogen) was added to the medium to a final concentration of 0.5mg/ml, and then the cells were cultured (with this medium) for 1 week. After trypsinization, the adherently growing cells were released and cloned by limiting dilution under conditions of a final concentration of 0.3mg/ml of Zecocin to give a number of cell clones. The most efficient cells expressing human FGF23H were identified using Western blotting (Western blotting) technique. The culture supernatant of each cell clone was collected and subjected to SDS-polyacrylamide gel electrophoresis, and then the (electrophoresed) protein was transferred onto a PVDF membrane (Millipore). By using an anti-His-tag (carboxy-terminal) antibody and ECL photoluminescence system (photo-luminescence, ge healthcare Bioscience), a signal from FGF23H protein near about 32kDa was detected. Thus, a cell clone designated as #20 having the highest expression ability was found, which was named CHO-OST311H and deposited at 11.8.2000 at the National Institute of Advanced Industrial Science and Technology (AIST), International Patent Organism Depositary (IPOD) National Institute of Advanced Industrial Science and technology (Japan), Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki (accession number: FERM BP-7273). In the present specification, CHO-OST311H was referred to as CHO-hFGF 23H.

(2) Obtaining of human FGF23 expressing cells

Transfection of CHO Ras clone-1 cells with pEAK8/IRES/EGFP/hFGF23 vector was accomplished by gene transfection method using membrane-fused lipids. When CHO Ras clone-1 cells cultured in 6-well plates had covered the bottom of the 6-well plates by about 60%, the medium was removed and 1ml of serum-free MEM α medium was added. Mu.g of the transferred vector and 10. mu.l of Transfectam (registered trademark) (Promega) were mixed with 50. mu.l of MEM. alpha. serum-free medium, and the two solutions were mixed and allowed to stand for 10 min. The mixture was added to culture wells of a 6-well plate prepared in advance. After 2 hours of incubation, the medium containing the DNA was removed and replaced with medium containing 10% FCS and the culture was incubated overnight. The following day, puromycin (Sigma) was added to a final concentration of 5. mu.g/ml to select drug resistant cells. The thus obtained drug-resistant cells were cloned by the limiting dilution method. In addition, a cell line expressing the target protein with the highest efficiency is obtained by using a western blotting technique. The cells were designated CHO-hFGF 23.

(3) Expression and detection of recombinant human FGF23 protein in animal cells.

Western blotting of the CHO-hFGF23H recombinant in the culture supernatant with an antibody against the carboxy-terminal His6 tag sequence detected two bands of approximately 32kDa and 10 kDa. These two bands were excised from the gel, and the N-terminal amino acid sequences thereof were determined. In the larger molecular weight band (approximately 32kDa), the detected sequence is represented by SEQ ID NO:4, which can be regarded as human FGF23 protein from which the signal peptide sequence has been cleaved off during secretion. On the other hand, in the smaller molecular weight band, the detected sequence was confirmed to be a sequence represented by SEQ ID NO:4 starting at amino acid 180, and the fact that this fragment is a carboxy-terminal fragment generated by cleavage between amino acids 179 and 180. Further, the presence of a polypeptide having an amino acid sequence starting from the 179 th amino acid to the N-terminus (amino acid-terminal peptide fragment) was also confirmed by detecting the human FGF 23N-terminal side with a polyclonal antibody recognizing the same (International patent publication No. WO 02/14504).

Similarly, in the culture supernatant of CHO-hFGF23 without His6 tag sequence, the cleavage occurring between the 179 th and 180 th amino acid residues was also confirmed (International patent publication No. WO 02/14504). Thus, the following procedure was used to isolate and purify non-cleaved full length human FGF23 protein, the full length human FGF23 protein having the amino acid sequence of SEQ ID NO:4 from position 25 to position 251 (sometimes referred to as full length FGF 23).

(4) Purification of recombinant full-length human FGF23 protein

The CHO-hFGF23 culture supernatant was filtered using SuperCap (registered trademark) (Pall Gelman laboratory) which was a membrane filtration device having a pore size of 0.2. mu.m, and the resulting filtrate was filtered through SP-Sepharose FF (GE Healthcare Bioscience). Substances having a weak binding ability to the column were washed and eluted by 50mM sodium phosphate buffer (pH 6.7). The (eluting) component includes the carboxy-terminal fragment resulting from the cleavage that occurs between amino acid residues 179 and 180. The protein adsorbed in the column was eluted by a NaCl solution having a concentration gradient of 0 to 0.7M, and the full length human FGF23 protein was observed in the eluted fraction of about 0.3M NaCl solution. Next, the full length human FGF23 protein was adsorbed to a metal affinity chromatography column talon superflow (registered trademark) (Clonetech) and rinsed with 50mM sodium phosphate buffer (ph6.7), and the full length human FGF23 protein was eluted and purified by adding imidazole at various concentrations.

The (eluted) fraction containing the target protein was adsorbed on an SP-Sepharose FF column and purified after elution.

Human FGF23 amino acid sequence (SEQ ID NO: 4)

MLGARLRLWV CALCSVCSMS VLRAYPNASP LLGSSWGGLIHLYTATARNS YHLQIHKNGH VDGAPHQTIY

SALMIRSEDA GFVVITGVMS RRYLCMDFRG NIFGSHYFDPENCRFQHQTL ENGYDVYHSP QYHFLVSLGR

AKRAFLPGMN PPPYSQFLSR RNEIPLIHFN TPIPRRHTRSAEDDSERDPL NVLKPRARMT PAPASCSQEL

PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG PEGCRPFAKFI

(example 3)

Generation of human antibody-producing mice (KM mice)

Fully human antibody-producing mice used to make human monoclonal antibodies have a homozygote genetic background in which both the endogenous Ig heavy and kappa-light chains are disrupted, along with a chromosome 14 fragment containing the human Ig heavy chain gene locus (SC20) and a human Ig kappa-light chain transgene (KCo 5). The mice were obtained by cross breeding of a strain a mice having a human Ig heavy chain gene locus and a strain B mice having a human Ig kappa-light chain transgene (KCo 5). The a strain mice are homozygotes in which both the endogenous Ig heavy and kappa-light chains are disrupted, and have chromosome 14 fragment (SC20) that can be transmitted to offspring. This mouse line is described in reports such as those made by Tomizuka et al (Tomizuka et al, Proc Natl.Acad.Sci.USA., 97: 722-. Furthermore, the B strain mice are homozygous for both endogenous Ig heavy and kappa-light chain disrupted, and are transgenic mouse strains harboring the human Ig kappa-light chain transgene (KCo 5). This mouse line is described in reports such as those made by Fishwild et al (nat. Biotechnol., 14; 845. sup. 851, 1996).

The mouse individuals used in the following immunization experiments were mice obtained by crossing male A strain mice and female B strain mice or male B strain mice and female A strain mice, and human Ig heavy chains and kappa-light chains were simultaneously detected in the serum of the mice used herein [ Ishida & Lonberg, IBC's 11th Antibody Engineering, Abstract 2000 ]. In addition, the human antibody-producing mouse can be obtained from Kirin Beer Company by a method of association contract.

(example 4)

Preparation of human monoclonal antibody against human FGF23

(1) Obtaining a hybridoma producing a human monoclonal antibody to human FGF 23.

The monoclonal antibody used in this example can be prepared by a conventional method, for example, "laboratory instructions for monoclonal antibody", written by Tamio Ando et al (Kodansha, 1991). The full-length human FGF23 protein prepared in example 2 was used as an immunogen, and mice were generated using human antibodies that can produce the human immunoglobulin prepared in example 3.

To prepare a human anti-FGF 23 monoclonal antibody, first, the purified full-length human FGF23 protein obtained in example 2 was mixed intraperitoneally with RIBI adjuvant (Corixa), and was intraperitoneally inoculated to human antibody-producing mice at a dose of 20 μ g per mouse at the time of primary immunization. Similar to the first immunization, mice were co-inoculated 3 times with a mixture of purified FGF23 protein and RIBI adjuvant (Corixa) at two week intervals. Five mice were used for immunization, and after the third immunization, blood samples were taken and the presence of anti-FGF 23 human IgG antibody in serum was confirmed by the enzyme-labeled immunosorbent assay (ELISA) described below. Mice having the highest concentration value (anti-FGF 23 human IgG antibody) in serum were selected by ELISA using FGF23 immobilized with anti-FGF 23 protein monoclonal mouse antibody- -3CIE antibody, wherein the anti-FGF 23 protein monoclonal mouse antibody- -3CIE antibody used was disclosed in International patent publication No. WO03/057733 (anti-FGF 23 antibody produced by hybridoma deposited as FERM BP-7839). Mice were immunized with 20 μ g of full-length human FGF23 protein per mouse administered via the rat tail vein (vaccination) 3 days before spleen isolation as described below.

The spleen was surgically isolated from the immunized mice and immersed in 10mL of serum-free DMEM medium (Invitrogen, hereinafter referred to as serum-free DMEM medium) containing 350mg/mL of sodium bicarbonate, 50 units/mL of penicillin, 50. mu.g/mL of streptomycin, and then crushed on a sieve (mesh) with a spatula (cell filter: Falcon). The cell suspension passing through this mesh screen (mesh) was centrifuged to generate a cell pellet, and then, the cells were rinsed twice with serum-free DMEM medium, followed by cell counting of the cells suspended in the serum-free DMEM medium. When myeloma cells SP2/0(ATCC No. CRL-1581) were cultured in DMEM (Invitrogen) medium (hereinafter referred to as serum-containing DMEM medium) containing 10% FCS (Sigma) at 37 ℃ in the presence of 5% CO₂Culturing under the condition that the cell density is not higher than 1 × 10⁶cells/mL. Similarly, the myeloma cells were rinsed with serum-free DMEM medium and suspended in serum-free DMEM medium for cell counting. The recovered spleen cell suspension was mixed with the mouse myeloma cell suspension at a ratio of 5: 1 in terms of cell number and centrifuged, and then the supernatant was completely removed. To the cell mass, 1mL of 50% (w/v) polyethylene glycol 1500 (Boehringer-Mannheim) as a fusion medium was slowly added while stirring the cell mass with a pipette tip, and then 1mL of serum-free DMEM medium preheated to 37 ℃ was slowly added in two portions, followed by addition of 7mL of serum-free DMEM medium. After centrifugation, the supernatant was removed to obtain fused cells. The fused cells thus obtained were screened by the limiting dilution method as described below. Screening of hybridomas was performed by culturing in DMEM containing 10% FCS, IL-6(10ng/mL) (or 10% hybridoma cloning factor (hereinafter referred to as HCF): Biobase), hypoxanthine (H), aminopterin (A) and thymidine (T) (hereinafter referred to as HAT Sigma)Cultured in medium. In addition, individual clones were performed in DMEM medium containing HT (Sigma), 10% FCS and 10% HCF by limiting dilution method. The (cell) cultures were performed in 96-well microtiter plates (Becton, Dickinson). Selection (screening) of hybridoma clones producing the human monoclonal antibody against FGF23 and quality identification of the individual hybridoma-producing human monoclonal antibodies were carried out by an enzyme-labeled immunoabsorbent method (ELISA method) as described below. As a result, a number of hybridomas were obtained which contain a human immunoglobulin gamma chain (higg γ) and a human immunoglobulin light chain κ and which produce human monoclonal antibodies having specific reactivity with human FGF 23. From among many of these resultant hybridomas, 2 clones (C10 and C15) were obtained in particular as hybridomas producing antibodies recognizing FGF23 protein. Furthermore, in all of the following examples, including this example, each hybridoma clone producing the anti-FGF 23 human monoclonal antibody of the present invention is represented by a symbol. "antibody" appearing before or after the symbol means an antibody produced by a hybridoma or a recombinant antibody produced by a host cell harboring an antibody gene (full-length or variable region) isolated from the hybridoma. Within the ranges explicitly indicated herein, sometimes the name of a hybridoma clone may refer to the name of an antibody. On 2.2.2007, C10 hybridoma clone was deposited at National Institute of Advanced Industrial Science and Technology (AIST), National Institute of Advanced Industrial science and Technology (IPOD), Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki (accession number FERM ABP-10772) (ID tag: C10) in that respect

(2) Purification of C10 and C15 antibodies from the hybridoma culture supernatant

The C10 and C15 hybridomas obtained in example 4(1) were restricted from culture in eRDF medium (Kyokuto Seiyaku) containing insulin (5. mu.g/ml, Invitrogen), human transferrin (5. mu.g/ml, Invitrogen), diphosphatidylglycerol (0.01mM, Sigma), sodium selenite (2.5X 10-5mM, Sigma), 1% Low IgG fetal bovine serum (Hyclone). The hybridomas are cultured in flasks, and the resulting culture supernatant is recovered. The culture supernatant was affinity purified using Protein G Fast Flow gel (GE Healthcare, Bioscience) using PBS (-) as adsorption buffer and 0.1M glycine buffer as elution buffer (pH 2.8). The pH of the eluted fractions was adjusted to about pH 7.2 by adding 1M Tris (pH 9.0). The antibody solution thus prepared was replaced with PBS using Sephadex G25 desalting chromatography column (NAP column; GE Healthcare Bioscience), and then sterilized by filtration sterilization through a membrane filter MILLEX-GV (Millipore) having a pore size of 0.22. mu.m, thereby obtaining purified C10 and C15 antibodies. The concentration of the purified antibody was determined by measuring its absorbance at 280nm, wherein the calculated standard was 1.4 OD-1 mg/mL.

(example 5)

Obtaining of antibody gene encoding C10 antibody and determination of its sequence

(1) Synthesis of cDNA sequence of the C10 antibody

To obtain the DNA fragment containing the antibody variable regions of the heavy and light chains of a human antibody expressed in the C10 hybridoma, cloning was performed using the 5 'RACE method (5' cDNA terminal rapid amplification technique) using primers that specifically bind to the heavy and light chain constant regions of a human antibody. Specifically, the kit used for cloning was a BD SMART RACE cDNA amplification kit (Becton Dickinson Bioscience Clonetech), and cloning was performed according to the instructions attached to the kit.

According to the protocol, the RNA extraction reagent ISOGEN (Nippon Gene) was added to the C10 hybridoma, and 15. mu.g of purified total RNA was used as a starting material for cDNA synthesis. First strand cDNA was prepared using about 1. mu.g each of the purified total RNA as a template. All reagents and enzymes except RNA were provided by BD SMART RACE cDNA amplification kit (cDNA amplification kit).

In the synthesis of the first strand cDNA,

total RNA 1. mu.g/3. mu.l

5′CDS 1μl

SMART Oligo 1μl

The reaction mixture consisting of the above components was incubated at 70 ℃ for 2 minutes, and then:

subsequently, the cells were incubated at 42 ℃ for 1.5 hours.

Further, 50. mu.l of Tricine-EDTA buffer was added, followed by incubation at 72 ℃ for 7 minutes to obtain first strand cDNA.

(2) Amplifying the heavy chain and light chain genes by a PCR method and confirming the nucleotide sequences thereof

(2) -1; PCR method for amplifying the heavy and light chain genes

To amplify cDNA of the gene encoding the C10 antibody, a reaction mixture was prepared and used for PCR using a set of PCR primers including a3 ' primer and a 5 ' primer, wherein the 3 ' primer sequence has a sequence specific to a human antibody gene (the specific sequence will be described below), the 5 ' primer (universal primer A mixture) specifically hybridizes to a sequence added to the 5 ' end of the cDNA synthesized using the BD SMART RACE cDNA amplification kit, and further, the enzyme used for PCR was KOD-Plus-DNA polymerase (Toyobo).

For the amplification reaction of the heavy chain gene, the primers used were the UPM primer and IgG1p primer (SEQ ID NO: 5) in the SMART RACE cDNA amplification kit, while for the amplification of the light chain gene, the primers used were the UPM primer and hk-2 primer (SEQ ID NO: 6) in the SMART RACE cDNA amplification kit.

IgG 1p：TCTTGTCCACCTTGGTGTTGCTGGGCTTGTG(SEQ ID NO：5)

hk-2: GTTGAAGCTCTTTGTGACGGGCGAGC (SEQ ID NO: primer 6)

In addition, the reaction conditions used were as follows:

repeat 5 cycles, each cycle comprising: 94 ℃/30 seconds, 72 ℃/3min,

repeat 5 cycles, each cycle comprising: 94 ℃/30 seconds, 70 ℃/30 seconds, 72 ℃/3min

Repeating 25 cycles, each cycle comprising: 94 ℃/30 seconds, 68 ℃/30 seconds, 72 ℃/3min

Further, 2. mu.l of the reaction mixture was diluted by adding 98. mu.l of Tricine-EDTA buffer, and 5. mu.l of the resulting dilution was used as a template for the second (nested) PCR reaction

The composition of the PCR reaction solution was as follows:

in the above reaction, one set of primers used for the heavy chain gene amplification was the NUP primer (in SMART RACE cDNA amplification kit; Becton Dickinson Bioscience Clonetech) and the hh2 primer (SEQ ID NO: 7). One set of primers used for the light chain gene amplification is the UPM primer and the hk-5 primer (SEQ ID NO: 8). The reaction temperature conditions were as follows: the starting temperature was 94 ℃ for 1min, and then 20 cycles were repeated, each cycle consisting of: 94 ℃/5sec, 68 ℃/10 sec and 72 ℃/3min, and finally, heating at 72 ℃ for 7 min.

hh2：GCTGGAGGGCACGGTCACCACGC(SEQ ID NO：7)

hk-5：AGGCACACAACAGAGGCAGTTCCAGATTTC(SEQ ID NO：8)

(2) -2; determination of nucleotide sequence of the antigen Gene

The amplified heavy chain PCR fragment (hereinafter referred to as HV [ C ]: consisting of the leader sequence of the 5 '-untranslated region of the H chain, the variable region (HV) and the partial region of the constant region [ C ]) and the amplified light chain PCR fragment (hereinafter referred to as LV [ C ]: consisting of the leader sequence of the 5' -untranslated region of the L chain, the variable region (LV) and the partial region of the constant region [ C ]) were recovered by ethanol precipitation and then recovered by agarose gel electrophoresis. Further, the DNA was purified by using a membrane DNA purification kit (QIAquick Gel extraction kit (Qiagen)). The purified HV [ C ] amplified fragment or LV [ C ] amplified fragment was subcloned into PCR4Blunt-TOPO vector of Zero Blunt TOPO PCR cloning kit (Zero Blunt TOPO PCR cloning kit) (Invitrogen), respectively, and the obtained cloned plasmid DNA was subjected to nucleotide sequence analysis of the inserted DNA. The primers used for nucleotide sequencing of the DNA were M13-20FW (SEQ ID NO: 9) and M13RV (SEQ ID NO: 10).

M13-20FW：GTAAAACGAC GGCCAGTG(SEQ ID NO：9)

M13RV：CAGGAAACAGCTATGAC(SEQ ID NO：10)

The DNA nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the C10 antibody, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown below:

< C10 heavy chain nucleic acid sequence > (variable region from ATG initiation codon to DNA sequence encoding carboxyl terminal amino acid residue) (SEQ ID NO: 11)

10 20 30 40 50 60

ATGGACTGGA CCTGGAGGGT CTTCTGCTTG CTGGCTGTAG CTCCAGGTGC TCACTCCCAG

70 80 90 100 110 120

GTGCAGCTGG TGCAGTCTGG GGCTGAGGTG AAGAAGCCTG GGGCCTCAGT GAAGGTTTCC

130 140 150 160 170 180

TGCAAGGCAT CTGGATACAC CTTCACCAAC CACTATATGC ACTGGGTGCG ACAGGCCCCT

190 200 210 220 230 240

GGACAAGGGC TTGAGTGGAT GGGAATAATC AACCCTATTA GTGGTAGCAC AAGTAACGCA

250 260 270 280 290 300

CAGAAGTTCC AGGGCAGAGT CACCATGACC AGGGACACGT CCACGAGCAC AGTCTACATG

310 320 330 340 350 360

GAGCTGAGCA GCCTGAGATC TGAGGACACG GCCGTGTATT ATTGTGCGAG AGATATTGTG

370 380 390 400 408

GATGCTTTTG ATTTCTGGGG CCAAGGGACA ATGGTCACCG TCTCTTCA

< C10 heavy chain amino acid sequence > (to leader and variable region) (SEQ ID NO: 12) (underlined amino acid residues representing leader sequence as secretion signal)

10 20 30 40 50 60

MDWTWRVFCL LAVAPGAHSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN HYMHWVRQAP

70 80 90 100 110 120

GQGLEWMGII NPISGSTSNA QKFQGRVTMT RDTSTSTVYM ELSSLRSEDT AVYYCARDIV

130 136

DAFDFWGQGT MVTVSS

< C10 light chain nucleic acid sequence > (variable region from ATG initiation codon to DNA sequence encoding carboxyl terminal amino acid residue) (SEQ ID NO: 13)

10 20 30 40 50 60

ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTTCTGC TGCTCTGGCT CCCAGGTGCC

70 80 90 100 110 120

AGATGTGCCA TCCAGTTGAC CCAGTCTCCA TCCTCCCTGT CTGCATCTGT AGGAGACAGA

130 140 150 160 170 180

GTCACCATCA CTTGCCGGGC AAGTCAGGGC ATTAGCAGTG CTTTAGTCTG GTATCAGCAG

190 200 210 220 230 240

AAACCAGGGA AAGCTCCTAA GCTCCTGATC TATGATGCCT CCAGTTTGGA AAGTGGGGTC

250 260 270 280 290 300

CCATCAAGGT TCAGCGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG

310 320 330 340 350 360

CAGCCTGAAG ATTTTGCAAC TTATTACTGT CAACAGTTTA ATGATTACTT CACTTTCGGC

370 380 384

CCTGGGACCA AAGTGGATAT CAAA

< C10 light chain amino acid sequence > (to leader and variable regions) (SEQ ID NO: 14) (underlined amino acid residues representing leader sequence as secretion signal)

10 20 30 40 50 60

MDMRVPAQLL GLLLLWLPGA RCAIQLTQSP SSLSASVGDR VTITCRASQG ISSALVWYQQ

70 80 90 100 110 120

KPGKAPKLLI YDASSLESGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQFNDYFTFG

128

PGTKVDIK

In addition, in the gene sequence of the C10 antibody subcloned in the vector PCR4Blunt-TOPO, a part of the sequence of the constant region of the human antibody was cloned, and the DNA nucleotide sequence of this region was also analyzed. As a result, it was confirmed that the sequence encoding amino acid residues 118 to 191 of the heavy chain constant region shown in EU index written by Kabat et al was completely identical to the amino acid sequence of human IgG1 in this region, and the C10 antibody subclass was IgG 1. In addition, by using the same method, an antibody gene encoding the C15 antibody was obtained, and the sequence to which the antibody belongs was also determined.

(example 6)

Construction of recombinant C10 antibody expression vector

C10 production of expression vector (scheme shown in FIG. 1)

Using the PCR technique, using KOD-Plus-DNA polymerase, with the obtained plasmid DNA containing the LV [ C ] chain of the C10 antibody as a template, the LV (leader + variable region of the light chain) DNA of the C10 antibody was amplified using primers C10_ L5_ Bg1(SEQ ID NO: 15) and C10_ L3_ Bsi (SEQ ID NO: 16) designed to have restriction sites (5 '-terminal BgIII, 3' -terminal BgIII) attached to their ends. The reaction temperature conditions are as follows: initial temperature 94 ℃ for 1min, each cycle comprising: this cycle was repeated 35 times at 94 ℃/5sec and 68 ℃/45 sec and finally heated for 3min at 72 ℃. The amplified DNA fragment was digested with restriction enzymes BglII and BsiWI, purified by agarose gel electrophoresis, and recovered to obtain about 400bp DNA. On the other hand, the vector, N5KG1-Val Lark vector (IDEC Pharmaceuticals, N5KG1 modified vector (U.S. Pat. No. 6001358)) was similarly digested with restriction enzymes BglII and BsiWI, dephosphorylated with alkaline phosphatase (E.coli C75) (Takara Shuzo Co., Ltd.), purified with agarose gel and DNA purification kit, and recovered to obtain DNA slightly smaller than 9 kb. These two DNA fragments were ligated by T4DNA ligase and then transfected into E.coli DH10B to obtain a transformant. After DNA nucleotide sequence analysis of the plasmid DNA of the transformant containing the inserted DNA, plasmid DNA was obtained, N5KG1_ C10_ Lv, in which LV of C10 antibody was inserted into the frame 5' upstream of the human antibody light chain constant region encoding N5KG1-Val Lark. Next, the HV DNA (heavy chain leader + variable region) of the C10 antibody was inserted into a plasmid vector (N5KG1_ C10- _ Lv) into which LV had been inserted. HV was amplified by PCR using a plasmid DNA subcloned into pCR4Blunt-TOPO vector containing HV [ C ] of the C10 antibody as a template, and primers C10_ H5_ Sal (SEQ ID NO: 17) and C10_ H3_ Nhe (SEQ ID NO: 18) designed to be ligated to the ends of restriction sites (SalI at the 5 'end and NheI at the 3' end). The reaction temperature conditions are as follows: initial temperature 94 ℃ for 1min, each cycle comprising: this cycle was repeated 35 times at 94 ℃/5sec and 68 ℃/45 sec and finally heated at 72 ℃ for 7 min. The purified amplified HV DNA fragment was subcloned into pCR4Blunt-TOPO vector, and the cloned plasmid DNA thus obtained was subjected to nucleotide sequence analysis of the inserted DNA. The primers used for nucleotide sequencing of the DNA were M13-20FW and M13RV as described above. For subclones, analysis of the nucleotide sequence of the inserted part DNA was made, which was not different from HV as a template, and further, plasmid DNA (TOPO _ C10_ Hv) in which the primer part had the designed sequence was selected. These DNAs were digested with restriction enzymes SalI and NheI, purified by agarose gel electrophoresis, and about 420bp of DNA was recovered, and this DNA fragment was ligated with T4DNA ligase to N5KG 1-C10-Lv DNA (about 9kb) which had been similarly subjected to restriction enzyme treatment (SalI and NheI) and dephosphorylation treatment, and then the resulting ligation product was transferred to E.coli DH10B, and the plasmid DNA of interest was selected from the thus-obtained transformants. The antibody expression plasmid DNA obtained by the above-described method, N5KG1_ C10_ IH (clone #1), was purified in large quantities and it was confirmed that the entire region of the L chain and H chain and the DNA nucleotide sequence around the insertion site thereof were not altered during cloning (FIG. 2, FIG. 3). For confirmation of the DNA nucleotide sequence, SEQ ID NO: 19-25. The schematic diagram of the C10 antibody expression vector prepared is shown in FIG. 4. In addition, a recombinant C10 antibody expression vector was constructed using the same method.

C10_L5_Bgl：

GAGAGAGAGATCTCTCACCATGGACATGAGGGTCCCCGCT(SEQ IDNO：15)

C10_L3_Bsi：

AGAGAGAGAGCGTACGTTTGATATCCACTTTGGTCCCAGGGC(SEQ IDNO：16)

C10_H5_Sal：

AGAGAGAGAGGTCGACCACCATGGACTGGACCTGGAGGGTCTTC(SEQID NO：17)

C10_H3_Nhe：

AGAGAGAGAGGCTAGCTGAAGAGACGGTGACCATTGTCCC(SEQ IDNO：18)

hh-4：GGTGCCAGGGGGAAGACCGATGG(S EQ ID NO：19)

hh-1：CCAAGGGCCCATCGGTCTTCCCCCTGGCAC(SEQ ID NO：20)

CMVH903F：GACACCCTCATGATCTCCCGGACC(SEQ ID NO：21)

CMVHR1303：TGTTCTCCGGCTGCCCATTGCTCT(SEQ ID NO：22)

SEQU4618：TCTATATAAGCAGAGCTGGGTACGTCC(SEQ ID NO：23)

hk-1：TGGCTGCACCATCTGTCTTCATCTTC(SEQ ID NO：24)

SEQU1783：GGTACGTGAACCGTCAGATCGCCTGGA(SEQ ID NO：25)

(example 7)

Preparation of recombinant C10 antibody

C10 antibody expression cells are prepared by transferring the constructed C10 antibody expression vector into host cells. For the expression host CELLs, dihydrofolate reductase (DHFR) -deleted mutant CHO DG44 CELL line (hereinafter referred to as CHO CELLs, IDEC Pharmaceuticals) cultured in condition (conditioned) in serum-free medium- -EX-CELL325PF medium (JRH, containing 2mM glutamine, 100 units/ml penicillin, 100. mu.g/ml streptomycin, Hypoxanthine and Thymidine (HT) supplement (1: 100) (Invitrogen)) was used. The pair of carriers is carried out by electroporationAnd (4) transferring the host cell. About 2. mu. g C10 expression vector was linearized with the restriction enzyme AscI by electroporation, and the gene was transferred into 4X 106 CHO cells under 350V, 500. mu.F conditions using a BioRad Electroporator (BioRad Electroporator), and the resulting cells were seeded on 96-well cell culture plates. After the vector is transferred into the host cell, G418 is added and the culture is continued. And (4) after confirmation and inspection are carried out on the clone, an antibody expression strain is obtained by screening. The CHO CELL lines obtained from the screening were cultured in EX-CELL-325PF medium (containing 2mM glutamine, 100 units/ml penicillin, 100. mu.g/ml streptomycin, Hypoxanthine and Thymidine (HT) supplement (1: 100) (Invitrogen)) in 5% CO₂Culturing is carried out under the conditions. The resulting culture supernatant was absorbed in Mabselect Protein A column (GE Healthcare Bioscience), rinsed with PBS, and eluted with 20mM sodium citrate (citrate-Na) and 50mM NaCl (pH 3.4) buffer. The eluate was neutralized with 50mM sodium phosphate buffer pH 7.0. The eluate was diluted 1.5 times with deionized water, and the conductivity of the diluted eluate was adjusted to 4.0ms/cm or less. Immediately thereafter, the resulting sample was fed, absorbed onto a column composed of a Q-Sepharose (Hitrap Q HP, GE Healthcare Bioscience) and SP-Sepharose (Hitrap SP FF, GE Healthcare Bioscience) junction, rinsed with 20mM sodium phosphate salt buffer (pH 5.0), and then eluted with PBS (-). The antibody solution thus prepared was passed through a membrane filter having a pore size of 0.22 μm, MILLEX-GV (Millipore), and sterilized by filtration. The purified C10 antibody concentration was calculated by measuring its light absorbance at 280nm, calculated on the basis of: [1.4OD280 ═ 1mg/mL]. In addition, a recombinant C15 antibody was prepared by using the same method.

(example 8)

Construction of Actinidia chinensis FGF23 protein expression vector

The EDTA-treated macaque venous blood and 5% dextran T-2000(GE Healthcare Bioscience) suspended in PBS (-) were mixed in a ratio of 2: 1 to precipitate red blood cells. Subsequently, the supernatant was layered on top of a lymphocyte separation medium (Ficoll-plate) (GE Healthcare Bioscience), and centrifuged to obtain a lymphocyte fraction. The lymphocytes thus obtained were suspended in ISOGEN-LS (Nippon Gene) and macaque total lymphocyte RNA was obtained according to the attached experimental protocol. According to the attached experimental scheme, a macaque lymphocyte cDNA library is prepared by using the prepared macaque total lymphocyte RNA and a first strand cDNA synthesis kit (Invitrogen). Amplifying a cDNA encoding cynomolgus FGF23 by performing the following procedure: the cynomolgus lymphocyte cDNA library was used as a template, incubated at 94 ℃ for 5min using monkey yFGF23FW primer (SEQ ID NO: 26) and monkey FGF23RV primer (SEQ ID NO: 27), and KOD plus DNA polymerase (Toyobo), and then subjected to 45 PCR cycles each consisting of 94 ℃/20 sec, 55 ℃/30 sec, and 72 ℃/50 sec. The monkey FGF23FW primer was annealed and then ligated to a sequence present in the region 5 'upstream of the nucleotide sequence encoding human FGF23, and an EcoRI restriction enzyme site was added 5' to the coding region of the amplified DNA fragment FGF 23. The monkey FGF23RV primer contained a sequence that annealed to include a stop codon encoding the coding region of human FGF23 and contained a Not I restriction enzyme site. The amplified fragment was digested with EcoRI and NotI, and inserted into EcoRI and NotI restriction enzyme sites of pEAK8/IRES/EGFP vector, which had the ribosome entry sequence (IRES) and Enhanced Green Fluorescent Protein (EGFP) ligated into expression vector pEAKS (edge biosystem). The nucleotide sequence of the resulting plasmid was sequenced to determine that it encodes the cynomolgus FGF23 protein, and the vector was designated as pEAK 8/IRES/EGFP/monkey FGF 23. In addition, the nucleic acid sequence and amino acid sequence of cynomolgus monkey FGF23 obtained in this example are shown in SEQ ID NO: 28 and SEQ ID NO: 29 (b).

Monkey FGF23 FW: CGGAATTCCACCATGTTGGGGGCCCGCCTCAGGCT (SEQ ID NO: 26)

Monkey FGF23 RV: ATTTGCGGCCGCTAGATGAACTTGGCGAAGGGGC (SEQ ID NO: 27)

Kiwi FGF23 nucleic acid sequence (SEQ ID NO: 28)

ATGTTGGGGGCCCGCCTCAGGCTCTGGGTCTGTGCCTTGTGCAGCGTCTGCAGCATGAGCGTCATCAGAGCCTATCCCAATGCCTCCCCATTGCTCGGCTCCAGCTGGGGTGGCCTGATCCACCTGTACACAGCCACAGCCAGGAACAGCTACCACCTGCAGATCCACAAGAATGGCCACGTGGATGGCGCACCCCATCAGACCATCTACAGTGCCCTGATGATCAGATCAGAGGATGCTGGCTTTGTGGTGATTACAGGTGTGATGAGCAGAAGATACCTCTGCATGGATTTCGGAGGCAACATTTTTGGATCACACTATTTCAACCCGGAGAACTGCAGGTTCCGACACTGGACGCTGGAGAACGGCTACGACGTCTACCACTCTCCTCAGCATCACTTTCTGGTCAGTCTGGGCCGGGCGAAGAGGGCCTTCCTGCCAGGCATGAACCCACCCCCCTACTCCCAGTTCCTGTCCCGGAGGAACGAGATCCCCCTCATCCACTTCAACACCCCCAGACCACGGCGGCACACCCGGAGCGCCGAGGACGACTCGGAGCGGGACCCCCTGAACGTGCTGAAGCCCCGGGCCCGGATGACCCCGGCCCCGGCCTCCTGCTCACAGGAGCTCCCGAGCGCCGAGGACAACAGCCCGGTGGCCAGCGACCCGTTAGGGGTGGTCAGGGGCGGTCGGGTGAACACGCACGCTGGGGGAACGGGCCCGGAAGCCTGCCGCCCCTTCGCCAAGTTCATCTAG

Kiwi FGF23 amino acid sequence (SEQ ID NO: 29)

MLGARLRLWV CALCSVCSMS VIRAYPNASP LLGSSWGGLI

HLYTATARNS YHLQIHKNGH VDGAPHQTIY SALMIRSEDA

GFVVITGVMS RRYLCMDFGG NIFGSHYFNP ENCRFRHWTL

ENGYDVYHSP QHHFLVSLGR AKRAFLPGMN PPPYSQFLSR

RNEIPLIHFN TPRPRRHTRS AEDDSERDPL NVLKPRARMT PAPASCSQEL

PSAEDNSPVA SDPLGVVRGG RVNTHAGGTG PEACRPFAKFI

(2) Preparation of supernatant of cynomolgus FGF 23-expressing cells

Their culture supernatants were obtained by transient transfection of pEAK 8/IRES/EGFP/monkey FGF23 into PEAK fast cells (Edge biosystems) by the calcium phosphate method.

(example 9)

Study of the binding ability of C10 antibody to cynomolgus FGF 23.

By the following method using sandwich ELISA, study was madeThe C10 antibody binds not only to human FGF23, but also to cynomolgus monkey FGF 23. The C10 antibody, 2C3B antibody, and human IgG1 control antibody prepared in example 4 were diluted in 50mM NaHCO₃In the solution, a diluted solution having a concentration of 5. mu.g/ml was obtained, and then added to each well of a 96-well microplate and incubated at 4 ℃ for 12 hours for ELISA (Maxisorp (registered trademark), Nunc) reaction. Thereby, the C10 antibody, the 2C3B antibody, and the human IgG1 control antibody as a control were absorbed into the microplate. Next, these solutions were removed, and a blocking agent (SuperBlock (registered trademark) blocking buffer, PIERCE) was added to each well, incubated at room temperature for 30min, and then each well was rinsed twice with Tris-buffered saline (T-TBS) containing 0.1% Tween 20. The full-length human FGF23 protein purified in example 2 or the supernatant of cynomolgus monkey FGF 23-expressing cells prepared in example 8 was diluted to an appropriate concentration, added to each well of a microplate covered with an anti-FGF 23 antibody, reacted with the immobilized antibody for two hours, and then each well was rinsed twice with Tris buffered saline (T-TBS) containing 0.1% Tween 20. Next, 3 μ g/ml of biotin-labeled 3C1E antibody was added and incubated at room temperature for 1.5 hours to allow binding of the biotin-labeled 3C1E antibody to human or cynomolgus monkey FGF23, wherein the human or cynomolgus monkey FGF23 was bound to the immobilized antibody. After rinsing with T-TBS, a 5000-fold dilution of horseradish peroxidase-labeled streptavidin (DAKO) was added, allowed to react for 1 hour, and rinsed 3 times with T-TBS. Substrate buffer containing tetramethylbenzidine (DAKO) was added to each well and incubated at room temperature for 30 min. The reaction was terminated by adding 0.5M sulfuric acid to each well. The absorbance at a wavelength of 450nm was measured using a microplate reader (MTP-300, Colona electric Co.) using a wavelength of 570nm as a reference wavelength. The reactivity of the human full-length FGF23 protein and cynomolgus monkey FGF23 expressing cell culture supernatants when diluted at a 3-fold dilution ratio was compared. The results obtained are shown in FIGS. 5A and B. As is clear from fig. 5A, the reactivity of the C10 antibody or 2C3B antibody to the human full-length FGF23 protein was the same when immobilized. Under these conditions, no significant difference was observed between the reactivity of the C10 antibody and the reactivity of the 2C3B antibody for the reactivity of the dilution series of cynomolgus FGF23 expressing cell culture supernatantOtherwise (fig. 5B). That is, the C10 antibody, like the 2C3B antibody, was shown to be able to bind to human and cynomolgus FGF 23.

(example 10)

The effects of the C10 antibody and the 2C3B antibody on the concentration of phosphorus in the blood of a normal macaque and the concentration of 1 alpha, 25-dihydroxyvitamin D in the blood are compared.

FGF23 has the following effects: promoting renal phosphorus excretion and lowering blood phosphorus concentration, inhibiting vitamin D activating enzyme in kidney and lowering blood 1 alpha, 25-dihydroxyvitamin D (hereinafter referred to as 1,25D) concentration (International patent publication No. WO 02/14504). Administration of FGF23, such as 2C3B antibody, having an inhibitory effect, i.e., neutralizing activity, to normal mice has been shown to result in the inhibition of endogenous FGF23 activity, an increase in serum phosphate concentration and serum 1,25D concentration (International patent publication No. WO 03/057733). Thus, it is strongly suggested that an antibody having neutralizing activity to FGF23 has therapeutic effects on human diseases including tumor-induced osteomalacia, XLH, etc. caused by excessive FGF 23. Therefore, the neutralizing activity of the human antibody, C10 antibody, obtained in the present invention against FGF23 in vivo was investigated. In particular, due to the expectation of its pharmacological effects in humans, neutralizing activity in monkeys, which are evolutionarily closer to humans than other animal species such as rodents and the like, was determined by using, as indices, inhibition of the function of endogenous FGF23 in monkeys, and an increase in serum phosphate concentration and serum 1,25D concentration of monkeys. For the experiments, the mouse antibody, 2C3B, was used as a comparative control for the C10 antibody.

The effects of the C10 antibody and 2C3B on the increase in serum phosphate concentration in untreated normal macaques were compared using the following method. The C10 antibody produced in example 4 was used. The experimental animal is female macaque of 2-3 years old, and the weight is 2-4 kg. 3 animals each were used for the solvent administration group and the 2C3B antibody administration group, and 4 animals were used for the C10 antibody administration group. The C10 antibody and 2C3B antibody were each prepared as a solution of 3mg/ml antibody in PBS (-), and the solution was used as a solution for administration. The PBS (-) solvent was used as a negative control. CThe 10 and 2C3B antibodies were administered once through the brachiocephalic vein at a flow rate of 1mL/min, respectively, in an amount of 1mL/kg (equivalent to 3 mg/kg). Serum phosphate concentrations were measured by Wako inorganic phosphorus reagent type L (Wako Pure Chemical Industries) and Hitachi clinical Analyzer Model 7180(Hitachi, Ltd.). By 1,25(OH)₂D RIA Kit[TFB](Immunodiagnostic System) serum 1,25D concentrations were measured. The assays were performed 0.5, 1,2, 3, 5, 7, 10, 14, 21, 28, 35, 42, and 49 days after antibody administration, respectively. Data are presented as mean +/-standard error. The data in figure 6 show the change in serum phosphorus concentration in blood samples collected periodically over 10 days after administration of each antibody. During the test, the PBS (-) administered group had almost no change in serum phosphorus concentration, whereas in the C10 antibody administered group and the 2C3B antibody administered group, a significant increase in serum phosphorus concentration was observed compared to the pre-administration and PBS (-) administered groups. In the groups administered with the C10 antibody and 2C3B antibody, the time of the highest serum phosphorus concentration appeared was 5 days after antibody administration. At this time point, the serum phosphorus concentrations of the PBS (-) group, the 2C3B antibody group, and the C10 antibody group were 5.28mg/dl, 8.10mg/dl, and 9.59mg/dl, respectively. Comparing the rise in serum phosphorus concentration 5 days after antibody administration with that of the contemporary PBS (-) group for the 2C3B antibody group and the C10 antibody group, the increase in serum phosphorus concentration in the 2C3B antibody group was 2.82mg/dl, while the increase in serum phosphorus concentration in the C10 antibody group was 4.31mg, (these results) indicate that the increase in serum phosphorus concentration induced by the C10 antibody was 1.5 times or more the increase in serum phosphorus concentration induced by the 2C3B antibody compared with the 2C3B antibody group (fig. 7). Therefore, the effect of the C10 antibody administration group on the increase in serum phosphorus concentration was significantly higher than that of the 2C3B antibody administration group. Furthermore, 10 days after the administration, the serum phosphorus concentration of the 2C3B antibody-administered group was at the same level as that of the PBS (-) group, while the serum phosphorus concentration (8.76mg/dl) of the C10 antibody-administered group was maintained at a level higher than the highest level (8.10mg/dl) of the serum phosphorus concentration of the 2C3B antibody-administered group (FIG. 6). In addition, the retention time of serum phosphorus concentration after increase caused by the C10 antibody was longer than that after increase caused by the 2C3B antibody, which was 7 days with the 2C3B antibody, which is significantly different from the PBS (-) administration group,the retention time with the C10 antibody was surprisingly 35 days, which differed by about 5-fold. Similarly, for the 1,25D concentration after antibody administration, the 1,25D concentration after C10 antibody administration was significantly increased and its retention time was significantly prolonged compared to 2C3B antibody administration (fig. 8).

These results indicate that the C10 antibody has stronger serum phosphorus concentration-promoting and serum 1,25D concentration-increasing activities, that is, has stronger FGF 23-neutralizing activity in cynomolgus monkeys than the 2C3B antibody, which is an existing FGF 23-neutralizing antibody. Currently, for treating rickets with hypophosphatemia such as XLH, a large dose of phosphorus and vitamin D is required to be administered for multiple times every day so as to maintain the phosphorus concentration within a normal range. It has been reported that multiple administrations result in poor compliance in patients. The fact that a single administration of the C10 antibody resulted in sustained activity against serum phosphorus levels and serum 1,25D levels in this study indicates that the C10 antibody is likely to have significant advantages in therapeutic efficacy as a therapeutic agent for hypophosphatemia, as compared to conventional therapeutic methods.

(example 11)

Determination of the reactivity of the C15 antibody to human and cynomolgus FGF23

Transient genes pEAK8/IRES/EGFP/hFGF23 prepared in example 11 or pEAK 8/IRES/EGFP/monkey FGF23 prepared in example 8 were transfected into PEAK express cells (Edge Biosystem) by the calcium phosphate method. After 3 days of transfection, each culture supernatant was collected. The collected culture supernatant was subjected to western blotting using the C15 antibody prepared in example 13 as a primary antibody (FIG. 9). The results show that: similar to C15 binding to human FGF23, C15 also binds to cynomolgus monkey FGF 23.

(example 12)

The effects of the C10 antibody and the C15 antibody on the blood phosphorus concentration of normal macaques and the concentration of 1 alpha, 25-dihydroxy vitamin D in blood are compared.

Example 11 shows that: the C15 antibody has binding activity to human and cynomolgus FGF23 recombinant proteins as does the C10 antibody. Subsequently, for the C10 antibody and the C15 antibody, their neutralizing activity against FGF23 in vivo was compared by administering them to normal cynomolgus monkeys. The neutralization activity of cynomolgus monkey endogenous FGF23 was evaluated using the C10 antibody and C15 antibody prepared in example 7, using the increase in blood phosphorus concentration as an index. The experimental animal is a normal macaque of 2-3 years old and 2-3kg weight. A total of 3 animals were used for 2 males and one female per experimental group. The dilution medium used was PBS (-). The concentrations of C10 antibody were prepared at 1mg/ml and 3mg/ml, and the concentration of C15 antibody was 3 mg/ml. The antibody was administered once in a volume of 1mL/kg through the saphenous vein at a flow rate of 1mL/min, thereby obtaining 1mg/kg and 3mg/kg of C10 antibody administration doses and 3mg/kgC15 antibody administration doses. Serum phosphorus concentrations were determined by Wako model L inorganic phosphorus reagent (Wako pure chemical Industries) and Hitachi automatic analyzer 7180(Hitachi Clinical Analyzer model 7180(Hitachi, Ltd.)). Blood collection was performed before administration of the antibody and 1, 3, 5, 7, 10, 14, 21 and 28 days after administration. Serum phosphorus concentrations were determined at all blood collection points. The serum phosphorus concentrations of the C10 antibody-1 mg/kg-administered group, the C10 antibody-3 mg/kg-administered group and the C15 antibody-3 mg/kg-administered group before administration were 5.37, 5.70 and 5.58mg/dL, respectively, and there was no significant difference between the groups. After administration, an increase in serum phosphorus concentration was observed in all macaques. Therefore, not only the C10 antibody but also the C15 antibody showed neutralizing activity against cynomolgus endogenous FGF 23. The serum phosphorus concentrations 3 days after administration were 9.03, 9.10 and 8.64mg/dL in the C10 antibody 1 mg/kg-administered group, the C10 antibody 3 mg/kg-administered group and the C15 antibody 3 mg/kg-administered group, respectively. At this time point, the serum phosphorus concentrations of the C10 antibody-1 mg/kg-administered group and the C15 antibody-3 mg/kg-administered group reached the highest values. On the other hand, the serum phosphorus concentration of the C10 antibody 3 mg/kg-administered group continued to increase and reached a maximum value 5 days after administration, which was 9.75 mg/dL. In the C10 antibody 1mg/kg administration group, the C10 antibody 3mg/kg administration group and the C15 antibody 3mg/kg administration group, the maximum difference of the serum phosphorus concentration before and after administration was 3.67, 4.65 and 3.06mg/dL, respectively. From these results, it was found that the C10 antibody exhibited a higher effect of increasing serum phosphorus concentration than the C15 antibody (effect of increasing serum phosphorus concentration) at the same dose of 3 mg/kg. Furthermore, it was surprising that the 1mg/kg dose of the C10 antibody had a higher effect on the increase in serum phosphorus concentration than the 3mg/kg dose of the C15 antibody (which increased serum phosphorus concentration). Next, the duration of the increase in serum phosphorus concentration compared to the pre-dose level was compared. The result is: the durations of increase in serum phosphorus concentration of the C10 antibody 1 mg/kg-administered group, the C10 antibody 3 mg/kg-administered group and the C15 antibody 3 mg/kg-administered group were 14, 28 and 7 days, respectively. From these results, it was found that at the same 3mg/kg dose, the C10 antibody not only showed higher persistence of activity against serum phosphorus concentration increase than the C15 antibody (duration of serum phosphorus concentration increase). Furthermore, it was surprising that a 1mg/kg dose of the C10 antibody maintained serum phosphorus levels at higher levels for a longer period of time than a 3mg/kg dose of the C15 antibody. These facts indicate that, in cynomolgus monkeys, the C10 antibody has stronger activity of increasing serum phosphorus concentration and persistence of activity of increasing serum phosphorus concentration than C15 antibody (effect on serum phosphorus concentration) obtained at the same time. That is, the C10 antibody has a significantly strong neutralizing activity against cynomolgus FGF23, compared to the C15 antibody.

(example 13)

Preparation of human FGF23DNA fragment (without signal peptide sequence).

A reaction solution was prepared using KOD-plus-DNA polymerase (Toyobo) according to the protocol. 50pmol each of FGF23(-SP) FW primer (SEQ ID NO: 34) and FGF23(-SP) RV primer (SEQ ID NO: 35), human FGF23-cDNA (756 bp in total from start codon to stop codon, SEQ ID NO: 36) as a template was added to 50. mu.l of the reaction solution, and after incubation at 94 ℃ for 3min, amplification was performed for 30 cycles each consisting of 98 ℃/15 sec, 63 ℃/15 sec, and 68 ℃/2min30 sec. Incubate at 72 ℃ for 3 min. The 684bp amplified fragment was collected by 0.8% gel separation. The amplified fragment was recovered from the recovered Gel using QIAquick Gel Extraction Kit (Qiagen) according to the instructions. The recovered amplified fragment was subjected to enzymatic digestion with FseI (New England Biolabs Japan), and the enzyme-treated fragment was recovered with QIAquick PCR purification kit (Qiagen) according to the instructions. This gave a partial DNA fragment corresponding to the mature form of human FGF23 without the signal sequence of human FGF 23.

FGF23(-SP)FW：TATCCCAATGCCTCCCCACTGCTCGGCTCCAGCTG(SEQ ID NO：34)

FGF23(-SP)RV：

TTGGCCGGCCCTAGATGAACTTGGCGAAGGGGCGGCAGCCTTCCG (SEQ ID NO: 35, including the FseI site)

The human FGF23 nucleotide sequence (underlined marked as part of the signal sequence and rectangular boxes marked with nucleotides from the full length sequence (FGF23) not containing the mature form of human FGF23 region of the signal sequence) (SEQ ID NO: 36).

ATGTTGGGGGCCCGCCTCAGGCTCTGGGTCTGTGCCTTGTGCAGC GTCTGCAGCATGAGCGTCCTCAGAGCC

The amino acid sequence of human FGF23 is based on SEQ ID NO: 36 is the standard (underlined amino acid residues marked as part of the signal sequence, rectangular boxes marked with amino acid residues from the mature form region of human FGF23 without the signal sequence from the full length sequence (FGF 23)) (SEQ ID NO: 37).

MLGARLRLWVCALCSVCSMSVLRA

(example 14)

Construction of pPSs FGF23 vector

pPSs5.5 described in examples 1-8 of WO2006/78072 was digested with SfoI and FseI and its ends were dephosphorylated with alkaline phosphatase from E.coli. The DNA fragment containing the human FGF23 gene prepared in example 13 was inserted and then transfected into DH5 α. DNA was prepared from the obtained transformant, and the nucleotide sequence of the joining region was determined, thereby obtaining the vector pPSs FGF23 (FIG. 10).

(example 15)

Construction of pUS FGF23KI vector

pCk loxPV. DELTA.P described in example 43-1 of WO2006/78072 was digested with SalI and FseI enzymes, and the ends thereof were dephosphorylated with alkaline phosphatase derived from E.coli C75. After inserting an about 1.5kb fragment, which was obtained by digesting the pPSs FGF23 vector prepared in example 14 above with SalI and FseI enzymes and separating and recovering it on a 0.8% agarose gel, the vector was transfected into E.coli XL10-Gold super competent Cells (Ultracompetent Cells) (STRA TAGENE). DNA was obtained from the obtained transformant. The nucleotide sequence of the linker region was determined, thereby obtaining a pUS FGF23KI vector (FIG. 11).

The 985bp sequence (SEQ ID NO: 38) and the amino acid sequence (SEQ ID NO: 39) are shown below. The 985bp sequence (SEQ ID NO: 38), wherein the polynucleotide sequence from the start codon to the stop codon of the pUS FGF23KI vector human FGF23 expression unit (FGF23 signal sequence) is replaced by a murine Ig kappa signal sequence (underlined in SEQ ID NO: 38) containing the coding region, and downstream thereof contains the mature form of FGF23 sequence; the amino acid sequence (SEQ ID NO: 39) is that encoded by the cDNA (247 amino acids, underlined representing the murine Ig kappa signal sequence, SEQ ID NO: 39). Sequence information for the murine Ig κ signal sequence, including the intron region, was based on MUSIGKVR1 (accession number K02159) available from GenBank, the upstream genomic sequence of which was obtained from the UCSC murine genomic database.

SEQ ID NO：38

ATGGAGACAGACACACTCCTGTTATGGGTACTGCTGCTCTGGGTT CCAGGTGAGAGTGCAGAGAAGTGTTGGATGCAACCTCTGTGGCCATTA TGATACTCCATGCCTCTCTGTTCTTGATCACTATAATTAGGGCATTTGT CACTGGTTTTAAGTTTCCCCAGTCCCCTGAATTTTCCATTTTCTCAGAG TGATGTCCAAAATTATTCTTAAAAATTTAAATAAAAAGGTCCTCTGCT GTGAAGGCTTTTATACATATATAACAATAATCTTTGTGTTTATCATTCC AGGTTCCACTGGCTATCCCAATGCCTCCCCACTGCTCGGCTCCAGCTGGGGTGGCCTGATCCACCTGTACACAGCCACAGCCAGGAACAGCTACCACCTGCAGATCCACAAGAATGGCCATGTGGATGGCGCACCCCATCAGACCATCTACAGTGCCCTGATGATCAGATCAGAGGATGCTGGCTTTGTGGTGATTACAGGTGTGATGAGCAGAAGATACCTCTGCATGGATTTCAGAGGCAACATTTTTGGATCACACTATTTCGACCCGGAGAACTGCAGGTTCCAACACCAGACGCTGGAAAACGGGTACGACGTCTACCACTCTCCTCAGTATCACTTCCTGGTCAGTCTGGGCCGGGCGAAGAGAGCCTTCCTGCCAGGCATGAACCCACCCCCGTACTCCCAGTTCCTGTCCCGGAGGAACGAGATCCCCCTAATTCACTTCAACACCCCCATACCACGGCGGCACACCCGGAGCGCCGAGGACGACTCGGAGCGGGACCCCCTGAACGTGCTGAAGCCCCGGGCCCGGATGACCCCGGCCCCGGCCTCCTGTTCACAGGAGCTCCCGAGCGCCGAGGACAACAGCCCGATGGCCAGTGACCCATTAGGGGTGGTCAGGGGCGGTCGAGTGAACACGCACGCTGGGGGAACGGGCCCGGAAGGCTGCCGCCCCTTCGCCAAGTTCATCTAG

SEQ ID NO：39

METDTLLLWVLLLWVPGSTGYPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRRHTRSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI

(example 16)

Preparation of pUS FGF23KI vector for electroporation

Mu.g of pUS FGF23KI vector was digested with a buffer (Roche diagnostics, restriction enzyme with H buffer (H buffer for restriction enzyme)) to which spermidine (speramine-extended) (1mM pH7.0, Sigma Aldrich Japan) was added and NotI (Takara Bio, Inc.) for 5 hours at 37 ℃. After phenol/chloroform extraction, 2.5 volumes of 100% ethanol and 0.1 volume of 3M sodium acetate were added and the resulting mixture was left at-20 ℃ for 16 hours. The vector was not I linearized, collected by centrifugation and sterilized with 70% ethanol. The 70% ethanol was removed in a clean bench and air dried for 1 hour. HBS solution was added to prepare 0.5. mu.g/. mu.LDNA solution, and the resulting solution was used to prepare pUSGFGF 23KI vector for electroporation after being left at room temperature for 1 hour.

(example 17)

Knockout of the murine embryonic cell line with pUS FGF23KI vector and RS factor gave PL FGF23 murine embryonic cell line.

To obtain PL FGF23 murine embryonic cell line in which human FGF23-cDNA was inserted downstream of immunoglobulin kappa light chain Gene by homologous recombination, pUS FGF23KI vector (prepared as shown in example 16) treated with restriction enzyme NotI linearization was transferred into RS factor-knocked murine embryonic cells according to established methods (Shinichi Aizawa, "Biotechnology Manual Series 8, Gene Targeting," Yodosha, 1995.) by using the method described in example 10 of WO2006/78072, RS factor-knocked murine embryonic cells were obtained.

Culture method of RS factor-knocked out murine embryonic cells the feeder cells used were G418 resistant primary culture cells (purchased from Invitrogen) cultured in a mitomycin C (Sigma Aldrich Japan) treated medium according to the method already described (Shinichi Aizawa, supra). First, RS factor knock-out murine embryonic cells were cultured and trypsinized at 3X 10⁷The concentration of cells/ml was suspended in HBS. 0.5ml of the cell suspension was mixed with 10. mu.g of vector DNA. Then, electroporation (capacitance: 960. mu.F, voltage: 250V, room temperature) was carried out using a Gene pulse tube (Gene Pulser Cuvette) (electrode distance: 0.4cm, Bio Rad laboratories). The electroporated cells were suspended in 10ml of embryonic cell culture medium (Shinichi Aizawa, supra), and then these fine cells were culturedThe cells were seeded in plastic culture dishes (Falcon, Becton Dickinson) for 100mm tissue culture, in which trophoblast cells had previously been pre-plated. After 36 hours, the medium was changed to embryonal cell culture medium (Sigma Aldrich Japan) containing 0.8. mu.g/ml puromycin. Each of the colonies picked up after 7 days of culture was inoculated into a 24-well plate culture to complete confluence. Two thirds of the cells obtained were suspended in 0.2ml of inoculation medium (FBS + 10% DMSO, SigmaAldrich Japan) and the resulting suspension was stored at-80 ℃. The remaining one third was seeded in 12-well gelatin-coated plates. After the cells were cultured for 2 days, 10 were extracted using the Puregene DNA extraction kit (Qiagen)⁶-10⁷Genomic DNA of the cell. The genomic DNA of the puromycin-resistant RS factor-knocked-out murine embryonic cells thus obtained was digested with restriction enzyme EcoRI (Takara Bio, Inc.) and separated by agarose gel electrophoresis. Subsequently, Southern blot detection was performed using a Ck3 ' probe as a probe to detect homologous recombinants, wherein the Ck3 ' probe was used in the present invention and a DNA fragment at the 3 ' -end of Ig light chain J.kappa.Ckappa.Ckappa.genomic DNA (XhoI cleavage site to EcoRI cleavage site, about 1.4kb in length, WO00/10383, FIG. 5) was described in WO00/10383 (see example 48). After EcoRI digestion, a 15.1kb band was detected in wild type RS factor knock-out mouse embryonic cells. In the homologous recombinants, in addition to the 15.1kb band, it was expected that a new band (12.8kb) appeared below the 15.1kb band (FIG. 12), and the new band was detected in puromycin-resistant lines. That is, these clones were confirmed to have human FGF23-cDNA inserted at one of the allelic loci downstream of the immunoglobulin kappa chain gene.

(example 18)

The US FGF23 mouse embryonic stem cell line is obtained by knocking out a drug resistance gene in a PL FGF23 mouse embryonic stem cell line.

2 resistance genes (Puro) in the obtained US FGF23 mouse embryonic stem cell line from PL FGF23 mouse embryonic stem cell line^r，Neo^r) Is knocked out, which is in reference to the existing mature practiceThe assay method (Shinichi Aizawa, "Biotechnology Manual" Series 8, Gene Targeting technology "Yodosha, 1995, Shinichi Aizawa," Biotechnology Manual Series 8, Gene Targeting, "Yodosha, 1995) was performed in which the pCAGGS-Cre vector (Sunaga et al, MolReprod Dev., 46: 109-.

Method for culturing PL FGF23 mouse embryonic stem cells referring to the existing method (Shinichi Aizawa, above-mentioned document), G418-resistant primary culture cells (purchased from Invitrogen) treated with mitomycin c (sigma Aldrich japan) were added to the culture broth as trophoblast cells. First, cultured PL FGF23 mouse embryonic stem cells were trypsinized at 3X 10⁷The cells/ml were suspended in HBS, and 0.5ml of the cell suspension was mixed with 10. mu.g of carrier DNA and subjected to electroporation (capacitance: 960. mu.F, voltage: 250V, room temperature operation) using a Gene pulse tube (Gene Pulser Cuvette) (electrode spacing: 0.4cm, Bio Rad laboratories). The electroporated cells were suspended in 10ml of embryonic stem cell culture medium (Shinichi Aizawa, supra) and 2.5ml of the cell suspension was seeded onto 60mm plastic culture dishes for tissue culture (Falcon, Becton Dickinson) previously plated with trophoblast cells. After 30 hours of culture, 1000 embryonic stem cells were seeded into a plastic culture dish for 100mm tissue culture (Falcon, Becton Dickinson) previously plated with trophoblast cells. Clones that appeared after 6 days were picked and each clone was inoculated into a 24-well plate culture to complete confluence. Two thirds of the cells were suspended in 0.2ml of frozen medium (FBS + 10% and subjected to Southern blot detection with Ck3 ' probe to detect homologous recombinants, wherein the Ck3 ' probe was used in the present invention and has been described in WO00/10383 (see example 48) the 3 ' end DNA fragment of Ig light chain J κ -C κ genomic DNA (XhoI cleavage site to EcoRI cleavage site, about 1.4kb in length, WO00/10383, FIG. 5). DMSO (Sigma Aldrich Japan) was stored at-80 ℃ and the remaining third was plated on 12-well gelatin-coated plates, and after the cells were cultured for 2 days, 10 minutes were extracted using Puregene DNA extraction kit (Qiagen) to obtain 10⁶-10⁷Genes of cellsGroup DNA. The genomic DNA of the mouse embryonic stem cell thus produced was digested with restriction enzyme EcoRI (Takara Bio, Inc.), and the band of interest was separated by agarose gel electrophoresis. Subsequently, Southern blot detection was performed using the Ck 3' probe as a probe to detect Puro among them, which is located only between the loxPV gene sequences^rEmbryonic stem cell lines with knocked-out genes using the detection of probe Ck3 ', used in the present invention, the 3' terminal DNA fragment (XhoI to EcoRI cleavage site, about 1.4kb long, WO00/10383, fig. 5) of Ig light chain jk-Ck genomic DNA, described in WO00/10383 (see example 48). When Puro is retained in the genome of embryonic stem cells^rGene, two bands (15.1kb and 12.8kb) were detected by EcoRI digestion; while only when Puro is knocked out from the genome of the embryonic stem cell^rThe gene was digested with EcoRI, and two bands were detected, 15.1kb and 10.9kb, respectively (FIG. 12). Furthermore, only loxpVPuro is located in genomic DNA of embryonic stem cell line detected by Southern hybridization membrane obtained in the above process using 3' KO probe method in WO2006/78072 shown in patent No. 9^rNeo between gene sequences^rThe gene was knocked out and detected by using the probe 3' KO, when Neo remained in the embryonic stem cell genome^rGene, two bands (7.4k and 5.7k) were detected by EcoRI digestion; and only when the genome of the embryonic stem cell knocks out Neo^rThe gene was digested with EcoRI to detect two bands of 5.7k and 4.6k, respectively (FIG. 12). Through the above experiment, 2 drug-resistant genes (Puro) can be obtained from PL FGF23 mouse embryonic stem cell line^r，Neo^r) A knockout embryonic stem cell line at the same time (US FGF23 mouse embryonic stem cell line.

(example 19)

US FGF23KI chimeric mice were prepared using US FGF23 mouse embryonic stem cell line and a host embryo from a B lymphocyte deficient mouse strain.

In a mouse homozygous for an immunoglobulin u-chain gene knockout, B lymphocytes are deficient in function and are unable to produce antibodies (Kitamura et al, Nature, 350: 423-426, 1991). In this example, host embryos were bred by crossing homozygous male and female mice cultured in a clean environment as described above for the preparation of chimeric mice. In this case, most of the B lymphocyte function of the chimeric mouse is from the embryonic stem cell into which it was injected. In this example, immunoglobulin μ chain gene knockout homozygous mice (Proc. Natl. Acad. Sci. USA, 97: 722-7, 2000), reported by Tomizuka et al, were used for host embryo preparation by backcrossing with MCH (ICR) line (CLEA Japan, Inc.) 3 or more times.

The US FGF23 embryonic stem cell lines obtained by the above example 18 were recovered, and these embryonic stem cell lines were confirmed to have FGF23-cDNA inserted therein the downstream sequence of human immunoglobulin kappa chain gene. The US FGF23 embryonic stem cells were injected into 8-cell stage host embryos bred by crossing homozygous male and female mice with the immunoglobulin μ chain knockout described above, at an injection cell rate of 8-10 embryonic stem cells/host embryos. Injected embryos were cultured overnight in embryonic stem cell culture (Shinichi Aizawa, "Biotechnology Manual" series 8, Gene targeting technology "Yodosha, 1995), and after the injected embryos developed into blastocysts, the injected embryos were transplanted into the uterus of 2.5 day-pseudopregnant surrogate MCH (ICR) mother mice (CLEA Japan, Inc.), and approximately 10 injected embryos were implanted into each lateral uterus. Chimeric mice were obtained by embryo injection using the US FGF23 mouse embryonic stem cell line of example 18 above. Chimeric mice are identified by differences in coat color, where the presence or absence of wild-type color (dark brown) of embryonic stem cell origin can be identified in the host embryonic (white) coat color. In the offspring of chimeric mice, each mouse has a hair color distribution with a significant portion of wild-type derived hair color, that is, chimeric mice can be distinguished from homozygous mice by identifiable wild-type color (dark brown) of embryonic stem cell origin. From these experimental results, the US FGF23 embryonic stem cell line, into which FGF23-cDNA of a sequence downstream of a human immunoglobulin kappa chain gene was inserted, had the ability to maintain production of chimeric mice. That is, in a mouse individual, USFGF23 embryonic stem cells have the ability to differentiate into normal tissues. Furthermore, the blood of US FGF23KI chimeric mice had a high concentration of FGF23, which we further mention in example 21 later that US FGF23KI chimeric mice have characteristics similar to rickets with hypophosphites, which can be used as an animal model for the study of this disease.

(example 20)

Preparation of control chimeric mice

Chimeric mice (derived from wild-type mice) without insertion of a gene having the function of human FGF23-cDNA were used as respective control chimeric mice in the experiment of administration of C10 antibody to US FGF23KI chimeric mice in example 21 described below, according to the method in example 11 described previously (patent WO 2006/78072).

(example 21)

Demonstration of efficacy of pathological improvement of C10 antibody using US FGF23 KI-chimeric mice

In examples 10 and 12, it was demonstrated that the C10 antibody significantly inhibited the effect of endogenous FGF23 and increased the phosphorus concentration in serum and the serum 1,25D concentration in cynomolgus monkeys (cynomolgus monkey) compared to the 2C3B antibody and the C15 antibody. It has been strongly suggested that: an antibody having neutralizing activity against human FGF23 has therapeutic effects on human diseases such as tumor-induced osteomalacia, hypophosphatemic rickets such as XLH, and osteomalacia induced by excessive FGF 23. Thus, the improvement of the condition caused by excess FGF23 by the C10 antibody was investigated herein. Therapeutic test of the C10 antibody was conducted using US FGF23KI chimeric mouse (hereinafter abbreviated as hFGF23KI mouse) prepared in example 19. 12 hFGF23KI mice were used as disease model animals, and 6 normal mice of the same week age (wild type mice, prepared in example 20) were used as control animals for comparative experiments. Sera of 7-week-old hFGF23KI mice were collected and assayed for FGF23 concentration (FGF-23 ELISA KIT, Kainos Laboratories, Inc) and blood phosphorus concentration, respectively. Compared with wild-type mice, the serum FGF23 concentration of the hFGF23KI mice is obviously increased (wild-type mice; n is6, 163pg/mL, hFGF23KI mice; n is 12, 1467 pg/mL). This result indicates that the introduction of human FGF23 gene in hFGF23KI mice was performed correctly and also indicates that there was an excess of exogenous human FGF23 in hFGF23KI mouse serum. Furthermore, the serum phosphorus concentration of hFGF23KI mice was significantly reduced compared to wild type mice (wild type mice; n 6, 5.82mg/dL, hFGF23KI mice; n 12, 2.62 mg/dL). The hFGF23KI mice developed hypophosphatemic symptoms due to the effect of excess human FGF 23. At this time, 12 hFGF23KI mice were divided into two groups of a C10 antibody-administered group and a control IgG 1-administered group, six in each group, each having an equivalent FGF23 concentration (fig. 13). Then, starting from the eighth week, the mice in the two groups were repeatedly injected with the purified human IgG1 (control antibody) for the intravenous injection of the C10 antibody or isotype control at a dose of 30mg/kg at a frequency of once a week for five times in total. Blood samples were taken before the first dose and three days after the dose, and serum was extracted. The force of grasping the limbs of the mice 24 hours after the fourth administration was evaluated using a Saitoh hand-grasping device (Saitoh-GRIP STRENGTH METER, MK-380S, Muromachi Kikai Co., Ltd.). The limb gripping force is the maximum force (gripping force) of the mouse when the mouse is full, wherein the mouse is placed in a measured grid, the grid is held by the mouse, and then the tail of the mouse is horizontally pulled by hand until the mouse cannot bear the tensile force to loosen the grid. The bone condition of the mice was evaluated 24 hours after the fifth dose. Mice were anesthetized, sacrificed by cardiac hemorrhaging, and their femur (fermur) and tibia (tibia) were removed and fixed in 70% ethanol. Serum phosphorus concentrations were measured before, three days after and 24 hours after the first dose, respectively. The calcemized femur was encapsulated in resin and stained with Villanueva-Goldner for histological evaluation. Mineral content in the tibia was measured after ashing.

The experimental results showed that hFGF23KI mice of the control antibody administration group, at the time of grouping and at the time of sacrifice together (i.e., at the time before the administration and at the time 24 hours after the fifth administration), had significantly lower phosphorus concentrations in the serum than wild type mice of the control antibody administration group, which indicated a sustained low blood phosphorus condition in hFGF23KI mice (fig. 14). On the other hand, three days after the administration of the hFGF23KI mouse in the C10 antibody administration group, the phosphorus concentration in the serum was increased to the same level as that in the wild-type mouse in the control antibody administration group (fig. 14). In addition, the hFGF23KI mouse of the C10 antibody-administered group was at the same level of phosphorus concentration in serum after the fifth administration as the wild-type mouse of the control antibody-administered group, which indicates that the effect of the C10 antibody on increasing the serum phosphorus concentration was still present even after the five administrations (fig. 15).

Skeletal muscle weakening has been reported clinically as a symptom in patients with low blood phosphorus (Baker and Worthley, Crit Care Resusc., 4: 307-315, 2000). In this study, we expected that hFGF23KI mice develop muscle weakening symptoms due to hypo-phosphatemia. Therefore, the limb grasping power of each group of mice was measured and compared as an index of muscle weakness by the above method. As a result, the gripping force of the hFGF23KI mice of the control antibody administration group was significantly lower than that of the wild-type mice to which the control antibody was administered, and symptoms of muscle weakening were observed in the disease model mice (fig. 16). In contrast, the grip strength of hFGF23KI mice in the C10 antibody administration group was significantly improved (fig. 16).

Then, the calcoactivated femur tissue was stained by the Villaneuva-Goldner method and histologically observed. As a result, a large amount of osteoid (osteopoid, indicated by red in fig. 17) was observed in the bone of the hFGF23KI mouse of the control antibody administration group, compared to the wild type mouse of the control antibody administration group, indicating that the mice of this group were induced with calcification defect. This is the characteristic symptom of rickets. In contrast, in the human femur bone tissue of hFGF23KI mouse in the group to which the C10 antibody was administered, a decrease in the region occupied by the osteoid was observed, and the osteoid was presumed to be replaced by calcified bone (shown in green in fig. 17). These results indicate that the C10 antibody can ameliorate the reduction in bone calcification caused by excess FGF 23. Minerals in the tibia were measured by the method of ashing and compared between groups. The mineral content in the tibia of the hFGF23KI mice of the control antibody administration group was significantly lower than that of the wild-type mice of the control antibody administration group (fig. 18). In contrast, the experiment demonstrated that the mineral content in the tibia was increased in the hFGF23KI mice in the group to which the C10 antibody was administered (fig. 18). Through the experiments, the C10 antibody proves that in a hFGF23KI mouse, the effect of the over-expressed human FGF23 in vivo is inhibited, and various symptoms of the hypophosphatemic rickets, such as hypophosphatemia, muscle weakening, bone calcification disorder and the like, are improved. These results indicate that the C10 antibody is an effective therapeutic agent for various human diseases associated with FGF 23.

Industrial applicability

The C10 antibody is an anti-FGF 23 antibody, and compared with other anti-FGF 23 antibodies, the C10 antibody has high activity in vivo for continuously increasing phosphorus concentration and/or continuously increasing serum 1 and 25D concentration. The C10 antibody of the present invention is a drug having a significant therapeutic effect on the prevention or treatment of diseases caused by excessive FGF23 activity, or diseases in which the pathology can be improved by controlling FGF23 activity.

Claims (7)

1. An isolated anti-human FGF23 antibody or a functional fragment of the antibody comprising as heavy chain CDRs the CDR1 of SEQ ID NO:40, the CDR2 of SEQ ID NO:41 and the CDR3 of SEQ ID NO:42, and as light chain CDRs the CDR1 of SEQ ID NO:43, the CDR2 of SEQ ID NO:44 and the CDR3 of SEQ ID NO:45, wherein the functional fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Peptide fragments of disulfide-stabilized Fv and single chain Fv (scFv).

2. The anti-human FGF23 antibody of claim 1, wherein the antibody is of the IgG, IgA, IgE, or IgM class.

3. The anti-human FGF23 antibody of claim 2, wherein the subclass of the antibody is IgG1, IgG2, IgG3, or IgG 4.

4. A pharmaceutical composition comprising as an active ingredient an anti-human FGF23 antibody according to any one of claims 1 to 3, or a functional fragment of such an antibody.

5. Use of an anti-human FGF23 antibody according to any one of claims 1 to 3, or a functional fragment of such an antibody, for the manufacture of a medicament for the control of phosphorus metabolism and/or vitamin D metabolism by FGF 23.

6. The use according to claim 5, wherein the disease associated with metabolism is selected from the group consisting of neoplastic osteomalacia, ADHR due to an excess of FGF23, XLH, bone fibrotic dysplasia due to an excess of FGF23, Mao-Aus syndrome due to an excess of FGF23 and autosomal recessive hypophosphatemia due to an excess of FGF 23.

7. The use according to claim 5, wherein the disease associated with the metabolism is selected from the group consisting of diseases caused by an excess of FGF 23: osteoporosis, rickets, hypercalcemia, hypocalcemia, ectopic calcification, bone sclerosis, Pagey's disease, hyperparathyroidism, hypoparathyroidism and pruritus.