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  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present invention relates to anti-FGF23 antibodies or antibody fragments thereof.
• Fibroblast growth factor (hereinafter referred to as FGF) forms a family of structurally similar polypeptides, and it not only has fibroblast proliferation activity but also mesoderm and neuroectoderm proliferation.
• FGF Fibroblast growth factor
• Various effects have been reported, including angiogenic effects, angiogenic effects, and limb bud formation during developmental stages. In adults, it also functions as a homeostasis factor, such as tissue maintenance, repair, regeneration, and metabolism (Non-Patent Document 1).
• FGF family In mammals, 22 types of proteins belonging to the FGF family are known. In humans, 22 types of FGF1 to FGF23, excluding FGF15, have been identified. Furthermore, the human FGF family is composed of about 150 to 300 amino acids, and the core sequence of about 120 amino acids has an identity of about 30 to 60%. The FGF family is classified into those that are expressed as secreted proteins and act on receptor tyrosine kinases, and those that are expressed as intracellular proteins and act on voltage-dependent sodium channels and other molecules (Non-patent Document 2). .
• FGF23 is a secreted protein that was identified from mice using a database search and PCR method using homology with FGF15, and was subsequently identified by homology search.
• Human FGF23 is composed of a polypeptide of 251 amino acid residues, and it is known that the N-terminal 24 residues function as a secretion signal and are cleaved during the protein maturation process (Non-Patent Document 3).
• FGF23-related hypophosphatemic diseases for which the causative gene is known include X-linked hypophosphatemic rickets caused by phosphate-regulating endopeptidase homolog, X-linked (PHEX) mutations. rickets, hereinafter XLH) has the highest frequency, and a large number of PHEX gene mutations have been reported so far (Non-Patent Document 5).
• PHEX is a single-transmembrane protein and is known to be expressed in large numbers in cartilage, osteoblasts, and odontoblasts (Non-patent Document 6 and Non-Patent Document 7), and XLH affects 20,000 people. It is said that the disease occurs in only one person (Non-Patent Document 8).
• Tumor-induced osteomalacia (TIO) is a known example of an acquired disease.
• Non-Patent Document 9 Non-Patent Document 10
• Burosumab an FGF23 neutralizing antibody
• Burosumab an FGF23 neutralizing antibody
• Patent Document 1 the antibody described in Patent Document 1 is also known as a human FGF23 neutralizing antibody.
• the present invention aims to provide a novel anti-FGF23 antibody whose degradation at a lower pH is suppressed than that of the anti-human FGF23 antibody described in WO 2008/099969.
• the present inventors have developed an anti-FGF23 antibody in which the 100th amino acid residue or the 105th amino acid residue of the VH of the anti-FGF23 antibody described in WO 2008/099969 is substituted.
• the inventors have discovered that the above-mentioned problems can be solved, and have completed the present invention.
• VH heavy chain variable region
• VL light chain variable region
• the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is an alanine residue, an asparagine residue, a glycine residue, a tyrosine residue, an arginine residue, an aspartic acid residue, a histidine residue, and a tryptophan residue.
• 3. The antibody or antibody fragment according to 1 or 2 above, wherein the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is substituted with an alanine residue or a tyrosine residue. 4.
• the 105th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH is an alanine residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue. group, proline residue, glutamine residue, arginine residue, valine residue, tryptophan residue, tyrosine residue, threonine residue, asparagine residue, and serine residue. , the antibody or antibody fragment according to any one of 1 to 3 above. 5. 5. The antibody or antibody fragment according to any one of 1 to 4 above, wherein the antibody further comprises 1 substitution selected from (a1) to (a4) below.
• At least one substitution selected from substitution with a histidine residue, substitution of the 57th amino acid residue with a threonine residue, and substitution of the 58th amino acid residue with a phenylalanine residue (a2) Substitution of the 91st amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL with a methionine or leucine residue, substitution of the 92nd amino acid residue with a tyrosine residue, substitution of the 94th amino acid residue with a tyrosine residue, at least one substitution selected from the substitution of an amino acid residue with an aspartic acid residue, and the substitution of the 96th amino acid residue with an asparagine residue or an aspartic acid residue; (a3) Substitution of the 28th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL with an aspartic acid residue, substitution of the 29th amino acid residue with a valine residue, and 31st amino acid residue at least one substitution selected from substitution of a threon
• (d1) Fc containing a substitution of the 252nd amino acid residue in the EU index with a tyrosine residue, the 254th amino acid residue with a threonine residue, and the 256th amino acid residue with a glutamic acid residue region
• (d2) an Fc region comprising a substitution of the 428th amino acid residue in the EU index with a leucine residue and a substitution of the 434th amino acid residue with a serine residue
• (d3) an Fc region containing a substitution of the 308th amino acid residue of the EU index with a proline residue
• (d4) Fc region containing the substitution of the 250th amino acid residue of the EU index with a glutamine residue and the substitution of the 428th amino acid residue with a leucine residue, and (d5) the 434th amino acid residue of the EU index
• a method for producing the antibody or antibody fragment according to any one of 1 to 10 above which comprises culturing the transformed cell according to 13 above in a medium and collecting the antibody or antibody fragment from the culture. 15.
• a composition comprising the antibody or antibody fragment according to any one of 1 to 10 above.
• a therapeutic agent for human FGF23-related diseases comprising the antibody or antibody fragment according to any one of 1 to 10 above.
• a method for treating human FGF23-related diseases comprising the antibody or antibody fragment according to any one of 1 to 10 above.
• the anti-FGF23 antibody of the present invention exhibits superior stability with suppressed decomposition at low pH compared to the anti-FGF23 antibody described in WO 2008/099969.
• an anti-FGF23 antibody or the antibody fragment a nucleic acid having a base sequence encoding the antibody or the antibody fragment, a vector containing the nucleic acid, a transformed cell containing the vector, an anti-FGF23 antibody or the antibody fragment, Methods of production and compositions comprising the antibodies or antibody fragments can be provided.
• the present invention provides an antibody that binds to fibroblast growth factor 23 (hereinafter referred to as FGF23) described in International Publication No. 2008/099969 (hereinafter referred to as a heavy chain variable antibody comprising the amino acid sequence represented by SEQ ID NO: 1). (hereinafter referred to as an antibody containing a light chain variable region (hereinafter referred to as VL) containing the amino acid sequence represented by SEQ ID NO: 2), at least the amino acid sequence represented by SEQ ID NO: 1 in VH.
• the present invention relates to an antibody or an antibody fragment thereof that binds to FGF23 in which the 100th or 105th amino acid residue of the amino acid sequence is substituted with another amino acid residue (hereinafter referred to as the antibody of the present invention).
• FGF23 is a type of fibroblast growth factor and a hormone derived from bone cells.
• Human FGF is a protein consisting of 251 amino acids including a 24 amino acid secretion signal at the N-terminus, and the secretion signal is cleaved during the maturation process of the protein.
• the functions of FGF23 are mainly through the FGF receptor 1 (hereinafter also referred to as FGFR1)/ ⁇ -Klotho complex on renal proximal tubular cells, and promotes phosphorus reabsorption and vitamin D in the kidney. Examples include suppressing activation.
• human FGF23 includes a polypeptide comprising the amino acid sequence of NCBI Accession No. NP_065689, an amino acid sequence consisting of an amino acid sequence with one or more amino acids deleted, substituted, or added to the amino acid sequence of NCBI Accession No. NP_065689, and From an amino acid sequence having 60% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more homology with a polypeptide having the function of human FGF23, or the amino acid sequence of NCBI accession number NP_065689.
• Examples include polypeptides that have the same structure as human FGF23 and have the functions of human FGF23.
• a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown by NCBI accession number NP_065689 can be obtained by site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 ( 1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. It can be obtained by introducing site-directed mutations into DNA encoding a polypeptide containing the amino acid sequence shown by NP_065689.
• the number of amino acids to be deleted, substituted or added is not particularly limited, but preferably 1 to several tens of amino acids, for example 1 to 20, more preferably 1 to several, for example 1 to 5 amino acids. It is.
• Examples of the gene encoding human FGF23 include the base sequence with NCBI accession number NM_020638.
• a gene consisting of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of NM_020638, and containing DNA encoding a polypeptide having the function of human FGF23, at least 60% or more of the base sequence of NM_020638 , preferably 80% or more homology, more preferably 90% or more homology, and most preferably 95% or more homology.
• a gene encoding human FGF23 of the present invention also includes a gene encoding human FGF23 of the present invention.
• DNA that hybridizes under stringent conditions colony hybridization method, plaque hybridization method, Southern blot hybridization method, DNA microarray method, etc. using DNA containing the base sequence of NM_020638 as a probe can be used. Refers to the resulting hybridizable DNA.
• hybridizable DNA examples include DNA that has at least 60% homology with the base sequence of NM_020638, preferably DNA that has 80% or more homology, and more preferably 95% or more homology. be able to
• Genetic polymorphisms are often observed in the base sequences of genes encoding eukaryotic proteins. Genes used in the present invention that have small-scale mutations in their base sequences due to such polymorphisms are also included in the gene encoding human FGF23 of the present invention.
• the numerical value of homology in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art.
• amino acid sequences such as numerical values calculated using default parameters in ., 215, 403 (1990)]
• BLAST2 Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649 (1997). )] using default parameters.
• the default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, -E (Cost to extend gap) is 2 if it is a base sequence, and 1 if it is an amino acid sequence.
• -q (Penalty for nucleotide mismatch) is -3
• -r (reward for nucleotide match) is 1
• -e (expect value) is 10
• -W (wordsize) ) is the base sequence
• there are 11 residues and the amino acid sequence , 3 residues for -y [Dropoff(X) for blast extensions in bits], 20 for blastn, 7 for programs other than blastn
• -X(X dropoff value for gapped al ignment bits) is 15
• - Z final X dropoff value for gapped alignment in bits
• a polypeptide comprising a partial sequence of the amino acid sequence of NCBI Accession No. NP_065689 can be produced by methods known to those skilled in the art. Specifically, it can be produced by deleting a portion of the DNA encoding the amino acid sequence of NP_065689 and culturing a transformant into which an expression vector containing the deletion is introduced. Furthermore, a polypeptide having an amino acid sequence with one or more amino acids deleted, substituted, or added to the amino acid sequence of NCBI Accession No. NP_065689 can be obtained by a method similar to that described above. Furthermore, polypeptides consisting of the amino acid sequence of NCBI Accession No.
• NP_065689 or polypeptides having an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of NCBI Accession No. NP_065689, are It can also be produced by chemical synthesis methods such as the oxycarbonyl (Fmoc) method and the t-butyloxycarbonyl (tBoc) method.
• Fmoc oxycarbonyl
• tBoc t-butyloxycarbonyl
• amino acid deletion, substitution, addition, etc. are also referred to as amino acid modifications.
• the antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and oligoclonal antibodies.
• Polyclonal antibodies refer to a population of antibody molecules secreted by antibody-producing cells of different clones.
• a monoclonal antibody is an antibody secreted by a single clone of antibody-producing cells, which recognizes only one epitope (also called an antigenic determinant) and whose amino acid sequence (primary sequence) that constitutes the monoclonal antibody is uniform.
• An oligoclonal antibody refers to a population of antibody molecules that is a mixture of multiple different monoclonal antibodies.
• Monoclonal antibodies in the present invention include antibodies produced by hybridomas or genetically recombinant antibodies produced by transformants transformed with expression vectors containing antibody genes.
• the epitope includes a single amino acid sequence, a three-dimensional structure consisting of an amino acid sequence, an amino acid sequence modified by post-translational modification, and a three-dimensional structure consisting of the amino acid sequence, which are recognized and bound by a monoclonal antibody.
• Amino acid sequences modified by post-translational modification include O-linked sugar chains that have OH substituents and are bonded to Tyr and Ser, N-linked sugar chains that are bonded to Gln and Asn that have NH2 substituents, and Examples include amino acid sequences in which a sulfuric acid molecule has a sulfuric acid group bonded to Tyr and Ser having an OH substituent group.
• the binding of the antibody of the present invention to human FGF23 can be confirmed by measuring the binding of the antibody of the present invention to human FGF23 using ELISA, surface plasmon resonance, and the like.
• known immunological detection methods [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] and so on.
• the amino acid residues or epitopes of human FGF23 to which the antibody of the present invention binds include a deletion form in which a part of the domain of human FGF23 is deleted, a mutant in which a domain derived from another protein is substituted, and a partial peptide fragment of human FGF23. It can be determined by conducting an antibody binding experiment using, for example,
• the amino acid residue or epitope of human FGF23 to which the antibody of the present invention binds can be determined by adding the antibody of the present invention to a peptide fragment of human FGF23 digested with a proteolytic enzyme, and performing epitope mapping using known mass spectrometry. It can also be determined by doing
• Antibody molecules are also called immunoglobulins (hereinafter referred to as Ig), and human antibodies are classified into IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM isotypes according to differences in molecular structure. be done.
• IgG1, IgG2, IgG3 and IgG4, which have relatively high amino acid sequence homology, are also collectively referred to as IgG.
• Antibody molecules are composed of polypeptides called heavy chains (hereinafter referred to as H chains) and light chains (hereinafter referred to as L chains).
• the H chain consists of VH and H chain constant regions (also referred to as CH) from the N-terminal side
• the L chain consists of VL and L chain constant regions (also referred to as CL) from the N-terminal side.
• CH is further composed of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain from the N-terminal side.
• a domain refers to a functional structural unit that constitutes each polypeptide of an antibody molecule.
• the CH2 domain and CH3 domain are collectively referred to as the Fc region or simply Fc.
• CL is known as a C ⁇ chain and a C ⁇ chain.
• the CH1 domain, hinge domain, CH2 domain, CH3 domain and Fc region in the present invention are referred to as the EU index [Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] (hereinafter simply EU index). It can be specified by the amino acid residue number from the N-terminus. Specifically, CH1 is the amino acid sequence of EU index numbers 118 to 215, Hinge is the amino acid sequence of EU index numbers 216 to 230, CH2 is the amino acid sequence of EU index numbers 231 to 340, and CH3 is the amino acid sequence of EU index numbers 341 to 447. Each amino acid sequence is specified.
• the antibodies of the present invention include, in particular, genetically engineered recombinant mouse antibodies, recombinant rat antibodies, recombinant rabbit antibodies, human chimeric antibodies (hereinafter simply referred to as chimeric antibodies), and humanized antibodies (human Also included are recombinant antibodies such as complementarity determining region CDR-grafted antibodies) and human antibodies.
• the antibodies of the present invention also include genetically recombinant antibodies produced by recombining H chains (or VH) and L chains (or VL) derived from two different types of antibodies. The two different types of antibodies may be any of hybridoma-derived monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
• the antibodies of the present invention also include genetically recombinant antibodies in which appropriate amino acid residues are substituted when producing the above-mentioned genetically recombinant antibodies.
• Chimeric antibody refers to an antibody consisting of the VH and VL of an antibody from a non-human animal (non-human animal) and the CH and CL of a human antibody.
• non-human animal any animal can be used as long as it is possible to produce a hybridoma, such as mouse, rat, hamster, rabbit, etc.
• a hybridoma is a cell that produces a monoclonal antibody with desired antigen specificity and is obtained by fusing B cells obtained by immunizing a non-human animal with an antigen and myeloma cells derived from a mouse or the like. means. Therefore, the variable region of the antibody produced by the hybridoma consists of the amino acid sequence of a non-human animal antibody.
• a chimeric antibody is produced by obtaining cDNA encoding the VH and VL of a monoclonal antibody from a hybridoma derived from non-human animal cells that produce the monoclonal antibody, and using an expression vector for animal cells having DNA encoding the CH and CL of a human antibody. It is possible to construct a human chimeric antibody expression vector by inserting the antibody into each antibody, and to express and produce the antibody by introducing it into animal cells.
• a humanized antibody refers to an antibody in which the amino acid sequences of the VH and VL CDRs of a non-human animal antibody are grafted onto the corresponding CDRs of the VH and VL of a human antibody.
• the area other than the CDR of VH and VL is called a framework area (hereinafter referred to as FR).
• a humanized antibody consists of a cDNA encoding a VH amino acid sequence consisting of the amino acid sequence of the VH CDR of a non-human animal antibody and the amino acid sequence of the FR of the VH of any human antibody, and the amino acid sequence of the VL CDR of the non-human animal antibody.
• a cDNA encoding the amino acid sequence of VL consisting of the sequence and the amino acid sequence of the FR of VL of an arbitrary human antibody is constructed, and cDNA is inserted into an expression vector for animal cells containing DNA encoding CH and CL of a human antibody, respectively, to produce a human antibody.
• the antibody can be expressed and produced by constructing a modified antibody expression vector and introducing it into animal cells.
• Human antibodies originally refer to antibodies that naturally exist in the human body, but human antibody phage libraries and human antibody-producing transducers created through recent advances in genetic engineering, cell engineering, and developmental engineering technologies are also used. Also included are antibodies obtained from genetic animals.
• Human antibodies can be obtained by immunizing mice carrying human immunoglobulin genes (Tomizuka K. et al., Proc Natl Acad Sci U S A. 97, 722-7, 2000) with the desired antigen. I can do it.
• human antibodies can be obtained without immunization by selecting human antibodies with the desired binding activity ( Winter G. et al., Annu Rev Immunol.12:433-55. 1994).
• immortalizing human B cells using EB virus cells that produce human antibodies with desired binding activity can be created and human antibodies can be obtained (Rosen A. et al., Nature 267, 52-54.1977).
• Antibodies present in the human body can be obtained, for example, by immortalizing lymphocytes isolated from human peripheral blood by infecting them with EB virus, etc., and then cloning them to obtain lymphocytes that produce the antibodies.
• the antibody can be purified from a culture in which the lymphocytes are cultured.
• a human antibody phage library is a phage library in which antibody fragments such as Fab and scFv are expressed on the surface by inserting antibody genes prepared from human B cells into phage genes. Phage expressing antibody fragments having a desired antigen-binding activity can be recovered from the library using the binding activity to a substrate on which an antigen is immobilized as an indicator.
• the antibody fragment can also be converted by genetic engineering techniques into a human antibody molecule consisting of two complete H chains and two complete L chains.
• a human antibody-producing transgenic animal is an animal in which a human antibody gene has been integrated into the chromosome of a host animal.
• human antibody-producing transgenic animals can be produced by introducing human antibody genes into mouse ES cells, transplanting the ES cells into early embryos of other mice, and then allowing them to develop.
• the method for producing human antibodies from human antibody-producing transgenic animals is to obtain human antibody-producing hybridomas using the hybridoma production method normally used in mammals other than humans, and to culture them to produce human antibodies in the culture. It can be produced and accumulated.
• the amino acid sequences of VH and VL of the antibody of the present invention may be any of the amino acid sequences of VH and VL of a human antibody, a non-human animal antibody, or a humanized antibody.
• the amino acid sequence of CL in the antibody of the present invention may be the amino acid sequence of a human antibody or a non-human animal antibody, but preferably the amino acid sequence of human antibody C ⁇ or C ⁇ .
• the CH of the antibody of the present invention may be of any type as long as it belongs to immunoglobulin, but subclasses belonging to the IgG class, such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), and ⁇ 4 (IgG4), are preferable.
• subclasses belonging to the IgG class such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), and ⁇ 4 (IgG4), are preferable.
• the antibodies of the present invention include Fc fusion proteins in which Fc is bound to an antibody fragment, Fc fusion proteins in which Fc is bound to a naturally occurring ligand or receptor (also referred to as immunoadhesin), and antibodies in which multiple Fc regions are fused. It also includes Fc fusion proteins and the like.
• the antibodies or antibody fragments of the present invention include antibodies or antibody fragments containing any post-translationally modified amino acid residues.
• Post-translational modifications include, for example, deletion of a lysine residue at the C-terminus of the H chain (lysine clipping) or conversion of a glutamine residue to pyroglutamine (pyroGlu) at the N-terminus of a polypeptide. [Beck et al, Analytical Chemistry, 85, 715-736 (2013)].
• an antibody fragment refers to a human antibody whose decomposition at low pH is suppressed compared to an antibody containing a VH containing the amino acid sequence represented by SEQ ID NO: 1 and a VL containing the amino acid sequence represented by SEQ ID NO: 2.
• This is an antibody fragment that binds to FGF23.
• antibody fragments include Fab, Fab', F(ab') 2 , single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv), or multiple CDR Examples include peptides containing.
• Fab is a fragment obtained by treating an IgG antibody with the protease papain (cleaved at the 224th amino acid residue of the H chain), and approximately half of the N-terminal side of the H chain and the entire L chain have disulfide bonds. It is an antibody fragment with a molecular weight of approximately 50,000 and having antigen-binding activity, which is bonded through (SS bond).
• F(ab') 2 is one of the fragments obtained by treating IgG with the protease pepsin (cleaved at the 234th amino acid residue of the H chain). It is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 100,000, which is slightly larger than the antibody fragment bound to the antibody.
• Fab' is an antibody fragment with a molecular weight of about 50,000 and an antigen-binding activity obtained by cleaving the SS bond in the hinge region of F(ab') 2 .
• the scFv is produced using an appropriate peptide linker (P) such as a linker peptide that connects one VH and one VL with an arbitrary number of linkers (G4S) consisting of 4 Gly and 1 Ser residues. It is a VH-P-VL or VL-P-VH polypeptide linked together, and is an antibody fragment having antigen-binding activity.
• P peptide linker
• G4S arbitrary number of linkers
• Diabody is an antibody fragment in which scFvs with the same or different antigen binding specificities form a dimer, and is an antibody fragment that has bivalent antigen-binding activity to the same antigen or specific antigen-binding activity to different antigens.
• dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is replaced with a cysteine residue, and the polypeptides are linked via an SS bond between the cysteine residues.
• a CDR-containing peptide is comprised of at least one region of a VH or VL CDR.
• the CDRs can be linked directly or via a suitable peptide linker.
• a DNA encoding the VH and VL CDRs of the modified antibody of the present invention is constructed, the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is introduced into the prokaryote or eukaryote. It can be expressed and manufactured by Peptides containing CDRs can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
• One embodiment of the antibody of the present invention includes the following antibodies (i) and (ii) or antibody fragments thereof.
• (i) In an antibody that binds to FGF23 containing a VH containing the amino acid sequence represented by SEQ ID NO: 1 and a VL containing the amino acid sequence represented by SEQ ID NO: 2, at least the amino acid sequence represented by SEQ ID NO: 1 in the VH
• the 100th amino acid residue is selected from alanine residue, asparagine residue, glycine residue, tyrosine residue, arginine residue, aspartic acid residue, histidine residue, tryptophan residue, and methionine residue.
• an antibody that binds to FGF23 which includes a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2, at least the amino acid sequence represented by SEQ ID NO: 1 in the VH
• the 105th amino acid residue is an alanine residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, a proline residue, a glutamine residue, an arginine residue.
• An antibody or an antibody fragment thereof that binds to FGF23 substituted with one amino acid residue selected from a valine residue, a tryptophan residue, a tyrosine residue, a threonine residue, an asparagine residue, and a serine residue.
• the antibody has higher stability at low pH and suppresses antibody degradation compared to antibodies containing a VH containing the amino acid sequence represented by SEQ ID NO: 1 and a VL containing the amino acid sequence represented by SEQ ID NO: 2.
• the antibody has at least the 100th position of the amino acid sequence represented by SEQ ID NO: 1 in the VH.
• the antibody binds to FGF23 in which the amino acid residue is replaced with an alanine residue or a tyrosine residue.
• low pH refers to weak acidity or acidity with a pH of less than 6, and includes, for example, pH 5, pH 4.5, pH 4, etc., but is not particularly limited.
• the antibody of the present invention has suppressed decomposition at low pH and has excellent stability compared to antibodies containing a VH containing the amino acid sequence represented by SEQ ID NO: 1 and a VL containing the amino acid sequence represented by SEQ ID NO: 2. Show your gender.
• antibody degradation can be measured by size exclusion chromatography (hereinafter referred to as SEC) or SDS polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE). can.
• the antibody of the present invention degradation of the antibody at low pH is suppressed compared to an antibody containing a VH containing the amino acid sequence represented by SEQ ID NO: 1 and a VL containing the amino acid sequence represented by SEQ ID NO: 2.
• This can be confirmed, for example, by a method including the following steps (I) to (III), but is not particularly limited to this method.
• the solvent of the antibody solution containing the antibody of the present invention or the antibody containing the VH containing the amino acid sequence represented by SEQ ID NO: 1 and the VL containing the amino acid sequence represented by SEQ ID NO: 2 is NAP (trademark). Using a column such as No.
• an antibody solution with pH 4, 4.5, or 5 substituted with an appropriate solvent is prepared.
• (II) After allowing the antibody solution prepared in (I) above to stand at 40 degrees for 1 month or 2 weeks or at 25 degrees for 3 months, the peak (%) corresponding to the antibody degradation products was detected using SEC. To detect.
• SEC SEC
• the peak (%) corresponding to the decomposition product of the antibody detected by SEC in (II) above contains the VH containing the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2. If the antibody of the present invention is reduced in intensity than the antibody containing VL, or if the density of the band around 40 kDa in SDS-PAGE is lower than that of the antibody containing VL, the concentration of the band near 40 kDa is lower than that of the antibody containing VL, or if the density of the band around 40 kDa is lower than that of the antibody containing VL, If the antibody of the present invention is lower than the antibody containing the VL containing the amino acid sequence represented by SEQ ID NO: 1, the VL containing the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2. It can be confirmed that the antibody of the present invention suppresses the decomposition of the antibody at low pH compared to the antibody including the above-menti
• the peak area ratio (%) of a band corresponding to a degraded product of the antibody (for example, 40 to 60 kDa) detected at a size about 10 to 20 kDa smaller than the peak of the full length of the H chain is , when the antibody of the present invention is lowered than the antibody comprising the VH comprising the amino acid sequence represented by SEQ ID NO: 1 and the VL comprising the amino acid sequence represented by SEQ ID NO: 2, It can be confirmed that the antibody of the present invention suppresses the degradation of the antibody at low pH compared to the antibody containing the VH containing the amino acid sequence represented by SEQ ID NO: 2 and the VL containing the amino acid sequence represented by SEQ ID NO: 2.
• examples of aspects showing the stability of the antibody of the present invention at low pH include the following. - At a low pH such as pH 4, 4.5 or 5 and at 40 degrees, compared to an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2. After standing for 1 month, 2 weeks, or 3 months at 25 degrees, the density of a band with a molecular weight around 40 kDa as determined by SDS-PAGE has decreased. The density of the band can be evaluated visually.
• the peak (%) corresponding to the decomposed product of the antibody detected by SEC has decreased.
• the peak (%) is 5% or more, 10% or more, and 20% compared to the antibody containing the VH containing the amino acid sequence represented by SEQ ID NO: 1 and the VL containing the amino acid sequence represented by SEQ ID NO: 2.
• the reduction is preferably 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
• a low pH such as pH 4, 4.5 or 5 and at 40 degrees, compared to an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2.
• H The peak area ratio (%) of a band (eg, 40 to 60 kDa) corresponding to a decomposition product of the antibody detected to be about 10 to 20 kDa smaller than the peak of the full chain length is decreased.
• the peak area ratio (%) is 5% or more, 10% or more, 20% higher than that of an antibody containing a VH containing the amino acid sequence represented by SEQ ID NO: 1 and a VL containing the amino acid sequence represented by SEQ ID NO: 2.
• the reduction is preferably 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.
• One embodiment of the antibody of the present invention includes the above-mentioned antibody further comprising one substitution selected from (a1) to (a4) below.
• At least one substitution selected from substitution with a histidine residue, substitution of the 57th amino acid residue with a threonine residue, and substitution of the 58th amino acid residue with a phenylalanine residue (a2) Substitution of the 91st amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL with a methionine or leucine residue, substitution of the 92nd amino acid residue with a tyrosine residue, substitution of the 94th amino acid residue with a tyrosine residue, at least one substitution selected from the substitution of an amino acid residue with an aspartic acid residue, and the substitution of the 96th amino acid residue with an asparagine residue or an aspartic acid residue; (a3) Substitution of the 28th amino acid residue of the amino acid sequence represented by SEQ ID NO: 2 in VL with an aspartic acid residue, substitution of the 29th amino acid residue with a valine residue, and 31st amino acid residue at least one substitution selected from substitution of a threon
• the antibody of the present invention can improve the FGF23-binding activity and FGF23-neutralizing activity of the antibody by substituting one amino acid residue selected from (a1) to (a4) above.
• FGF23 neutralizing activity refers to an activity that inhibits a signal generated when FGF23 binds to a receptor. Examples of receptors for FGF23 include a complex between FGFR1 and ⁇ Klotho.
• the FGF23 neutralizing activity of the antibody of the present invention can be confirmed by the reporter assay (also referred to as promoter assay) described in Nature 2006 Dec 7; 444 (7120). Furthermore, the FGF23 neutralizing activity of the antibody of the present invention can be confirmed by reporter assay using ⁇ Klotho stably expressing HEK293 cells transformed with a luciferase expression vector having a promoter derived from the mouse Egr1 gene.
• antibodies of the present invention include any one antibody selected from the following (b1) to (b5).
• antibodies of the present invention include antibodies selected from the following (c1) to (c10), with (c1), (c8), (c9) or (c10) being preferred, and (c1) or (c10) being preferred. c9) is more preferred.
• (c1) an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 39 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2;
• (c2) an antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 47 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2;
• (c5) an antibody comprising a V
• an Fc region in which amino acid residues are substituted can also be used in order to control binding to FcRn for the purpose of controlling blood half-life.
• Examples of the Fc region in which amino acid residues are substituted to improve the binding of antibodies to FcRn include any one of the Fc regions (d1) to (d5) below.
• the Fc region (d1) is preferred.
• (d1) Fc containing a substitution of the 252nd amino acid residue in the EU index with a tyrosine residue, the 254th amino acid residue with a threonine residue, and the 256th amino acid residue with a glutamic acid residue region
• (d3) an Fc region containing a substitution of the 308th amino acid residue of the EU index with a proline residue
• (d4) Fc region containing the substitution of the 250th amino acid residue of the EU index with a glutamine residue and the substitution of the 428th amino acid residue with a leucine residue, and
• amino acid sequences of heavy chain constant regions containing Fc regions in which amino acid residues have been substituted in order to improve binding of antibodies to FcRn include SEQ ID NOs: 48, 49, 50, Examples include amino acid sequences represented by 51 and 52. Among these, the amino acid sequence represented by SEQ ID NO: 48 is preferred.
• the monoclonal antibody of the present invention or the antibody fragment of the present invention that binds to human FGF23 of the present invention may be chemically or Includes genetically engineered antibodies or derivatives of antibody fragments thereof.
• the antibody or the derivative of the antibody fragment is a monoclonal antibody that binds to human FGF23 of the present invention, or the N-terminal side or C-terminal side of the H chain or L chain of the antibody fragment, an appropriate substituent or side chain in the antibody molecule, or Binding of radioactive isotopes, low-molecular drugs, high-molecular drugs, immunostimulants, proteins, antibody drugs, nucleic acid drugs, etc. to sugar chains etc. by chemical methods [Introduction to Antibody Engineering, Chijinshokan (1994)] It can be manufactured by
• a DNA encoding a monoclonal antibody that binds to human FGF23 of the present invention or an antibody fragment thereof and a DNA encoding a protein or antibody drug to be bound are ligated and inserted into an expression vector, and the expression vector is inserted into an appropriate host. It can be produced by genetic engineering techniques in which it is introduced into cells and expressed.
• radioactive isotope examples include 111 In, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, and 211 At. Radioactive isotopes can be directly attached to antibodies, such as by the chloramine T method. Furthermore, a substance that chelates a radioactive isotope may be bound to the antibody. Examples of the chelating agent include 1-isothiocyanatebenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).
• low-molecular drugs examples include alkylating agents, nitrosoureas, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, and platinum.
• Complex derivatives, anticancer drugs such as M-phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy Co., Ltd.
• anticancer drugs examples include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), and doxorubicin.
• Examples of methods for bonding a low-molecular drug and an antibody include bonding the amino group of the drug and antibody via glutaraldehyde, or bonding the amino group of the drug and the carboxyl group of the antibody via water-soluble carbodiimide. Examples include a method of binding.
• polymeric drugs examples include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, or hydroxypropyl methacrylamide.
• methods for binding PEG and antibodies include a method of reacting with a PEGylation modification reagent [Bioconjugate Pharmaceuticals, Hirokawa Shoten (1993)].
• PEGylation modification reagents include modifiers for modifying the ⁇ -amino group of lysine (Japanese Unexamined Patent Publication No. 61-178926), modifying agents for the carboxyl group of aspartic acid and glutamic acid (Japanese Unexamined Patent Publication No. 56-23587); (Japanese Unexamined Patent Publication No. 2-117920), or a modifier for the guanidino group of arginine (Japanese Unexamined Patent Publication No. 2-117920).
• the immunostimulant may be a natural product known as an immunoadjuvant; for example, the immunostimulatory agent may be ⁇ (1 ⁇ 3) glucan (e.g. lentinan or schizophyllan) or ⁇ -galactosylceramide (KRN7000). ), etc.
• the immunostimulatory agent may be ⁇ (1 ⁇ 3) glucan (e.g. lentinan or schizophyllan) or ⁇ -galactosylceramide (KRN7000). ), etc.
• proteins include cytokines, growth factors, and toxin proteins that activate immunocompetent cells such as NK cells, macrophages, and neutrophils.
• cytokines or growth factors examples include interferon (hereinafter referred to as IFN)- ⁇ , IFN- ⁇ , IFN- ⁇ , interleukin (hereinafter referred to as IL)-2, IL-12, IL-15, IL- 18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (M-CSF).
• IFN interferon
• IFN- ⁇ interleukin-2
• IL-12 interleukin
• IL-15 interleukin
• IL-15 interleukin- 18, IL-21
• IL-23 granulocyte colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• GM-CSF granulocyte/macrophage colony stimulating factor
• M-CSF macrophage colony stimulating factor
• examples of the toxin protein include ricin, diphtheria tox
• antibody drugs include antibodies against antigens that induce apoptosis upon antibody binding, antigens that are involved in tumor pathogenesis, antigens that regulate immune function, and antigens that are involved in angiogenesis at diseased sites.
• antigens that induce apoptosis upon antibody binding include cluster of differentiation (hereinafter referred to as CD) 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA)-Class II or Ep idermal Growth Factor Receptor (EGFR ), etc.
• CD cluster of differentiation
• antigens involved in tumor pathogenesis or antigens of antibodies that regulate immune function include CD4, CD40, CD40 ligand, B7 family molecules (e.g., CD80, CD86, CD274, B7-DC, B7-H2, B7- H3 or B7-H4), ligands of the B7 family of molecules (e.g. CD28, CTLA-4, ICOS, PD-1 or BTLA), OX-40, OX-40 ligand, CD137, the tumor necrosis factor (TNF) receptor family molecules (e.g.
• cytokine e.g., IL-1 ⁇ , IL-1 ⁇ , IL-4, IL-5, IL-6 , IL -10, IL-13,
• Antigens for antibodies that inhibit angiogenesis at lesion sites include, for example, Vascular Endothelial Growth Factor (VEGF), angiopoietin, Fibroblast Growth Factor (FGF), EGF, Hepatocyte te Growth Factor (HGF), Platelet-Derived Growth Factor (PDGF) , Insulin-like Growth Factor (IGF), erythropoietin (EPO), TGF ⁇ , IL-8, ephrin or SDF-1, or their receptors.
• VEGF Vascular Endothelial Growth Factor
• FGF Fibroblast Growth Factor
• EGF Hepatocyte te Growth Factor
• HGF Hepatocyte te Growth Factor
• PDGF Platelet-Derived Growth Factor
• IGF Insulin-like Growth Factor
• EPO erythropoietin
• TGF ⁇ IL-8, ephrin or SDF-1, or their receptors.
• nucleic acid medicines include medicines containing nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA, which act on living bodies by controlling gene functions.
• nucleic acids such as small interference ribonucleic acid (siRNA) or microRNA, which act on living bodies by controlling gene functions.
• siRNA small interference ribonucleic acid
• microRNA microRNA
• a conjugate with a nucleic acid drug that suppresses the master transcription factor ROR ⁇ t of Th17 cells can be considered.
• the drug that binds to the antibody is a drug that is used in ordinary immunological detection or measurement methods.
• labeled substances include enzymes such as alkaline phosphatase, peroxidase, or luciferase, luminescent substances such as acridinium ester or lophine, or fluorescent substances such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC). Can be mentioned.
• one embodiment of the present invention is a composition comprising the antibody of the present invention or the antibody fragment.
• the composition include compositions containing a monoclonal antibody that binds to human FGF23 or a fragment thereof as an active ingredient.
• a composition comprising the antibody of the present invention or the antibody fragment can be used to treat human FGF23-related diseases.
• a therapeutic agent for human FGF23-related diseases is provided, which comprises the antibody of the present invention.
• the present invention also relates to a method for treating human FGF23-related diseases, which includes administering a monoclonal antibody that binds to human FGF23 or an antibody fragment thereof.
• the human FGF23-related disease may be any disease that involves human FGF23 or human FGF23 receptors, such as tumor osteomalacia (TIO), autosomal dominant hypophosphatemic rickets, Osteomalacia (ADHR), X-linked hypophosphatemia (XLH), fibrous dysplasia, McCune-Albright syndrome, autosomal recessive hypophosphatemic rickets/osteomalacia (ARHR), osteoporosis, rickets (including hypophosphatemic rickets and vitamin D-resistant rickets), hypercalcemia, hypocalcemia, ectopic calcification, osteosclerosis, Paget's disease, hyperparathyroidism, parathyroid glands
• diseases associated with renal failure and artificial dialysis during renal failure such as hypofunction, pruritus, renal osteodystrophy, dialysis osteopathy, and renal tubular dysfunction.
• the present invention also provides a method for treating hypophosphatemia found in diseases such as TIO, ADHR, XLH, fibrous dysplasia, McCune-Albright syndrome, and ARHR, which contains a monoclonal antibody that binds to human FGF23 or a fragment thereof as an active ingredient. It also includes therapeutic agents for symptoms such as bone mineralization deficiency, bone pain, muscle weakness, skeletal deformities, growth disorders, and low 1,25 vitamin D levels.
• Therapeutic agents containing the antibody or antibody fragment of the present invention may contain only the antibody or antibody fragment as an active ingredient, but usually together with one or more pharmacologically acceptable carriers. It is preferable to provide a pharmaceutical preparation prepared by any method known in the pharmaceutical field.
• examples include oral administration, and parenteral administration such as intraorally, intratracheally, intrarectally, subcutaneously, intramuscularly, or intravenously, and preferably intravenous administration.
• parenteral administration such as intraorally, intratracheally, intrarectally, subcutaneously, intramuscularly, or intravenously, and preferably intravenous administration.
• examples include internal administration.
• dosage forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, and tapes.
• the dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 ⁇ g/kg to 10 mg/kg per day for adults.
• composition of the present invention includes a reagent for detecting or measuring FGF23, which contains a monoclonal antibody that binds to human FGF23 or a fragment thereof.
• the present invention also relates to a method for detecting or measuring FGF23 using a monoclonal antibody that binds to human FGF23 or a fragment thereof.
• methods for detecting or measuring human FGF23 include any known method. Examples include immunological detection or measurement methods.
• An immunological detection or measurement method is a method of detecting or measuring the amount of antibodies or antigens using labeled antigens or antibodies.
• Immunological detection or measurement methods include, for example, radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescent immunoassay, Western Examples include blotting method or physicochemical method.
• composition of the present invention includes a diagnostic agent for FGF23-related diseases, which includes a monoclonal antibody that binds to human FGF23 or a fragment thereof.
• the present invention also relates to a method for diagnosing FGF23-related diseases, which includes detecting or measuring FGF23 using a monoclonal antibody that binds to human FGF23 or a fragment thereof. By detecting or measuring human FGF23 using the monoclonal antibody of the present invention or the antibody fragment according to the method described above, a disease related to human FGF23 can be diagnosed.
• biological samples to be detected or measured for human FGF23 may include tissues, cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluids, or culture fluids that may contain human FGF23. There is no particular limitation as long as it is.
• a diagnostic agent containing the monoclonal antibody of the present invention or an antibody fragment thereof may include a reagent for performing an antigen-antibody reaction and a reagent for detecting the reaction, depending on the intended diagnostic method.
• Reagents for performing antigen-antibody reactions include buffers, salts, and the like.
• Detection reagents include reagents used in conventional immunological detection or measurement methods, such as a labeled secondary antibody that recognizes the monoclonal antibody or its antibody fragment, or a substrate compatible with a label.
• One embodiment of the present invention relates to the use of anti-human FGF23 monoclonal antibodies or antibody fragments for the production of therapeutic or diagnostic agents for FGF23-related diseases. Further, one embodiment of the present invention relates to a method for treating or diagnosing FGF23-related diseases.
• Human FGF23 as an antigen is obtained by introducing an expression vector containing a cDNA encoding full length human FGF23 or a partial length thereof into Escherichia coli, yeast, insect cells, animal cells, etc. be able to.
• human FGF23 can also be obtained by purifying human FGF23 from various human cell lines, human cells, human tissues, etc. that express large amounts of human FGF23. Further, these human cell lines, human cells, human tissues, etc. can also be used as antigens as they are.
• a synthetic peptide having a partial sequence of human FGF23 can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method and used as an antigen.
• a known tag such as FLAG or His may be added to the C-terminus or N-terminus of human FGF23 or a synthetic peptide having a partial sequence of human FGF23.
• Human FGF23 used in the present invention can be obtained by methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols In Molecular Biology, John Wiley & Sons (1987-1997), etc.
• the human FGF23-encoding DNA can be expressed and produced in a host cell using, for example, the following method.
• a recombinant vector is constructed by inserting a full-length cDNA containing a portion encoding human FGF23 downstream of the promoter of an appropriate expression vector.
• a DNA fragment of an appropriate length containing a polypeptide-encoding portion prepared based on the full-length cDNA may be used.
• a transformant that produces the polypeptide can be obtained.
• Any expression vector can be used as long as it is capable of autonomous replication in the host cell used or integration into the chromosome, and contains an appropriate promoter at a location where the DNA encoding the polypeptide can be transcribed. Can be done.
• the host cell any cell that can express the gene of interest can be used, such as microorganisms belonging to the genus Escherichia such as Escherichia coli, yeast, insect cells, or animal cells.
• the recombinant vector When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in the prokaryote, and at the same time contains a promoter, a ribosome binding sequence, a DNA containing a portion encoding human FGF23, and a transcription termination sequence.
• a vector containing the following is preferred.
• the recombinant vector does not necessarily have a transcription termination sequence, it is preferable to place the transcription termination sequence immediately below the structural gene.
• the recombinant vector may contain a gene that controls a promoter.
• the recombinant vector it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also referred to as SD sequence), which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
• SD sequence also referred to as SD sequence
• start codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
• bases can be substituted so as to become optimal codons for expression within the host, thereby improving the production rate of the target human FGF23. Can be done.
• Any expression vector can be used as long as it can function in the host cell used, such as pBTrp2, pBTac1, pBTac2 (manufactured by Roche Diagnostics), pKK233-2 ( pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Unexamined Patent Publication No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad.
• Any promoter may be used as long as it can function in the host cell used.
• promoters derived from E. coli or phage such as the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter.
• examples include artificially designed promoters such as a tandem promoter in which two Ptrps are arranged in series, a tac promoter, a lacT7 promoter, or a let I promoter.
• Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, or E. coli DH5 ⁇ .
• any method that introduces DNA into the host cell to be used can be used.
• a method using calcium ions Proc. Natl. Acad. Sci. USA , 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].
• any expression vector can be used as long as it can function in animal cells, such as pcDNAI, pCDM8 (manufactured by Funakoshi), pAGE107 [Japanese Patent Publication No. 3] -22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Unexamined Patent Publication No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (manufactured by Invitrogen) , pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), pAGE103 [J.
• Any promoter can be used as long as it can function in animal cells; for example, the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the early promoter of SV40, and the promoter of retroviruses. , metallothionein promoter, heat shock promoter, SR ⁇ promoter, or Moloney murine leukemia virus promoter or enhancer. Furthermore, the enhancer of the human CMV IE gene may be used together with the promoter.
• IE immediate early
• CMV cytomegalovirus
• host cells examples include human leukemia cells Namalwa cells, monkey cells COS cells, Chinese hamster ovary cells CHO cells [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129(1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260(1997); Proc. Natl. Acad Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci.,60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp.
• CHO cells CHO/DG44 cells deficient in the dihydrofolate reductase gene (hereinafter referred to as dhfr) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)]
• CHO-K1 ATCC CCL-61
• DUkXB11 ATCC CCL-9096
• Pro-5 ATCC CCL-1781
• CHO-S Life Technologies, Cat#11619
• Pro-3 rat myeloma cells YB2/3HL. P2.
• mice 20 also referred to as YB2/0
• mouse myeloma cells NSO mouse myeloma cells SP2/0-Ag14
• Syrian hamster cells BHK or HBT5637 Japanese Patent Application Laid-Open No. 63-000299.
• any method for introducing DNA into animal cells can be used, such as electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method. (Japanese Unexamined Patent Publication No. 2-227075) or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
• a transformant derived from a microorganism or animal cell carrying a recombinant vector incorporating the DNA encoding human FGF23 obtained as described above is cultured in a medium, and the human FGF23 is produced and accumulated in the culture solution.
• Human FGF23 can be produced by culturing and collecting from the culture solution.
• the transformant can be cultured in a medium according to a conventional method used for culturing hosts.
• human FGF23 When expressed in cells derived from eukaryotes, human FGF23 with added sugars or sugar chains can be obtained.
• an inducer may be added to the medium as necessary.
• an inducer may be added to the medium as necessary.
• isopropyl- ⁇ -D-thiogalactopyranoside is used to culture a microorganism transformed with a recombinant vector using the trp promoter.
• indole acrylic acid or the like may be added to the medium.
• Examples of media for culturing transformants obtained using animal cells as hosts include the commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)] or Iscove's Modified Examples include Dulbecco's Medium (IMDM) medium and a medium prepared by adding fetal bovine serum (FBS) or the like to these mediums.
• IMDM Dulbecco's Medium
• FBS fetal bovine serum
• Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40°C, and the presence of 5% CO2 .
• an antibiotic such as kanamycin or penicillin may be added to the medium as necessary during culturing.
• Methods for expressing the gene encoding human FGF23 include, for example, in addition to direct expression, methods such as secretory production or fusion protein expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] are available. It will be done.
• Examples of methods for producing human FGF23 include a method for producing it within a host cell, a method for secreting it outside the host cell, and a method for producing it on the outer membrane of the host cell. By changing the structure, an appropriate method can be selected.
• human FGF23 When human FGF23 is produced within host cells or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci. ., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], by using the method described in Japanese Patent Application Publication No. 05-336963 or International Publication No. 94/23021, etc. Human FGF23 can be actively secreted outside host cells. Furthermore, the production amount of human FGF23 can also be increased using a gene amplification system using a dihydrofolate reductase gene (Japanese Unexamined Patent Publication No. 2-227075).
• the obtained human FGF23 can be isolated and purified, for example, as follows.
• human FGF23 When human FGF23 is expressed in a dissolved state within the cells, the cells are collected by centrifugation after the completion of culture, suspended in an aqueous buffer, and then homogenized using an ultrasonicator, French press, Manton-Gaulin homogenizer, Dynomill, etc. Disrupt the cells to obtain a cell-free extract.
• the supernatant obtained by centrifuging the cell-free extract is extracted using conventional protein isolation and purification methods, such as solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, and diethylaminoprotein extraction.
• a purified sample can be obtained by using either alone or in combination.
• human FGF23 is expressed by forming an insoluble form within cells, the cells are collected and disrupted in the same manner as above, and then centrifuged to recover the insoluble form of human FGF23 as a precipitate fraction.
• the recovered insoluble form of human FGF23 is solubilized with a protein denaturing agent. After returning the human FGF23 to its normal three-dimensional structure by diluting or dialyzing the solubilized solution, a purified preparation of the polypeptide can be obtained by the same isolation and purification method as described above.
• human FGF23 or a derivative such as a glycoform thereof When human FGF23 or a derivative such as a glycoform thereof is secreted extracellularly, the human FGF23 or a derivative such as a glycoform thereof can be recovered in the culture supernatant.
• a soluble fraction is obtained by treating the culture using techniques such as centrifugation in the same manner as above, and a purified sample can be obtained from the soluble fraction by using the same isolation and purification method as above. can.
• human FGF23 used in the present invention can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
• chemical synthesis can be performed using a peptide synthesizer manufactured by Advanced Chemtech, Perkin-Elmer, Pharmacia, Protein Technology Instruments, Synthecell-Vega, Perceptive, or Shimadzu Corporation. You can also.
• mice, rats, or hamsters aged 3 to 20 weeks are immunized with the antigen obtained in (1).
• a mouse FGF23 knockout mouse can also be used as an immunized animal.
• Immunization is carried out by administering the antigen subcutaneously, intravenously or intraperitoneally to the animal together with a suitable adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and Bordetella pertussis vaccine.
• a suitable adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and Bordetella pertussis vaccine.
• the antigen is a partial peptide
• a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin) is prepared and used as an immunogen.
• BSA bovine serum albumin
• KLH keyhole limpet hemocyanin
• the antigen is administered 5 to 10 times every 1 to 2 weeks.
• Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the antibody titer of the serum is measured using an enzyme immunoassay [Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
• An animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells for fusion.
• tissues containing antibody-producing cells such as the spleen
• the spleen is shredded and loosened, centrifuged, and red blood cells are removed to obtain antibody-producing cells for fusion.
• myeloma cells established cell lines obtained from mice are used, such as 8-azaguanine-resistant mouse (BALB/c-derived) myeloma cell line P3-X63Ag8-U1 (P3- U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1) [European J. Immunology, 6, 511 (1976)], SP2/0-Ag14 ( SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)] or P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] etc. are used.
• the myeloma cells were passaged in normal medium (RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine) and subcultured into normal medium 3-4 days before cell fusion. On the day of fusion, ensure a cell count of 2 x 10 7 or more.
• normal medium RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine
• HAT medium normal medium containing hypoxanthine, thymidine, and aminopterin. This suspension is cultured for 7-14 days at 37° C. in a 5% CO 2 incubator.
• a portion of the culture supernatant is removed, and a cell group that reacts with antigens containing human FGF23 and does not react with antigens that do not contain human FGF23 is selected using a hybridoma selection method such as binding assay described below.
• cloning is performed by the limiting dilution method, and hybridomas with stable and strong antibody titers are selected as monoclonal antibody-producing hybridomas.
• the supernatant is removed by centrifugation, suspended in Hybridoma SFM medium, and cultured for 3 to 7 days. It is also possible to obtain a purified monoclonal antibody by centrifuging the obtained cell suspension, purifying the obtained supernatant using a protein A column or protein G column, and collecting the IgG fraction. Note that 5% Daigo GF21 can also be added to the Hybridoma SFM medium.
• the antibody subclass is determined by enzyme immunoassay using a subclass typing kit. Quantification of protein amount is calculated by the Lowry method or absorbance at 280 nm.
• a test substance such as serum, hybridoma culture supernatant, or purified monoclonal antibody as a first antibody is dispensed and reacted.
• an anti-immunoglobulin antibody labeled with an enzyme reagent or the like as a second antibody is dispensed and reacted.
• a substrate is added and the extinction coefficient of each well is measured using a plate reader to select a monoclonal antibody that specifically reacts with human FGF23.
• a genetically recombinant antibody expression vector is an expression vector for animal cells into which DNA encoding CH and CL of human antibodies has been integrated. It can be constructed by cloning DNAs encoding CH and CL of antibodies, respectively.
• CH and CL of any human antibody can be used.
• CH of the ⁇ 1 subclass and CL of the ⁇ class of human antibodies are used.
• cDNA is used as the DNA encoding CH and CL of human antibodies
• chromosomal DNA consisting of exons and introns can also be used.
• Any animal cell expression vector can be used as long as it can integrate and express a gene encoding the C region of a human antibody. For example, pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci.
• Promoters and enhancers of expression vectors for animal cells include the SV40 early promoter [J. Biochem., 101, 1307 (1987)] and the Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 ( 1987)] or immunoglobulin heavy chain promoters [Cell, 41, 479 (1985)] and enhancers [Cell, 33, 717 (1983)].
• Recombinant antibody expression vectors are easy to construct, easy to introduce into animal cells, and have a well-balanced expression level of antibody H chain and L chain in animal cells. From the point of view of Immunol. Methods, 167, 271 (1994)], but a type in which the antibody H chain and L chain are present on separate vectors can also be used.
• vectors for expressing tandem recombinant antibodies pKANTEX93 (International Publication No. 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], etc. are used.
• mRNA is extracted from hybridoma cells producing non-human antibodies and cDNA is synthesized.
• the synthesized cDNA is cloned into a vector such as a phage or a plasmid to create a cDNA library.
• a recombinant phage or a recombinant plasmid having cDNA encoding VH or VL is isolated using DNA encoding the C region or V region of a mouse antibody as a probe, respectively.
• the entire nucleotide sequence of the VH or VL of the mouse antibody of interest on the recombinant phage or plasmid is determined, and the entire amino acid sequence of the VH or VL is deduced from the nucleotide sequence.
• the non-human animal used to produce hybridoma cells that produce non-human antibodies is a mouse, rat, hamster, rabbit, etc., but any animal can be used as long as it is possible to produce hybridoma cells.
• RNA from hybridoma cells use the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] or a kit such as RNA easy kit (manufactured by Qiagen).
• mRNA can also be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation (registered trademark) Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification (registered trademark) Kit (manufactured by Pharmacia).
• kit such as Fast Track mRNA Isolation (registered trademark) Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification (registered trademark) Kit (manufactured by Pharmacia).
• any vector that can incorporate cDNA synthesized using mRNA extracted from hybridoma cells as a template can be used as long as it can incorporate the cDNA.
• ZAP Express [Strategies, 5, 58 (1992)]
• pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)]
• ⁇ ZAPII (Stratagene)
• ⁇ gt10, ⁇ gt 11 [DNA Cloning:A Practical Approach, I, 49 (1985)]
• Lambda BlueMid (Clontech), ⁇ ExCell, pT7T3-18U (Pharmacia)
• pCD2 Mol. Cell. Biol., 3, 280 (1983)
• pUC18 Gene , 33, 103 (1985)
• E. coli that can introduce, express, and maintain the cDNA library can be used as the E. coli strain into which the cDNA library constructed using phage or plasmid vectors can be introduced.
• E. coli strain into which the cDNA library constructed using phage or plasmid vectors can be introduced.
• XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)], or JM105 [Gene, 38, 275 (1985)].
• primers are prepared and cDNA synthesized from mRNA or a cDNA library is used as a template to perform the Polymerase Chain Reaction method [hereinafter referred to as PCR method], Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). ), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)], cDNA encoding VH or VL can also be prepared.
• PCR method Polymerase Chain Reaction method
• telomere sequence analysis After cutting the selected cDNA with an appropriate restriction enzyme, etc., it is cloned into a plasmid such as pBluescript SK(-) (manufactured by Stratagene), and the base sequence of the cDNA is determined by a commonly used base sequence analysis method. .
• base sequence analysis for example, after performing a reaction such as the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], ABI PRISM3700 (manufactured by PE Biosystems) or A. L. F. An automatic base sequence analyzer such as a DNA sequencer (manufactured by Pharmacia) is used.
• the complete amino acid sequences of VH and VL are estimated from the determined base sequences, and compared with the complete amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] This confirms whether the obtained cDNA encodes the complete amino acid sequences of the antibody VH and VL, including the secretion signal sequence. Regarding the complete amino acid sequences of the VH and VL of the antibody, including the secretion signal sequence, compare them with the complete amino acid sequences of the VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept.
• Human chimeric antibody expression vectors can be constructed by cloning cDNAs encoding the VH or VL of non-human antibodies, respectively.
• the base sequence of the linking portion encodes an appropriate amino acid
• VH and VL cDNAs designed to have appropriate restriction enzyme recognition sequences are prepared.
• the prepared VH and VL cDNAs are placed upstream of the respective genes encoding CH or CL of the human antibody in the recombinant antibody expression vector obtained in (1) so that they are expressed in an appropriate form. Clone and construct a human chimeric antibody expression vector.
• the cDNA encoding the non-human antibody VH or VL is amplified by PCR using synthetic DNA having recognition sequences for appropriate restriction enzymes at both ends, and the recombinant antibody expression vector obtained in (1) is obtained. It can also be cloned into.
• a cDNA encoding the VH or VL of a humanized antibody can be constructed as follows.
• the amino acid sequences of the FRs of the VH or VL of the human antibody are selected, respectively, into which the amino acid sequences of the CDRs of the VH or VL of the non-human antibody are to be transplanted.
• Any amino acid sequence of the selected FR can be used as long as it is derived from a human antibody.
• the amino acid sequences of human antibody FRs registered in databases such as Protein Data Bank, or the common amino acid sequences of each subgroup of human antibody FRs [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services ( 1991)] etc.
• an FR amino acid sequence is selected that has as high a homology as possible (at least 60% or more) to the FR amino acid sequence of the VH or VL of the original antibody.
• the amino acid sequences of the CDRs of the original antibody are transplanted to the amino acid sequences of the FRs of the VH or VL of the selected human antibody, respectively, to design the amino acid sequences of the VH or VL of the humanized antibody, respectively.
• the designed amino acid sequence is converted into a DNA sequence taking into consideration the frequency of codon usage found in the base sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] to create a humanized antibody.
• a DNA sequence encoding the amino acid sequence of VH or VL is designed, respectively.
• each amplified product is cloned into a plasmid such as pBluescript SK(-) (Stratagene), the base sequence is determined by the same method as described in (2), and the desired humanized antibody is obtained.
• a plasmid having a DNA sequence encoding the amino acid sequence of VH or VL is obtained.
• a full-length VH and full-length VL each synthesized as a single long-strand DNA based on the designed DNA sequence can be used instead of the above PCR amplification product.
• cDNA encoding the VH or VL of the humanized antibody can be easily transferred to the recombinant antibody expression vector obtained in (1). can be cloned into.
• amino acid residues that are directly involved in binding to the antigen, amino acid residues that interact with amino acid residues in the CDRs, and amino acid residues in the FRs of the human antibody VH and VL are Reduced antigen binding by maintaining the tertiary structure and identifying amino acid residues that are indirectly involved in binding to the antigen, and substituting those amino acid residues with amino acid residues from the original non-human antibody. Activity can be increased.
• Amino acid residues in the FRs of VH and VL of a human antibody can be modified by performing the PCR reaction described in (4) using synthetic DNA for modification.
• the base sequence of the amplified product after the PCR reaction is determined by the method described in (2) to confirm that the desired modification has been made.
• a humanized antibody expression vector can be constructed by cloning the cDNA encoding each of the following.
• an antibody expression vector in which the H chain (or VH) and L chain (or VL) derived from two different types of antibodies are recombined may be used.
• a vector for expressing a VL-substituted chimeric antibody can be constructed.
• Any host cell that can express the recombinant antibody can be used as the host cell into which the expression vector is introduced, but for example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651] can be used. [Methods in Nucleic Acids Res., CRC press, 283 (1991)].
• the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant were measured using the enzyme-linked immunosorbent method [Monoclonal Antibodies-Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)].
• a transformed strain that stably expresses the recombinant antibody can be obtained by introducing the recombinant antibody into a suitable host cell.
• An electroporation method Japanese Unexamined Patent Publication No. 2-257891, Cytotechnology, 3, 133 (1990) is used to introduce the expression vector into host cells.
• any host cell that can express the genetically recombinant antibody can be used.
• CHO-K1 ATCC CCL-61
• DUKXB11 ATCC CCL-9096
• Pro-5 ATCC CCL-1781
• CHO-S Life Technologies, Cat #11619
• mice 20 (ATCC number: CRL1662, or also referred to as YB2/0), mouse myeloma cell NS0, mouse myeloma cell SP2/0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cell (ATCC number: CRL1580), dihydro CHO cells (CHO/DG44 cells) lacking the folate reductase gene (dihydroforate reductase (hereinafter referred to as dhfr)) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] are used.
• dhfr dihydro CHO cells
• proteins such as enzymes involved in the synthesis of the intracellular sugar nucleotide GDP-fucose, sugar chain modification in which the 1st position of fucose is ⁇ -linked to the 6th position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex type sugar chain.
• Host cells with reduced or deleted activity of proteins such as enzymes involved in the transport of intracellular sugar nucleotides GDP-fucose to the Golgi apparatus such as CHO with a deletion of the ⁇ 1,6-fucosyltransferase gene.
• Cells International Publication No. 2005/035586, International Publication No. 02/31140
• Lec13 that has acquired lectin resistance [Somatic Cell and Molecular genetics, 12, 55 (1986)], etc. can also be used.
• a transformed strain that stably expresses the recombinant antibody is selected by culturing it in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418). (National Patent Publication No. 2-257891).
• Media for animal cell culture include RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), or Hybridoma SFM medium (manufactured by Invitrogen). (manufactured by S.A. Co., Ltd.), or a medium prepared by adding various additives such as FBS to these mediums. By culturing the obtained transformed strain in a medium, the recombinant antibody is expressed and accumulated in the culture supernatant.
• the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or the like. Furthermore, the expression level of the recombinant antibody produced by the transformed strain can be improved by using a dhfr gene amplification system (Japanese Patent Publication No. 2-257891).
• the recombinant antibody is purified from the culture supernatant of the transformed strain using a protein A column [Monoclonal Antibodies - Principles and practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. It is also possible to combine methods used in protein purification such as gel filtration, ion exchange chromatography and ultrafiltration.
• the molecular weight of the H chain, L chain, or entire antibody molecule of a purified recombinant antibody can be determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting method [Monoclonal Antibodies - Principles and Practice, Third edition, Academic Press (1996), Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory (1988).
• Activity evaluation of purified monoclonal antibody or antibody fragment thereof Activity evaluation of the purified monoclonal antibody of the present invention or antibody fragment thereof can be performed as follows.
• the binding activity of the antibody of the present invention or the antibody fragment to human FGF23 can be measured using an ELISA method, a surface plasmon resonance method, or the like.
• the human FGF23 neutralizing activity of the antibody of the present invention or its antibody fragment can be measured using the reporter assay described above.
• the monoclonal antibody or antibody fragment thereof of the present invention can be used to treat diseases related to human FGF23.
• Therapeutic agents containing the monoclonal antibody or antibody fragment of the present invention may contain only the antibody or antibody fragment as an active ingredient, but usually include one or more pharmacologically acceptable carriers. It is provided as a pharmaceutical preparation prepared by mixing together with the pharmaceutical preparation by a method known in the technical field of pharmaceutical science.
• Examples of the administration route include oral administration, and parenteral administration such as intraoral, intratracheal, intrarectal, subcutaneous, intramuscular, or intravenous administration.
• Examples of dosage forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, and tapes.
• Preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
• Liquid preparations such as emulsions or syrups may contain water, sugars such as sucrose, sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid. It is manufactured using preservatives such as esters or flavors such as strawberry flavor or peppermint as additives.
• Capsules, tablets, powders or granules may contain excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxyl, etc. It is manufactured using additives such as a binder such as propyl cellulose or gelatin, a surfactant such as fatty acid ester, or a plasticizer such as glycerin.
• excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxyl, etc. It is manufactured using additives such as a binder such as propyl cellulose or gelatin, a surfactant such as fatty acid ester, or a plasticizer such as glycerin.
• Preparations suitable for parenteral administration include injections, suppositories, and sprays.
• Injectables are manufactured using a carrier consisting of a salt solution, a glucose solution, or a mixture of both.
• Suppositories are prepared with carriers such as cocoa butter, hydrogenated fats or carboxylic acids.
• the spray is manufactured using a carrier that does not irritate the recipient's oral cavity and respiratory tract mucous membranes, disperses the monoclonal antibody of the present invention or its antibody fragment as fine particles, and facilitates absorption.
• a carrier for example, lactose or glycerin is used. It can also be produced as an aerosol or dry powder.
• the components exemplified as additives can be added in formulations suitable for oral administration.
• Method for diagnosing diseases using the anti-human FGF23 monoclonal antibody or antibody fragment of the present invention Diagnosis of human FGF23-related diseases by detecting or measuring human FGF23 using the monoclonal antibody of the present invention or the antibody fragment Can be done.
• Diagnosis of human FGF23-related diseases can be performed, for example, by detecting or measuring human FGF23 present in the patient's body using immunological techniques.
• An immunological method is a method of detecting or measuring the amount of antibodies or antigens using labeled antigens or antibodies.
• a radioactive substance-labeled immunoantibody method an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay, a Western blotting method, a physicochemical method, or the like is used.
• an antigen or a cell expressing the antigen is reacted with the antibody of the present invention or the antibody fragment thereof, and further reacted with a radiolabeled anti-immunoglobulin antibody or the antibody fragment. Afterwards, measure using a scintillation counter.
• Enzyme immunoassay is performed by, for example, reacting an antigen or cells expressing the antigen with the antibody of the present invention or its antibody fragment, and then reacting it with an anti-immunoglobulin antibody or binding fragment labeled with an enzyme or the like. , add the substrate, and measure the absorbance of the reaction solution using an absorptiometer. For example, a sandwich ELISA method is used.
• a label used in enzyme immunoassay a known enzyme label [Enzyme Immunoassay, Igaku Shoin (1987)] can be used.
• an alkaline phosphatase label, a peroxidase label, a luciferase label, a biotin label, or the like is used.
• the sandwich ELISA method is a method in which an antibody is bound to a solid phase, an antigen to be detected or measured is trapped, and a second antibody is reacted with the trapped antigen.
• two types of antibodies or antibody fragments that recognize the antigen to be detected or measured and have different antigen recognition sites are prepared, and the first antibody or antibody fragment is plated in advance (for example, 96 Then, the second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin.
• the sandwich ELISA method As the antibody used in the sandwich ELISA method, either a polyclonal antibody or a monoclonal antibody may be used, and an antibody fragment such as Fab, Fab' or F(ab) 2 may be used.
• the combination of two types of antibodies used in the sandwich ELISA method may be a combination of monoclonal antibodies or antibody fragments that recognize different epitopes, or a combination of a polyclonal antibody and a monoclonal antibody or antibody fragment.
• Fluorescence immunoassay is measured using the method described in literature [Monoclonal Antibodies-Principles and Practice, Third Edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)], etc.
• a label used in the fluorescence immunoassay a known fluorescent label [Fluorescent antibody method, Soft Science Co., Ltd. (1983)] can be used.
• FITC or RITC is used.
• the luminescence immunoassay method is measured by the method described in the literature [Bioluminescence and Chemiluminescence Clinical Examination 42, Hirokawa Shoten (1998)]. Labels used in the luminescent immunoassay include known luminescent labels, such as acridinium ester or lophine.
• Western blotting involves fractionating antigens or antigen-expressing cells using SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel) [Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory (1988)], and then dividing the gel. Blotting is performed on a polyvinylidene fluoride (PVDF) membrane or nitrocellulose membrane, and the membrane is reacted with an antibody or antibody fragment that recognizes the antigen, and the antibody is then labeled with a fluorescent substance such as FITC, an enzyme label such as peroxidase, or a biotin label. The measurement is performed by visualizing the label after reacting with a mouse IgG antibody or binding fragment.
• SDS sodium dodecyl sulfate
• PAGE polyacrylamide gel
• the physicochemical method is performed, for example, by binding the antigen human FGF23 with the monoclonal antibody of the present invention or its antibody fragment to form an aggregate, and detecting this aggregate.
• a physicochemical method a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetric method, or a latex immunoturbidimetric method [Recommendation of Clinical Testing Methods, Kanehara Publishing (1998)], etc. can also be used.
• Latex immunoturbidimetry uses a carrier such as polystyrene latex with a particle size of about 0.1 to 1 ⁇ m sensitized with antibodies or antigens, and when an antigen-antibody reaction is caused by the corresponding antigen or antibody, the Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integrating sphere turbidity, the antigen concentration in the test sample is measured.
• a carrier such as polystyrene latex with a particle size of about 0.1 to 1 ⁇ m sensitized with antibodies or antigens
• Example 1 Detection of degraded product of antibody A
• the amino acid sequence of the heavy chain variable region of the anti-human FGF23 antibody described in WO 2008/099969 is SEQ ID NO: 1
• the amino acid sequence of the light chain variable region is SEQ ID NO: 2. Describe it.
• a human IgG1 antibody comprising a VH comprising the amino acid sequence represented by SEQ ID NO: 1 and a VL comprising the amino acid sequence represented by SEQ ID NO: 2 will be referred to as antibody A.
• Antibody A was prepared in the same manner as in Example 2, and the solvent of the antibody solution was replaced with a pH 4 solvent containing 10mM Sodium L-glutamate, 262mM D-Sorbitol, and 0.05mg/mL Polysorbate 80.
• the obtained antibody solution was allowed to stand at 40 degrees for one month (hereinafter referred to as 1M sample at 40 degrees) or at -80 degrees for one month and then thawed (hereinafter referred to as initial sample).
• band A located at a size approximately 10 kDa smaller than the H chain around 50 kDa was significantly darker in the 40° 1M sample than in the initial sample, and this correlated with The H chain band had become thinner. Therefore, it was suggested that band A is a decomposition product band of H chain, and that the 40° 1M sample is decomposed by H chain. As a result of analyzing the N-terminal amino acid sequence of band A, it was confirmed that it was cleaved at position 99D and position I of the heavy chain variable region of antibody A.
• Example 2 Preparation of I100 modified antibody Regarding antibody A, an antibody was prepared in which I, the 100th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 in VH, was replaced with the amino acid residue listed in Table 1. It was produced by the method described below. Hereinafter, some or all of the antibodies listed in Table 1 will also be referred to as I100 modified antibodies.
• the gene fragment corresponding to the base sequence encoding the amino acid sequence of the VH of each antibody listed in Table 1 was introduced into an expression vector using the seamless cloning method (contracted to Fasmac) to create the necessary plasmid.
• As the H chain expression vector pCI-OtCAG_hG1 vector having a signal sequence and human ⁇ chain constant region sequence was used. In all clones, human IgG1 was used for the heavy chain constant region.
• amino acid sequence of the VL of antibody A (SEQ ID NO: 2) was used as the amino acid sequence of the VL of the I100 modified antibody.
• L chain expression vector pCI-OtCMV_hK vector having a signal sequence and human ⁇ chain constant region sequence was used.
• the completed plasmid was prepared in large quantities using QIAGEN Plasmid Maxi Kit (QIAGEN).
• each antibody was transiently expressed using Expi293 Expression System Kit (Life Technologies).
• the method for introducing the plasmid was in accordance with the attached document.
• the L chain expression vector and the H chain expression vector were mixed and introduced at a ratio of 1:2.
• the cells after the plasmid introduction were cultured for 3 days under the conditions of 37 degrees, 5% CO 2 and 125 rpm. Thereafter, the cell culture suspension was centrifuged, and the culture supernatant was collected through a 0.2 ⁇ m filter (Thermo Scientific). Purified antibodies were obtained from the culture supernatant by affinity purification using MabSelect SuRe (GE Healthcare).
• the obtained antibody solution was neutralized by adding 1/10 amount of neutralization buffer (1M phosphoric acid-NaOH, pH 7.0), and the solvent of the antibody solution was replaced with PBS using NAP25 (GE Healthcare).
• the antibody solution after buffer replacement was concentrated by ultrafiltration using Amicon Ultra-4 Centrifugal Filter Units (Millipore), and absorbance A280 was measured using Nanodrop (Thermo Scientific) to determine the concentration of the antibody solution. Measurements and adjustments were made.
• the extinction coefficient is C. N. It was calculated from the amino acid sequence of each humanized antibody according to the method of Pace et al. (1995, Prot. Sci. 4:2411-2423).
• Example 3 Evaluation of antigen binding activity of I100 modified antibody
• the I100 modified antibody and A antibody obtained in Example 2 were evaluated against Recombinant Human FGF-23 (R&D Systems, Cat No. 2604-FG-025/CF). Binding activity was measured as follows.
• the antibody prepared at 1 ⁇ g/mL was added to the flow cell on which the Anti-human IgG antibody was immobilized at a flow rate of 10 ⁇ L/min for 30 seconds.
• Recombinant Human FGF-23 which was diluted stepwise from 130.5 ng/mL to 5 concentrations, was monitored at a flow rate of 30 ⁇ L/min for 2 minutes for binding reaction and 10 minutes for dissociation reaction. A single cycle method was used for the measurement.
• the obtained sensorgrams were analyzed using Bia Evaluation Software (Global Life Science Technologies Japan Co., Ltd.), and the kinetic constants of each antibody were calculated.
• A_1, A_2, and A_3 in Table 3 all refer to antibody A.
• antibodies I100W, I100S, I100K, and I100E have lower antigen-binding activity than antibody A, and that antibodies I100T, I100Q, I100M, and I100F have antigen-binding activity comparable to that of antibody A. did it.
• Example 4 Confirmation of the degradation suppressing effect of the I100 modified antibody Regarding the I100 modified antibody and A antibody obtained in Example 2, the solvent of the antibody solution was mixed with 10 mM Sodium L-glutamate and 262 mM D using NAP25 (GE Healthcare). - Substituted with a pH 4 solvent containing Sorbitol. The obtained antibody solution was allowed to stand at 40 degrees for one month or two weeks, and then subjected to SDS-PAGE under reducing conditions. Table 4 shows the results of comparing the density of the band around 40 kDa, which corresponds to the decomposed product of the antibody, between each antibody and the A antibody. When the density of the band in the modified antibody was lower than that in the A antibody, it was determined that the modified antibody suppressed the degradation compared to the A antibody.
• Example 5 Thermostability evaluation of antibody A and I100 modified antibody The thermal stability of each antibody domain (Fab, CH2, CH3) of antibody A and I100 modified antibody was evaluated by differential scanning calorimetry (hereinafter referred to as DSC). It was evaluated using the method described in
• the measurement sample was prepared with D-PBS buffer to a concentration of 0.5 mg/mL.
• Micro Cal VP-Capillary DSC system (Spectris Co., Ltd.) was used.
• measurements were performed using a program that raised the temperature from 25 degrees to 100 degrees per minute. The results obtained are shown in Tables 5 and 6.
• I100A, I100G, I100N, and I100R were detected with the Fab peak and CH2 peak overlapping, and the thermostability of Fab was improved by 3 to 8 degrees compared to antibody A. From the above, it was confirmed that I100A, I100G, I100N, and I100R have higher structural stability than antibody A.
• Example 6 Production of D105 modified antibody
• the 105th D of the amino acid sequence of VH containing the amino acid sequence represented by SEQ ID NO: 1 of antibody A was replaced with the amino acid residue shown in Table 7.
• An antibody hereinafter, part or all of the antibody will also be referred to as a D105 modified antibody
• Example 7 Evaluation of antigen binding activity of D105 modified antibody The binding activity of the D105 modified antibody obtained in Example 6 to Recombinant Human FGF-23 (R&D Systems, Cat No. 2604-FG-025/CF) was evaluated. It was measured in the same manner as in Example 3. The results obtained are shown in Tables 8 and 9. Both A_1 and A_2 in Table 8 are the measurement results for antibody A.
• Example 8 Confirmation of the decomposition-inhibiting effect of the D105 modified antibody Regarding the D105 modified antibody obtained in Example 6, the decomposition-inhibiting effect of the antibody was confirmed in the same manner as in Example 4. Note that the antibody solution was allowed to stand at 40 degrees for two weeks.
• I100 modified antibody and D105 modified antibody against human FGF23 I100 modified antibody and D105 modified antibody (I00A , I100N, I100G, I100Y, I100R, I100D, I100H, D105A, D105F, D105G, D105H, D105I, D105K, D105L, D105M, D105P, D105Q, D105R, D105V, D105W, D105Y, D1 05T, D105N, and D105S) 24 types
• the neutralizing activity against human FGF23 was measured using the method described below.
• mEgr1/ ⁇ KL/HEK293 ⁇ Klotho stably expressing HEK293 cells transformed with a luciferase expression vector having a promoter derived from the mouse Egr1 gene was used.
• mEgr1/ ⁇ KL/HEK293 was produced by a method similar to that described in Nature 2006 Dec 7; 444 (7120).
• the antibody was diluted to 100 ⁇ g/ml with a buffer containing 10 mM Sodium L-glutamate and 262 mM D-Sorbitol, and used as a stock solution.
• a buffer containing 10 mM Sodium L-glutamate and 262 mM D-Sorbitol As a standard culture medium for mEgr1/ ⁇ KL/HEK293, DMEM medium (Thermofisher) supplemented with 10 (vol%) FetalBovine Serum (Thermofisher) and 1 (vol%) penicillin/streptomycin was used.
• the maximum concentration was a concentration obtained by diluting the antibody 1000 times with the above standard culture medium (100 ng/ml), and an 8-step dilution series with ⁇ 10 times dilution was prepared and used for evaluation.
• the evaluation was performed using a 384-well plate. The number of cells was 2000 cells/well, and the concentration of FGF23 added was 4 ng/ml. The same FGF23 as in Example 3 was used after being diluted with a standard culture medium.
• Example 10 Production of antibody with modified heavy chain constant region of antibody A An antibody with amino acid modified heavy chain constant region of antibody A (hereinafter referred to as CH modified antibody) was produced by the method described below.
• the CH-modified antibody replaces the CH (human IgG1) of antibody A with human IgG2, IgG2AAAS (for the Fc of human IgG2, valine at EU index position 234 is changed to alanine, glycine at position 237 is changed to alanine, and proline at position 331 is changed to human IgG2 modified antibody with serine substitution) (Michael, S., et al., J.Immunol., 1997, 159: 3613; J.Immunol.
• IgG4PE_R409K human IgG4 Fc isotype antibody in which CH is substituted for serine at position 228 of the EU index, leucine at position 235 is replaced with glutamic acid, and arginine at position 409 is replaced with CH (human IgG4 modified antibody) (International Publication No. 2006/033386).
• Fc regions (10-1) to (10-5) which are known to improve the binding activity to human and monkey FcRn, are added to the Fc region of each of the A antibody or three isotype antibodies of the A antibody. A total of 23 types of antibodies with amino acid modifications were produced.
• the CH-modified antibody in which the CH of antibody A is replaced with the CH of human IgG2 will be referred to as the A_G2 antibody.
• CH modified antibodies substituted with other subclasses are also described.
• CH modified antibodies in which the above amino acid modifications (10-1) to (10-5) are added to the Fc region of antibody A are referred to as A_YTE antibody, A_QL antibody, A_A antibody, A_P antibody, and A_LS antibody, respectively.
• CH modified antibodies obtained by adding the above amino acid modifications (10-1) to (10-5) to the A_G2 antibody, A_G2AAS antibody, and A_G4PE_R409K antibody are also described.
• the plasmid containing the base sequence encoding the amino acid sequence of the variable region and constant region of the H chain of antibody A prepared in Example 2 was treated with restriction enzymes with NheI and BamHI to prepare a vector fragment excluding the constant region portion. . Based on the plasmid fragment, a gene fragment containing the base sequence encoding the amino acid sequence of the above 23 types of CH modified antibodies was synthesized at Fasmac and introduced into an appropriate expression vector to produce the necessary plasmid. . Antibodies were prepared in the same manner as in Example 2 using the obtained plasmids.
• Example 11 Measurement of binding activity to FcRn of antibody A and CH-modified antibody of antibody A.
• Antibody A and CH-modified antibody of antibody A produced in Examples 2 and 10 were measured by the method described below.
• the binding activity to human and monkey FcRn was measured using Biacore.
• HBS-EP+ Global Life Science Technologies Japan Co., Ltd., Cat. No. BR-1006-69
• HBS-EP+ Global Life Science Technologies Japan Co., Ltd., Cat. No. BR-1006-69
• Each antibody was buffer exchanged using NAP25 into HBS-EP+ at pH 6 as described above.
• Human FcRn and monkey FcRn were also diluted using HBS-EP+ pH 6.
• Human FcRn and monkey FcRn used in the experiment were produced as follows.
• Human FcRn (only amino acids 1 to 297 of the full-length amino acid sequence of human FcRn are used to express it as a soluble molecule) and human FcRn-His tag with six histidines added to its C-terminus (
• a DNA sequence encoding human ⁇ 2 microglobulin (amino acid sequence: SEQ ID NO: 54) and human ⁇ 2 microglobulin (amino acid sequence: SEQ ID NO: 55) was inserted into a mammalian cell expression plasmid downstream of the CMV promoter. Using the plasmid, transient expression was performed using the Expi293 Expression System Kit (Life Technologies) in the same manner as in Example 2.
• Monkey FcRn (only amino acids 1 to 297 of the full-length amino acid sequence of monkey FcRn are used to express it as a soluble molecule) and monkey FcRn-His tag with six histidines added to its C-terminus ( Amino acid sequence: SEQ ID NO: 56) and monkey ⁇ 2 microglobulin (amino acid sequence: SEQ ID NO: 57) were also transiently expressed in the same manner as human FcRn-His tag.
• Tetra His Antibody BSA Free (QIAGEN) was immobilized on a CM5 sensor chip (Global Life Science Technologies Japan Co., Ltd., Cat. No. BR100530). Human FcRn and monkey FcRn adjusted to 10 ⁇ g/mL were added to a flow cell on which Tetra His Antibody BSA Free was immobilized at a flow rate of 10 ⁇ L/min for 120 seconds. Next, each Fc-modified antibody was prepared stepwise at 5 concentrations by 3-fold dilution from 450 ⁇ g/mL, and the binding reaction was monitored for 2 minutes and the dissociation reaction was monitored for 5 minutes at a flow rate of 30 ⁇ L/min.
• Example 12 Preparation of CH modified antibodies of I100 and D105 modified antibodies Among the I100 and D105 modified antibodies that were confirmed to suppress antibody degradation more than antibody A in Example 4 and Example 8, D105N , I100A, I100H, and I100Y antibodies, CH modified antibodies with the following amino acid modifications (12-1) to (12-5) were produced by the method described below.
• CH modified antibodies in which the above amino acid modifications (12-1) to (12-5) have been made are referred to as D105N_YTE, D105N_G2_YTE, D105N_G2_LS, D105N_G4PE_R409K_P, and D105N_G4PE_R409K_LS antibodies, respectively. Similar descriptions are given for other antibodies.
• the amino acid sequences of CH of the CH modified antibodies in which the above amino acid modifications (12-1) to (12-5) were carried out are SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively. Describe it in
• the plasmid produced in Example 10 was treated with restriction enzymes Bstz17I and NheI to prepare a vector fragment from which the variable region portion was removed. Based on the plasmid fragment, a gene fragment containing the nucleotide sequence encoding the amino acid sequence of the VH of antibodies D105N, I100A, I100H, and I100Y was synthesized at Fasmac and introduced into an appropriate expression vector to produce the necessary A plasmid was created. Each antibody was prepared in the same manner as in Example 2 using the obtained plasmids.
• Example 13 Measurement of FGF23 neutralizing activity of heavy chain constant region modified antibodies The FGF23 neutralizing activity of the 20 types of CH modified antibodies prepared in Example 12 was measured in the same manner as in Example 9. The results obtained are shown in Table 14.
• antibodies with the same variable region amino acid sequence exhibited similar FGF23 neutralizing activity even if the constant region amino acid sequence was changed. From the above, it was confirmed that the amino acid modification of the heavy chain constant region of each antibody did not affect the FGF23 neutralizing activity of the antibody.
• Example 14 Production of modified antibodies that improve antigen-binding activity
• Abwiz Bio conducted an affinity test using a phage display method. Maturation was performed to obtain the following amino acid sequence information (14-1) to (14-9).
• VH containing the amino acid sequence represented by SEQ ID NO: 1 the 50th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is L, the 54th amino acid is W, and the 55th amino acid is VH (J2H2B9_A, amino acid sequence: SEQ ID NO: 39) in which the 57th amino acid is replaced with H, the 57th amino acid is replaced with T, the 58th amino acid is replaced with F, and the 100th amino acid is replaced with A.
• VH a VH containing the amino acid sequence represented by SEQ ID NO: 1
• the 50th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is V
• the 54th amino acid is F
• the 55th amino acid is VH (J2H2E9_A, amino acid sequence: SEQ ID NO: 40) in which C is substituted
• the 57th amino acid is substituted with F
• the 58th amino acid is substituted with V
• the 100th amino acid is substituted with A.
• VH containing the amino acid sequence represented by SEQ ID NO: 1 the 51st amino acid of the amino acid sequence represented by SEQ ID NO: 1 is L, the 54th amino acid is W, and the 55th amino acid is VH (2H2E1_A, amino acid sequence: SEQ ID NO: 41) in which the 57th amino acid is replaced with T, the 57th amino acid is replaced with Y, the 58th amino acid is replaced with R, and the 100th amino acid is replaced with A.
• Example 15 Preparation of modified antibodies Using the amino acid sequence information obtained in Example 14 and the amino acid sequence of VH described in (15-1), modifications of A-1 to A-8 described in Table 15 were made. Antibodies were produced.
• a plasmid containing a base sequence encoding the amino acid sequence of each antibody was prepared in the same manner as in Example 12, and antibodies were prepared from the plasmid in the same manner as in Example 2.
• Example 16 Evaluation of modified antibodies Antigen binding activity, FGF23 neutralizing activity, and thermal stability were evaluated using the eight types of modified antibodies prepared in Example 15 and antibody A as a control.
• the buffer was D-PBS (Nacalai Tesque Co., Ltd., Code 14249-24), and the concentration was adjusted to 0.5 mg/mL.
• Various measurements were performed by diluting the sample according to the purpose. .
• Example 3 the antigen binding activity of the I100A antibody and I100Y antibody was lower than that of the A antibody. Furthermore, in all antibodies A-1 to A-8, the 100th amino acid residue of VH is A or Y.
• Example 9 the FGF23 neutralizing activity of the I100A antibody and I100Y antibody was lower than that of the A antibody. Furthermore, in all antibodies A-1 to A-8, the 100th amino acid residue of VH is A or Y.
• the isoelectric point of each antibody was determined using the iCE3 system (Protein Simple).
• Pharmalyte 3-10 for IEF Global Life Science Technologies Japan Co., Ltd., Cat. No. 17-0456-01
• pI Marker 5.12 Protein Simple Co., Ltd., Cat. No. .102224
• pI Marker 9.77 Protein Simple, Cat. No. 102219 was used as the basic marker.
• the measurement method followed a standard protocol.
• thermostability Six antibodies, A antibody, A_YTE antibody, A-1 antibody, A-3 antibody, A-5 antibody, and A-8 antibody, were tested using the method described in Example 5. Similarly, thermal stability evaluation was conducted. The results obtained are shown in Table 19.
• thermostability of each antibody site between antibody A and antibody A_YTE As shown in Table 19, there was no change in the thermostability of each antibody site between antibody A and antibody A_YTE. On the other hand, for antibodies A-1 and A-3, which have the same heavy chain constant region amino acid sequence as antibody A_YTE, it was confirmed that the thermostability of Fab was improved by approximately 3 degrees compared to antibody A and antibody A_YTE. .
• Example 17 Production of A-9 antibody and A-10 antibody A-9 antibody and A-10 antibody listed in Table 20 were produced in the same manner as in Example 2 (hereinafter referred to as A-9 antibody and A-10 antibody). -10 antibody is sometimes referred to as a modified antibody).
• the L chain expression vector for the A-9 antibody the pcDNA3.4 vector (Invitrogen) was used instead of the pCI vector of Example 2. Furthermore, for both the A-9 antibody and the A-10 antibody, the L chain expression vector and the H chain expression vector were mixed at a ratio of 1:1 and introduced into cells.
• affinity maturation was performed using the phage display method at Abwiz Bio for the I100A antibody, and the following amino acid sequence information was obtained.
• VL was designed and used for the A-10 antibody.
• Example 18 Evaluation of modified antibodies Antigen binding activity, FGF23 neutralizing activity, and thermal stability were evaluated using the two types of modified antibodies prepared in Example 17 and antibody A as a control.
• the buffer was D-PBS (Nacalai Tesque Co., Ltd., Code 14249-24), the concentration was adjusted to 1 mg/mL, and various measurements were performed by diluting the sample according to the purpose.
• the A-9 antibody had twice the antigen binding activity of the A antibody, and the A-10 antibody had the same antigen binding activity as the A antibody.
• the antigen binding activity of the I100A antibody and the I100Y antibody was lower than that of the A antibody.
• the 100th amino acid residue of VH is A and Y, respectively. From the above, replacing the 100th amino acid residue of VH of antibody A with A or Y reduces the antigen-binding activity of the antibody, but substitution of additional amino acid residues in antibody A-9 and I100Y causes It was confirmed that the binding activity of the antibody was improved to the same level or higher than that of antibody A.
• Example 9 the neutralizing activity of I100A and I100Y was lower than that of antibody A. Furthermore, in the A-9 antibody and the A-10 antibody, the 100th amino acid residue of VH is A and Y, respectively.
• the antibody used in this example was obtained by inserting the target gene sequence into a mammalian expression vector, introducing it into CHO cells, and then using MabSelect SuRe (Global Life Science Technologies Japan Co., Ltd.) from the mammalian cell culture medium supernatant. Purified antibodies were obtained by affinity purification using cation exchange chromatography. Using NAP25 (Global Life Science Technologies Japan Co., Ltd.), the solvent of the antibody solution was replaced with a pH 4 and pH 4.5 solvent containing 10 mM Sodium L-glutamate, 262 mM D-Sorbitol, and 0.05 mg/mL Polysorbase 80. The concentration was 1 mg/mL.
• the obtained antibody solution was left standing at 40 degrees for one month and at 25 degrees for three months, and then frozen at -80 degrees. After melting the antibody solution, decomposition of the antibody was confirmed using a biological product analysis system PA800Plus (SCIEX). Basic conditions such as sample preparation and analysis were performed according to the method of Oscar Salas-Solano et al. (2006, Anal. Chem.:6583-6594), and were modified as appropriate.
• the results at pH 4.5 are shown in Table 22, and the results at pH 5.0 are shown in Table 23.
• Initial sample frozen at -80 degrees immediately after solvent replacement sample frozen at -80 degrees after being left at 40 degrees for 1 month, 40 degrees Celsius 1M, left at 25 degrees for 3 months, then at -80 degrees. Frozen samples were labeled as 25°C 3M. All samples were melted under the same conditions and then measured.
• Example 20 Confirmation of degradation inhibition rate of modified antibody The degradation inhibition rate was confirmed using the two types of modified antibodies prepared in Example 17 and the A antibody and A-1 antibody as controls.
• the A antibody and A-1 antibody used in this example were obtained by inserting the target gene sequence into a mammalian expression vector, introducing it into CHO cells, and then using MabSelect SuRe (Global Life) from the mammalian cell culture medium supernatant. Purified antibodies were obtained by affinity purification and cation exchange chromatography (Science Technologies Japan Co., Ltd.). Using NAP25 (Global Life Science Technologies Japan Co., Ltd.), the solvent of the antibody solution was replaced with a pH 4, pH 4.5, and pH 5 solvent containing 10 mM Sodium L-glutamate, 262 mM D-Sorbitol, and 0.05 mg/mL Polysorbate. , the protein concentration was 1 mg/mL.
• the obtained antibody solution was allowed to stand at 40 degrees for one month, and then the antibody was extracted using a microchip electrophoresis system LabChip (PerkinElmer) and a Protein Clear Reagent kit (PerkinElmer, Cat. CLS960014). The decomposition of was confirmed. Regarding sample preparation and electrophoresis conditions, the experiment was conducted according to the protocol included with the kit.
• the results at pH 4.0 are shown in Table 24, the results at pH 4.5 are shown in Table 25, and the results at pH 5.0 are shown in Table 25.
• the results are shown in Table 26.
• the A-9 antibody and A-10 antibody were also tested for one month under the buffer conditions of pH 4.0, pH 4.5, or pH 5.0 and the storage condition of 40 degrees. It was confirmed that the degradation was suppressed compared to antibody A.
• Example 21 In order to confirm the sustainability of the pharmacological effect of a single subcutaneous administration of the test antibody to male cynomolgus monkeys, the serum inorganic phosphorus concentration (mg/dL) was measured. A8 antibody (1.8 mg/kg) was administered subcutaneously as a test antibody. Blood was collected over time for 56 days after administration, and the inorganic phosphorus concentration in the serum was measured at each time point. The phosphorus concentration was measured by the PNP-XDH method using a clarifier (JCA-BM6070).
• the A-8 antibody was shown to have a longer duration of serum inorganic phosphorus concentration in cynomolgus monkeys than burosumab.
• A-1 antibody and A-5 antibody were subcutaneously administered to cynomolgus monkeys at 3 mg/kg, and the inorganic phosphorus concentration in the serum was measured in the same manner as above.
• these antibodies like burosumab, increased the serum inorganic phosphorus concentration from the 3rd day after administration, and the inorganic phosphorus concentration decreased to the baseline on the 42nd day after administration.
• the amino acid sequence of the heavy chain constant region of burosumab is represented by SEQ ID NO: 53, and the amino acid sequence of the heavy chain constant region of A-1 antibody, A-5 antibody, and A-8 antibody is represented by SEQ ID NO: 48.
• SEQ ID NO: 53 amino acid sequence of the heavy chain constant region of A-1 antibody, A-5 antibody, and A-8 antibody is represented by SEQ ID NO: 48.
• antibodies A-1, A-5, and A-8 which have the same heavy chain constant region amino acid sequence, only antibody A-8 sustained serum inorganic phosphorus concentration longer than burosumab in cynomolgus monkeys.
• Example 22 Inorganic phosphorus concentration (mg/dL) in serum was measured in the same manner as in Example 21. A-9 antibody and A-10 antibody (3.0 mg/kg) were administered subcutaneously as test antibodies. Blood was collected over time for 57 days after administration, and the inorganic phosphorus concentration in the serum was measured at each time point.
• the serum inorganic phosphorus concentration increased from the third day after administration. Even on the 57th day, the inorganic phosphorus concentration was higher than the baseline inorganic phosphorus concentration (phosphorus concentration on Day 0 in Table 28).
• the amino acid sequence of the heavy chain constant region of the A-9 antibody and A-10 antibody is the same as the heavy chain constant region amino acid sequence of the A-1 antibody, A-5 antibody, and A-8 antibody, and is represented by SEQ ID NO: 48. It is an amino acid sequence.
• the duration of the inorganic phosphorus concentration in the serum of cynomolgus monkeys is longer than that for burosumab, A-1 antibody, and A-5 antibody. It was suggested that this was due to a difference in the amino acid sequence of the variable region, similar to the A-8 antibody, rather than a difference in sequence.
• SEQ ID NO: 1 Amino acid sequence of VH of antibody A SEQ ID NO: 2: Amino acid sequence of VL of antibody A SEQ ID NO: 3: Amino acid sequence of VH of I100A antibody SEQ ID NO: 4: Amino acid sequence of VH of I100L antibody SEQ ID NO: 5: Amino acid sequence of I100V antibody Amino acid sequence of VH of I100Y antibody SEQ ID NO: 7: Amino acid sequence of VH of I100W antibody SEQ ID NO: 8: Amino acid sequence of VH of I100T antibody SEQ ID NO: 9: Amino acid sequence of VH of I100S antibody Number 10: Amino acid sequence of VH of I100R antibody SEQ ID NO: 11: Amino acid sequence of VH of I100Q antibody SEQ ID NO: 12: Amino acid sequence of VH of I100N antibody SEQ ID NO: 13: Amino acid sequence of VH of I100M antibody SEQ ID NO: 14: Amino acid sequence of

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