WO2015129651A1 - Igm monomère modifié - Google Patents

Igm monomère modifié Download PDF

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Publication number
WO2015129651A1
WO2015129651A1 PCT/JP2015/055118 JP2015055118W WO2015129651A1 WO 2015129651 A1 WO2015129651 A1 WO 2015129651A1 JP 2015055118 W JP2015055118 W JP 2015055118W WO 2015129651 A1 WO2015129651 A1 WO 2015129651A1
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Prior art keywords
amino acid
igm
region
antibody
modified
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Japanese (ja)
Inventor
佐藤　英雄
和也 小見
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Fujirebio Inc
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Fujirebio Inc
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Priority to JP2016505215A priority Critical patent/JP6512214B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/528—CH4 domain

Definitions

  • the present invention relates to a modified monomer IgM, a transformed cell, and a method for measuring a target substance.
  • an antibody having a protein function can be prepared by conjugating an antibody with a predetermined protein.
  • a method of producing a labeled antibody for enzyme immunoassay by conjugating an antibody with an enzyme is generally performed. It is known that the antibody used for the production of the conjugate is preferable as it has a simple structure.
  • the F (ab ′) fragment obtained by pepsin digestion and reduction of IgG1 is converted via its free cysteine residue.
  • a method of conjugating with an enzyme is widely used.
  • Patent Documents 1 to 3 In addition, in order to produce an antibody having a protein function, an attempt is made to fuse the antibody with the protein instead of conjugating the antibody and the protein (Patent Documents 1 to 3).
  • JP 2010-17113 A JP-A-8-70875 Japanese Patent Laid-Open No. 10-323187
  • Multimeric IgM (present as pentamer or hexamer) is unstable, such as being easily aggregated. In addition, when used as an antibody for immunoassay, it tends to cause a non-specific reaction with a substance in a sample such as serum, causing false positives. Therefore, it is generally not preferred to use multimeric IgM as an antibody to be modified by a protein. In addition, the molecular weight of multimeric IgM is large (about 900,000 in the case of pentamer), and when IgM is labeled with a protein by a chemical technique, there are many sites that can be labeled.
  • an object of the present invention is to provide a protein-modified IgM that overcomes the problems peculiar to IgM that is a multimeric antibody.
  • the present inventors have demonstrated that a modified IgM in which a protein is fused to a predetermined modified constant region exhibits the function of an antibody as a monomer, despite the antibody that originally functions as a multimer, In addition, the present inventors have found that the function of the fused protein can be maintained well, and have completed the present invention.
  • the present invention is as follows. [1] comprising an IgM light chain, and a modified IgM heavy chain, A modified monomeric IgM, wherein the modified IgM heavy chain comprises A) a heavy chain region composed of a variable region and a C-terminal deleted constant region, and B) a protein fused to the heavy chain region. [2] The modified monomer IgM of [1], wherein the constant region is a constant region having an amino acid residue in a CH1 ⁇ CH2 adjacent region as a C-terminal amino acid residue. [3] The modified monomer IgM of [1], wherein the constant region is a constant region having an amino acid residue in a CH2 / CH3 adjacent region as a C-terminal amino acid residue.
  • the antibody of the present invention is secreted well into the culture supernatant and can retain the binding ability to the antigen. Since the antibody of the present invention can be easily prepared by purification from the culture supernatant, it is useful for greatly simplifying the production process.
  • FIG. 1 shows Western blotting of various modified antibodies having a fusion protein (modified IgM heavy chain) of IgM (antibody 1) heavy chain and ALP in which C-terminal regions having different lengths are deleted. Abbreviations are as follows (Conj: conjugate; ALP: alkaline phosphatase; S: culture supernatant; M: membrane fraction, and so on).
  • FIG. 2 shows Western blotting of various modified antibodies having a fusion protein (modified IgM heavy chain) of IgM (antibody 2) heavy chain and ALP in which C-terminal regions having different lengths are deleted.
  • FIG. 3 is a diagram showing the association of a modified IgM heavy chain with an IgM light chain.
  • FIG. 4 is a diagram showing measurement of an affinity complex (indirectly 25OH VD3) between 25OH VD3 and anti-25OH VD3 antibody using a modified antibody having a modified IgM heavy chain (derived from antibody 2).
  • pNPP was used as a substrate for ALP. pNPP is converted to paranitrophenol which absorbs a wavelength of 405 nm by an enzymatic reaction.
  • FIG. 5 shows Western blotting of various modified antibodies (derived from antibody 2) having modified IgM heavy chains.
  • FIG. 6 is a diagram showing measurement of affinity complex (indirectly, 25OH VD3) of 25OH VD3 and anti-25OH VD3 antibody using various modified antibodies (derived from antibody 2) having modified IgM heavy chains. is there.
  • AMPPD was used as a substrate for ALP.
  • AMPPD is decomposed by an enzymatic reaction to generate luminescence.
  • the luminescence count was measured.
  • FIG. 7 is a diagram showing measurement of human IgG using a modified antibody (derived from antibodies 3 and 4) having a modified IgM heavy chain.
  • AMPPD was used as a substrate for ALP.
  • the present invention provides a modified monomer IgM.
  • the modified monomer IgM of the present invention is abbreviated as the antibody of the present invention as necessary.
  • the antibody of the present invention can be derived from IgM.
  • IgM those derived from any animal having the ability to produce IgM can be used. Examples of such animals include birds (eg, chicken, quail, ostrich) and mammals (eg, human, monkey, mouse, rat, hamster, rabbit, cow, horse, sheep, donkey, pig). .
  • the antibody of the present invention contains an IgM light chain.
  • An IgM light chain is composed of a variable region (VL) and a constant region (CL). Examples of the light chain include ⁇ chain and ⁇ chain.
  • the antibody of the present invention also contains a modified IgM heavy chain.
  • a normal IgM heavy chain is composed of a variable region (VH) and a constant region (CH).
  • the CH of the IgM heavy chain is composed of four domains (CH1, CH2, CH3, and CH4). Assignment of domains (CH1, CH2, CH3, and CH4) in the constant region in the heavy chain of IgM antibody can be determined based on the amino acid sequence of the heavy chain according to common technical knowledge in the art.
  • the constant region of IgM is conserved in specific animal species. That is, the constant region of IgM for an antigen produced by a particular animal species can be the same as the constant region of IgM for another antigen produced by that animal species. This is because there is no subtype in IgM and only one species, and the ⁇ gene encoding the constant region of IgM is the same in a specific animal species.
  • the amino acid sequence of the constant region of chicken-derived IgM is disclosed with 1) X01613.1, 2) CAA25762.1, and 3) GenBank accession number or Swiss-prot accession number of P01875.2. The amino acid sequence of the constant region is consistent between these IgMs.
  • amino acid sequence such as a constant region disclosed by these accession numbers is shown as SEQ ID NO: 1.
  • CH1 (C ⁇ 1 domain) corresponds to an amino acid sequence consisting of amino acid residues at positions 1 to 105
  • CH2 (C ⁇ 2 domain) is an amino acid sequence consisting of amino acid residues at positions 106 to 209
  • CH3 (C ⁇ 3 domain) corresponds to an amino acid sequence consisting of amino acid residues at positions 210 to 316
  • CH4 (C ⁇ 4 domain) corresponds to an amino acid sequence consisting of amino acid residues at positions 317 to 427.
  • the amino acid sequence consisting of amino acid residues at positions 428 to 446 in the amino acid sequence of SEQ ID NO: 1 is referred to as the C-terminal region.
  • the constant region of IgM for an antigen produced by a particular animal species is not completely the same as the constant region of IgM for another antigen produced by that animal species, but is “substantially identical” There is also a possibility.
  • the expression “substantially identical” intends the amino acid sequence of the constant region containing a mutation of one or several amino acid residues caused by the mutation as described above.
  • the term “one or several amino acid residues” refers to 1 to 40, preferably 1 to 30, more preferably 1 to 20, even more preferably 1 to It means 10 amino acid residues, particularly preferably 1 to 5 amino acid residues.
  • the mutation include substitution, insertion, deletion and addition.
  • amino acid residue substitution may be a conservative substitution.
  • conservative substitution refers to substitution of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art.
  • such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g.
  • the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Or a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.
  • the constant region of IgM may be: a) a polypeptide comprising an amino acid sequence corresponding to amino acid residues 1 to 427 in the amino acid sequence of SEQ ID NO: 1; or b) corresponding to amino acid residues 1 to 427 in the amino acid sequence of SEQ ID NO: 1.
  • the constant region of IgM may be a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1.
  • the percent identity of amino acid sequences is preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more Good.
  • identity means a value (Identity) obtained using a default parameter in a NEEDLE program (J Mol Biol 1970; 48: 443-453) search.
  • the modified IgM heavy chain includes A) and B) below: A) a heavy chain region composed of a variable region and a C-terminal deleted constant region; and B) a protein fused to the heavy chain region.
  • the heavy chain region mentioned in (A) is composed of a variable region and a C-terminal deleted constant region.
  • a C-terminal deleted constant region is a constant region whose C-terminal region has been deleted so as to allow formation of monomeric IgM. Deletion of the C-terminal region is performed by deleting a continuous unit (region on the C-terminal side) from the C-terminal amino acid residue to a predetermined amino acid residue upstream thereof.
  • Monomeric IgM is not a multimer (pentamer or hexamer) formed by natural IgM, but a monovalent monomer (ie, a Fab-like molecule) or a divalent monomer (ie, F (Ab ′)-like molecule).
  • a cysteine residue (hereinafter sometimes referred to as “CH1 Cys”) involved in cross-linking of a heavy chain and a light chain by a disulfide bond may be located in CH1.
  • a cysteine residue involved in cross-linking between heavy chains by disulfide bonds (hereinafter sometimes referred to as “CH2 Cys”) may be located in CH2.
  • a cysteine residue (hereinafter sometimes referred to as “CH3 Cys”) involved in cross-linking between monomeric IgMs by disulfide bonds (ie, formation of multimeric IgM) may be located in CH3.
  • the C-terminal deletion constant region includes at least an essential region from the N-terminal amino acid residue to CH1 Cys as a constant region from the viewpoint of maintaining the antigen binding site, and also from the viewpoint of formation of the modified monomer IgM To at least the region from CH3 Cys to the C-terminal amino acid residue is deleted.
  • a modified monomer IgM having a C-terminal deleted constant region can have both an antibody function (eg, secretion ability and binding ability) and a protein function (eg, enzyme activity).
  • the C-terminal deletion constant region further includes a region from CH1 Cys to CH2 Cys in addition to the essential region as a constant region, the antibody of the present invention can be a divalent monomer.
  • the C-terminal deletion constant region does not further include the region from CH1 Cys to CH2 Cys in addition to the essential region as the constant region, in other words, the region from CH2 Cys to the C-terminal amino acid residue.
  • the antibody of the invention may be a monovalent monomer.
  • examples of the C-terminal deletion constant region in the antibody of the present invention include the following: i) a region that is deleted from a predetermined amino acid residue in CH1 on the C-terminal side of CH1 Cys to the C-terminal amino acid residue (that is, a region composed of a part of CH1); ii) a region from CH2 to the C-terminal amino acid residue (ie, a region composed of the entire CH1); iii) a region that is deleted from a predetermined amino acid residue to the C-terminal amino acid residue in CH2 (ie, a region composed of the entire CH1 and a part of CH2); iv) a region that deletes from CH3 to the C-terminal amino acid residue (ie, a region that is composed of the entire CH1 and the entire CH2); and v) a region that is deleted from CH3 Cys to the C-terminal amino acid residue ( That is, a region composed of the entire CH1, the entire CH2, and a part of
  • the C-terminal deletion constant region is a constant region having an amino acid residue in the CH1 ⁇ CH2 adjacent region, CH2 ⁇ CH3 adjacent region, or CH3 upstream region as the C-terminal amino acid residue.
  • the antibody of the present invention having such a C-terminal deleted constant region can be particularly excellent in both the antibody function (eg, secretion ability and binding ability) and the protein function (eg, enzyme activity).
  • the CH1 ⁇ CH2 adjacent region is defined as “ ⁇ 1” for the position of the C-terminal amino acid residue of CH1, and the N-terminal amino acid residue of CH2 relative to the CH1 ⁇ CH2 boundary (CH1-CH2). Assuming that the position is “+1” (there is no “0” amino acid residue, and the “ ⁇ 1” and “+1” amino acid residues are linked to each other by an amide bond) , A region consisting of 8 consecutive amino acid residues extending from the “ ⁇ 4” amino acid residue in the CH1 region to the “+4” amino acid residue in the CH2 region.
  • the C-terminal deletion constant region having an amino acid residue in the CH1 / CH2 adjacent region as the C-terminal amino acid residue may correspond to the above-mentioned i), ii) or iii).
  • the CH1 and CH2 flanking regions preferably consist of a) six consecutive amino acid residues ranging from a) the “ ⁇ 3” amino acid residue in the CH1 region to the “+3” amino acid residue in the CH2 region Region, b) a region consisting of 4 consecutive amino acid residues ranging from the “ ⁇ 2” amino acid residue in the CH1 region to the “+2” amino acid residue in the CH2 region, or c) in the CH1 region It may be a region consisting of two consecutive amino acid residues extending from the “ ⁇ 1” amino acid residue to the “+1” amino acid residue in the CH2 region.
  • the C-terminal deletion constant region may be derived from a chicken.
  • the C-terminal deletion constant region may be as follows. a1) a polypeptide comprising an amino acid sequence corresponding to amino acid residues 1 to X in the amino acid sequence of SEQ ID NO: 1; or b1) corresponding to amino acid residues 1 to X in the amino acid sequence of SEQ ID NO: 1.
  • X is any integer from 102 to 109, preferably any integer from 103 to 108, more preferably any integer from 104 to 107, and even more preferably 105 or 106 .
  • the C-terminal deletion constant region is a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence corresponding to the amino acid residues at positions 1 to X in the amino acid sequence of SEQ ID NO: 1. May be. X is as described above.
  • the percent identity of amino acid sequences is preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more Good. The determination of percent identity can be made as described above.
  • the CH2 ⁇ CH3 adjacent region refers to the position of the C-terminal amino acid residue of CH2 as “ ⁇ 1” and the N-terminal amino acid residue of CH3 based on the CH2 ⁇ CH3 boundary (CH2-CH3). Assuming that the position is “+1” (there is no “0” amino acid residue, and the “ ⁇ 1” and “+1” amino acid residues are linked to each other by an amide bond) , A region consisting of 8 consecutive amino acid residues extending from the “ ⁇ 4” amino acid residue in the CH2 region to the “+4” amino acid residue in the CH3 region.
  • the C-terminal deletion constant region having the amino acid residue in the CH2 ⁇ CH3 adjacent region as the C-terminal amino acid residue may correspond to the above-mentioned region iii), iv) or v).
  • the CH2 ⁇ CH3 flanking region preferably consists of a) 6 consecutive amino acid residues ranging from a) the “ ⁇ 3” amino acid residue in the CH2 region to the “+3” amino acid residue in the CH3 region Region, b) a region consisting of four consecutive amino acid residues ranging from the “ ⁇ 2” amino acid residue in the CH2 region to the “+2” amino acid residue in the CH3 region, or c) in the CH2 region It may be a region consisting of two consecutive amino acid residues extending from the “ ⁇ 1” amino acid residue to the “+1” amino acid residue in the CH3 region.
  • the C-terminal deletion constant region may be derived from a chicken.
  • the C-terminal deletion constant region may be as follows. a2) a polypeptide comprising an amino acid sequence corresponding to amino acid residues 1 to Y in the amino acid sequence of SEQ ID NO: 1; or b2) corresponding to amino acid residues 1 to Y in the amino acid sequence of SEQ ID NO: 1.
  • Y is any integer from 206 to 213, preferably any integer from 207 to 212, more preferably any integer from 208 to 211, and even more preferably 209 or 210.
  • the C-terminal deletion constant region is a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence corresponding to the amino acid residues at positions 1 to Y in the amino acid sequence of SEQ ID NO: 1. May be. Y is as described above.
  • the percent identity of amino acid sequences is preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more Good. The determination of percent identity can be made as described above.
  • the CH3 upstream region refers to the amino acid residue from the “+1” position to the “+18” amino acid residue in the CH3 region, assuming that the position of the N-terminal amino acid residue of CH3 is “+1”. , Refers to a region consisting of 18 consecutive amino acid residues.
  • the C-terminal deletion constant region having the amino acid residue in the CH3 upstream region as the C-terminal amino acid residue can correspond to the region v) above.
  • the CH3 upstream region is preferably, in the CH3 region, from the amino acid residue at “+1” position, “+17”, “+16”, “+15”, “+14”, “+13”, “+13” ”,“ +12 ”,“ +11 ”,“ +10 ”,“ +9 ”,“ +8 ”,“ +7 ”,“ +6 ”,“ +5 ”,“ +4 ”,“ +3 ” Or a region consisting of consecutive amino acid residues spanning the amino acid residue at the “+2 position”.
  • the C-terminal deletion constant region may be derived from a chicken.
  • the C-terminal deletion constant region may be as follows. a3) a polypeptide comprising an amino acid sequence corresponding to amino acid residues 1 to Z in the amino acid sequence of SEQ ID NO: 1; or b3) corresponding to amino acid residues 1 to Z in the amino acid sequence of SEQ ID NO: 1.
  • Z is an arbitrary integer from 210 to 227.
  • Z is 226 or less, 225 or less, 224 or less, 223 or less, 222 or less, 221 or less, 220 or less, 219 or less, 218 or less, 217 or less, 216 or less, 215 or less, 214 or less, 213 or less, 212 or less Or 211 or less.
  • the protein mentioned in B) is fused to the heavy chain region. Fusion is performed by linking the N-terminal amino acid residue of the protein to the C-terminal amino acid residue of the heavy chain region via an amide bond.
  • a polynucleotide encoding a protein is ligated downstream of the polynucleotide encoding the heavy chain region in accordance with the reading frame, and then the ligated polynucleotide is introduced into an expression vector, and the expression vector Can be prepared by introducing the protein into a cell and finally expressing the fusion protein in the cell.
  • the fusion is performed via a linker consisting of one or several (eg, 1-50, 1-30, 1-20, 1-10, or 1-5) amino acid residues. Also good.
  • a protein is an amino acid polymer that imparts some property to the antibody of the present invention.
  • Typical proteins include, for example, a protein (eg, an enzyme) that can generate a substance that can have a detectable physical property (eg, absorbance) or a substance that can generate a detectable signal (eg, luminescence, fluorescence). ), Or a protein (eg, a fluorescent protein) that can have a detectable physical property or can generate a detectable signal.
  • proteins examples include enzymes that can produce the aforementioned substances (eg, alkaline phosphatase, peroxidase, luciferase, ⁇ -galactosidase), and proteins that can generate signals (eg, green fluorescent protein, red fluorescent protein). It is done.
  • the protein is preferably a soluble protein.
  • the molecular weight of the protein may be, for example, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, or 50 kDa or more.
  • the molecular weight of the protein may also be, for example, 200 kDa or less, 180 kDa or less, or 160 kDa or less.
  • the antibody of the present invention can recognize any antigen having antigenicity.
  • antigens include low molecular weight substances, proteins, polysaccharides, and allergens.
  • the term “low molecular weight substance” refers to a compound having a molecular weight of less than 1,500.
  • the low molecular weight substance is a natural substance or a synthetic substance.
  • the molecular weight of the low molecular weight substance may be less than 1,200, less than 1,000, less than 800, less than 700, less than 600, less than 500, less than 400, or less than 300.
  • the molecular weight of the low molecular weight substance may also be 50 or more, 100 or more, 150 or more, or 200 or more.
  • low molecular weight substances include ligands, hormones, lipids, fatty acids, vitamins, opioids, neurotransmitters (eg, catecholamines), nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, amino acids, and oligopeptides, or pharmaceuticals. , Toxicants, and metabolites.
  • hormones include steroid hormones, thyroid hormones, and peptide hormones.
  • proteins include soluble proteins such as immunoglobulins and membrane proteins. Examples of the immunoglobulin include IgG, IgA, IgE, IgD, and IgY. Proteins such as immunoglobulins can be derived from any animal as described above (preferably, mammals such as humans).
  • the antibody of the present invention may recognize an affinity complex.
  • affinity complex refers to a complex formed by the association or aggregation (ie, non-covalent binding) of two or more factors (see, eg, WO2013 / 042426).
  • the at least one factor constituting the affinity complex may preferably be a protein. Examples of such proteins include proteins having affinity binding ability (eg, antibodies) and proteins having aggregation ability.
  • the protein that is a factor constituting the affinity complex is an antibody
  • the antibody of the present invention can be used as, for example, a secondary antibody.
  • the at least one factor constituting the affinity complex may also be a low molecular weight substance.
  • the low molecular weight substance may be a vitamin.
  • vitamins include vitamins A, B1, B2, B6, B12, C, D, E, and K.
  • the vitamin is a fat-soluble vitamin (eg, vitamins A, D, E, K), more preferably vitamin D as shown below.
  • the low molecular weight substance may also be a metabolite of a vitamin.
  • vitamin metabolites include compounds in which a hydroxyl group is added to the vitamin as described above, and conjugates (eg, glucuronic acid conjugates, sulfate conjugates, glutathione conjugates, acetyl conjugates, amino acid conjugates). Is mentioned.
  • the low molecular weight substance may further be a vitamin-like therapeutic drug or a metabolite thereof.
  • the low molecular weight substance may be a steroid compound.
  • a steroid compound refers to a compound having a steroid skeleton (cyclopentanoperhydrophenanthrene skeleton).
  • Steroid compounds include steroid hormones and derivatives thereof that retain the steroid skeleton (eg, synthetic steroids such as anabolic steroids, anti-androgenic and anti-follicular hormone agents).
  • steroid hormones include male hormones, follicular hormones, luteinizing hormones, and corticoids (eg, glucocorticoids and mineralocorticoids), with follicular hormones being preferred.
  • Examples of follicular hormones include estrone, estradiol, and estriol.
  • the low molecular weight substance may also be a metabolite of a steroid compound.
  • the metabolite of the steroid compound include a compound in which a hydroxyl group is added to the steroid compound as described above, and a conjugate.
  • the conjugate for example, a glucuronic acid conjugate, a sulfate conjugate (eg, a hydroxyl group at either the 3-position or 17-position of estradiol, or a hydroxyl group at both the 3-position and 17-position was conjugated with a sulfate group).
  • Compound glutathione conjugates, acetyl conjugates, and amino acid conjugates.
  • the low molecular weight substance may further be a steroid compound-like therapeutic drug (eg, estramustine) or a metabolite thereof (eg, estromustine). *
  • the low molecular weight substance may be an amino acid compound.
  • An amino acid compound refers to a compound having an amino group and a carboxyl group.
  • amino acid compounds include ⁇ -amino acids (eg, glycine, alanine, asparagine, cysteine, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, aspartic acid, glutamic acid, arginine, Histidine, lysine, ornithine, citrulline), ⁇ -amino acids (eg, ⁇ -alanine), ⁇ -amino acids (eg, ⁇ -aminobutyric acid), and derivatives thereof that retain amino and carboxyl groups.
  • ⁇ -amino acids eg, glycine, alanine, asparagine, cysteine, glutamine, isoleucine, le
  • the amino acid compound may be L-form or D-form.
  • the low molecular weight substance may also be a metabolite of an amino acid compound.
  • the metabolite of the amino acid compound include a compound in which a hydroxyl group is added to the amino acid compound as described above, and a conjugate as described above.
  • the low molecular weight substance may further be an amino acid compound-like therapeutic drug or a metabolite thereof.
  • the amino acid compound may be a tyrosine derivative biosynthesized from tyrosine.
  • the tyrosine derivative include thyroid hormone (eg, triiodothyronine, thyroxine).
  • the tyrosine derivative may also be a metabolite of thyroid hormone. Examples of thyroid hormone metabolites include compounds in which a hydroxyl group is added to thyroid hormone and conjugates as described above.
  • the low molecular weight substance may further be a thyroid hormone-like therapeutic drug or a metabolite thereof.
  • the antibody of the present invention may be provided in a form dissolved in a solution, or may be provided in a form fixed to a support.
  • the support include particles (eg, magnetic particles), membranes (eg, nitrocellulose membrane), glass, plastic, metal, plates (eg, multiwell plates), and devices.
  • the antibody of the present invention may also be provided in a form impregnated in a medium such as filter paper.
  • the antibody of the present invention can be obtained by preparing the transformed cell of the present invention that expresses the antibody of the present invention and then culturing the transformed cell of the present invention.
  • the host of the transformed cell is not particularly limited as long as, for example, the antibody of the present invention can be expressed.
  • prokaryotic cells eg, E. coli
  • eukaryotic cells eg, single cells such as yeast, and multicellular cells
  • Biological cells e.g., single cells such as yeast, and multicellular cells
  • cells derived from any animal can be used as the host. Examples of such animals include birds (eg, chickens, quails, ostriches), mammals (eg, humans, monkeys, mice, rats, hamsters, rabbits, cows, horses, sheep, donkeys, pigs).
  • the host may be a cell commonly used for commercial production of high-quality proteins (eg, protein drugs such as antibodies), such as Chinese hamster ovary (CHO) cells and sub-lines thereof (eg, CHO-K1, DG44), baby hamster kidney (BHK) cells and sub-lines thereof.
  • high-quality proteins eg, protein drugs such as antibodies
  • CHO Chinese hamster ovary
  • sub-lines thereof eg, CHO-K1, DG44
  • BHK baby hamster kidney
  • the transformed cell of the present invention can be prepared by introducing one or two vectors expressing IgM light chain and modified IgM heavy chain into the host. Specifically, such preparations involve introducing an IgM light chain expression vector and a modified IgM heavy chain expression vector together into a host or expressing a single IgM light chain and a modified IgM heavy chain. This can be done by introducing one expression vector into the host.
  • a single expression vector may be an expression vector in which the polynucleotide encoding the IgM light chain and the polynucleotide encoding the modified IgM heavy chain are each linked to different promoters, or the IgM light chain and the modified IgM heavy It may be an expression vector in which a polynucleotide encoding a chain is linked to one promoter.
  • expression vectors include plasmids, viral vectors (eg, adenovirus, retrovirus), artificial chromosomes, and integrated vectors (eg, retrovirus).
  • An expression vector is also a vector for transient expression or constitutive (ie, stable) expression.
  • the present invention also provides a method for measuring a target substance using the antibody of the present invention.
  • the measurement method of the present invention includes measuring a target substance in a sample.
  • the measurement method of the present invention is useful for tests such as clinical tests and biological assays. *
  • the sample is a sample containing or suspected of containing the target substance.
  • the origin of the sample is not particularly limited, and may be a biological sample derived from a living organism, or an environmental sample.
  • organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), insects, microorganisms, and plants.
  • the biological sample may also be serum, blood, plasma, saliva, tissue or cell extract.
  • the environmental sample include samples derived from soil, seawater, or fresh water.
  • the sample may be subjected to other processes before being subjected to the measurement method of the present invention. Examples of such treatment include centrifugation, extraction, filtration, precipitation, and fractionation.
  • the measurement method of the present invention can be performed qualitatively or quantitatively.
  • the measurement method of the present invention can also be performed by an immunological technique.
  • immunological techniques include enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Examples thereof include a magnetic particle method, an immunochromatography method, a luminescence immunoassay method, a spin immunoassay method, a Western blot method, and a latex agglutination method.
  • the heavy chain of the modified antibody is a complex (affinity complex) of 25OH VD3 and anti-25OH VD3 antibody (primary antibody) prepared by the method disclosed in WO2013 / 042426.
  • Two types of chicken-derived IgM to be recognized (antibody 1 and antibody 2) were prepared by fusing a heavy chain from which a C-terminal region having a different length was fused with a protein (ALP, molecular weight: 140 kDa).
  • two chicken-derived IgMs that recognize human IgG prepared using human IgG as an antigen and using the ADLib (Autonomous Diversifying Library) system (eg, see WO2004 / 011644) (antibody 3, In antibody 4), a heavy chain in which the C-terminal region having a different length was deleted and fused with a protein (ALP, molecular weight: 140 kDa) was used as the heavy chain of the modified antibody.
  • ALP molecular weight: 140 kDa
  • the capture description in Table 1 is as follows. 1) Although antibodies 1 to 4 have different amino acid sequences in the variable region (V ⁇ ), the amino acid sequences in the constant regions (C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4) are completely identical. For example, the relationship between antibodies 1 and 2 will be described. The number of amino acid residues constituting the variable regions of antibodies 1 and 2 is six different. Therefore, with reference to the start and end amino acid residue positions (position n) of each constant region of antibody 1, the start and end amino acid residue positions of antibody 2 constant positions are “(n + 6) position”. is there.
  • a cysteine residue involved in the cross-linking of the heavy chain and the light chain by disulfide bond is present in C ⁇ 1 (antibody 1: cysteine at position 160; antibody 2: cysteine at position 166: antibody 3: cysteine at position 158) Antibody 4: cysteine at position 157).
  • Cysteine residues involved in cross-linking between heavy chains by disulfide bonds are present in C ⁇ 2 (antibody 1: cysteine at position 347; antibody 2: cysteine at position 353: Antibody 3: Cysteine at position 345; Antibody 4: Cysteine at position 344).
  • Cysteine residues involved in cross-linking between IgM monomers by disulfide bonds are present in C ⁇ 3 (antibody 1: cysteine at position 426; antibody 2: cysteine at position 432) Antibody 3: cysteine at position 424; antibody 4: cysteine at position 423).
  • Example 1 Expression and secretion of a modified antibody having a modified IgM heavy chain
  • the antibody heavy chain (H chain) fragment gene lacking the C-terminal region of different length and the alkaline phosphatase (ALP) gene were linked (5 'Terminal side: H chain fragment: 3' terminal side: ALP.
  • ALP alkaline phosphatase
  • Expression vectors (6 types of expression vectors for antibodies 1 and 2, respectively), and IgM antibody light chain (L chain ( ⁇ )) full length
  • the expression vector was cotransfected into CHO cells to express the modified antibody transiently.
  • the culture supernatant of the obtained CHO cells was analyzed by Western blotting (WB).
  • the prepared modified antibody is indicated by “number + protein (ALP)” for the modified IgM heavy chain.
  • 250ALP is a "H chain composed of amino acid residues 1 to 250 of the IgM antibody H chain (in other words, deleted from the 251st amino acid residue to the C-terminal amino acid residue). It means a modified antibody consisting of “a fusion of H chain) and protein (ALP)” and “full length of IgM antibody L chain”. As a result, it was confirmed that at least a part of all of the prepared modified antibodies was localized in the membrane fraction (M) (FIGS. 1 and 2). This indicates that all modified antibodies are expressed.
  • modified antibodies that were found to be secreted into the culture supernatant (S) were three modified IgM heavy chains (derived from antibody 1) corresponding to 250 ALP, 342 ALP, and 366 ALP, and 256 ALP, 348 ALP, and 372 ALP. Only those with the corresponding three modified IgM heavy chains (derived from antibody 2) (FIGS. 1 and 2).
  • Example 2 Association of modified IgM heavy chain and IgM light chain
  • the culture supernatant of CHO cells obtained in Example 1 was subjected to WB after Native PAGE. As a result, the position of the band detected using the anti-ALP antibody was the same as the position of the band detected using the anti-L chain antibody (data not shown).
  • the following assay systems AD were then used to assess whether the modified IgM heavy chain was associated with the IgM light chain.
  • As the modified antibody the culture supernatant of CHO cells obtained in Example 1 containing antibody 1 (366ALP) or antibody 2 (372ALP) was used.
  • Anti-L chain antibody (derived from mouse) was immobilized in the well of an ELISA plate.
  • the modified antibody, followed by anti-L chain antibody (derived from mouse) was added to the well and reacted.
  • an HRP-labeled anti-rabbit Ig antibody and then TMB (substrate) were added to the well, an enzyme reaction with HRP was performed, and then the reaction was stopped with sulfuric acid. Finally, the absorbance at 450 nm was measured. As a result, signals were detected by assay systems A to D (FIG. 3). Therefore, it was confirmed that the modified IgM heavy chain is associated with the IgM light chain.
  • Example 3 Confirmation of function of modified antibody (1)
  • the same method as that disclosed in the examples of WO2013 / 042426 was used. Therefore, measurement of 25OH VD3 was performed by ELISA.
  • an expression vector (three types of expression vectors for antibody 2) in which a gene of an antibody heavy chain (H chain) fragment from which a C-terminal region having a different length is deleted and an alkaline phosphatase (ALP) gene are linked, and An IgM antibody light chain (L chain ( ⁇ )) full-length expression vector was co-transfected into CHO cells, and the modified antibody derived from antibody 2 was transiently expressed. Subsequently, the assay was performed using the culture supernatant of the obtained CHO cells. As a result, the modified antibody having two modified IgM heavy chains corresponding to 256ALP and 372ALP showed high absorbance values.
  • modified antibodies have both the function of the original antibody (multimeric IgM) (secretory ability and binding ability) and the function of protein (ALP) (enzyme activity) as monomers ( FIG. 4).
  • ALP protein
  • amino acid residues in the CH1 and CH2 adjacent regions located on the N-terminal side of the heavy chain bridged cysteine (position 353), and the CH3 upstream region located on the C-terminal side of the heavy chain bridged cysteine (position 353) It was confirmed that a modified monomeric antibody having a C-terminal deleted constant region having the amino acid residue in the middle as a C-terminal amino acid residue can have both the functions of the original antibody and the protein.
  • Example 4 Confirmation of function of modified antibody (2) Expression vector (11 types different from Examples 1 to 4 for antibody 2) in which the gene of antibody heavy chain (H chain) fragment lacking the C-terminal region of different length and the gene of alkaline phosphatase (ALP) are linked And six expression vectors similar to those in the previous examples), and an IgM antibody light chain (L chain ( ⁇ )) full-length expression vector were cotransfected into CHO cells and derived from antibody 2. The modified antibody was expressed transiently. 25OH VD3 was measured using the obtained culture supernatant of CHO cells. The 25OH VD3 was measured by ELISA according to the same method as disclosed in the examples of WO2013 / 042426.
  • the modified antibodies having modified IgM heavy chains were 357ALP, 362ALP, 367ALP, 372ALP, 373ALP, 374ALP, and 375ALP (FIGS. 5 and 6). Further, from the results of WB and ELISA (FIGS. 5 and 6), for these modified antibodies, a certain degree of correlation was confirmed between the expression level and the activity. Since 250ALP also showed a low count, it was confirmed that the function was retained slightly (FIGS. 5 and 6).
  • the position of the C-terminal amino acid residue in the heavy chain constant region is determined from the viewpoint of good retention of the function (secretory ability and binding ability) of the original antibody and the function (activity) of the protein fused to the antibody. It is thought that it is important to be in the region adjacent to CH1, CH2, or the region on the C-terminal side of the heavy chain bridged cysteine (particularly, the region adjacent to CH2 • CH3 or the upstream region of CH3). In particular, in order to obtain a large amount of a modified antibody that can have an excellent function in the culture supernatant, it is in the region on the C-terminal side of the heavy chain bridged cysteine (particularly, the CH2 ⁇ CH3 adjacent region or the CH3 upstream region). It can be important.
  • Example 5 Production and evaluation of modified antibodies derived from antibodies capable of binding to different antigens Genes of antibody heavy chain (H chain) fragments lacking the C-terminal region of different length and alkaline phosphatase (ALP) Expression vectors (5 ′ end side: H chain fragment: 3 ′ end side: ALP, one expression vector for each of antibodies 3 and 4) linked with the gene, and IgM antibody light chain (L chain ( ⁇ )) The full-length expression vector was co-transfected into CHO cells to transiently express the modified antibodies, antibody 3 (364ALP) and antibody 4 (363ALP). The constant regions of antibody 3 (364ALP) and antibody 4 (363ALP) are identical to those of antibody 2 (372ALP) (see Table 1).
  • ELISA was performed using the culture supernatant of the obtained CHO cells.
  • the ELISA was performed according to the following procedure. To the wells of the assay plate, only human IgG or buffer at a concentration of 5 ⁇ g / mL was added and incubated at 4 ° C. overnight. The wells were washed 3 times with TBST, then blocked with 1% BSA / TBS for 1 hour and then washed 3 times with TBST. The culture supernatant of CHO cells was added to the well and allowed to stand for a certain time, and then washed with TBST three times. AMPPD was added to the well, and a luminescence reaction was performed. Finally, the luminescence count was measured.
  • the modified monomeric antibody having a C-terminal deleted constant region as a group has functions of the original antibody (multimeric IgM) (secretory ability and ability to bind to human IgG) and protein (ALP) function (enzyme activity). It was confirmed to have both.
  • the modified monomer antibody of the present invention in which both the function of the original antibody and the function of the protein are maintained can be produced by modifying it so as to have a C-terminal deletion constant region as described above. From the above, the present invention is considered useful for producing modified monomeric antibodies against various antigens.

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Abstract

La présente invention concerne un IgM modifié par une protéine, qui résout les problèmes spécifiques à l'IgM, qui est un anticorps oligomère. Plus précisément, la présente invention concerne un IgM monomère modifié contenant une chaîne légère d'IgM et une chaîne lourde d'IgM, la chaîne lourde d'IgM modifié contenant A) une région de chaîne lourde configurée à partir d'une région variable et d'une région constante à délétion C-terminale (par exemple une région constante ayant en tant que résidu d'acide aminé C-terminal un résidu d'acide aminé dans le domaine adjacent à CH1/CH2, dans le domaine adjacent à CH2/CH3 ou dans les domaines en amont en CH3) et B) une protéine intégrée avec la région de chaîne lourde ; des cellules transformées (par exemple des cellules de mammifère) qui expriment un tel IgM monomère modifié ; et analogues.
PCT/JP2015/055118 2014-02-25 2015-02-24 Igm monomère modifié Ceased WO2015129651A1 (fr)

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WO2021193870A1 (fr) * 2020-03-26 2021-09-30 デンカ株式会社 Réactif contenant un anticorps ayant une délétion partielle de région fc

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KR20190137821A (ko) * 2017-04-07 2019-12-11 아이쥐엠 바이오사이언스 인코포레이티드 보체-의존성 세포용해 효과기 기능의 조정을 위한 변형된 인간 IgM 불변 영역
JP2020512820A (ja) * 2017-04-07 2020-04-30 アイジーエム バイオサイエンシズ インコーポレイテッド 補体依存性細胞溶解のエフェクター機能の調節のための修飾されたヒトＩｇＭ定常領域
US11401337B2 (en) 2017-04-07 2022-08-02 Igm Biosciences, Inc. Modified human IgM constant regions for modulation of complement-dependent cytolysis effector function
KR102696143B1 (ko) 2017-04-07 2024-08-21 아이쥐엠 바이오사이언스 인코포레이티드 보체-의존성 세포용해 효과기 기능의 조정을 위한 변형된 인간 IgM 불변 영역
WO2021193870A1 (fr) * 2020-03-26 2021-09-30 デンカ株式会社 Réactif contenant un anticorps ayant une délétion partielle de région fc
JPWO2021193870A1 (fr) * 2020-03-26 2021-09-30
CN115298544A (zh) * 2020-03-26 2022-11-04 电化株式会社 含有Fc区域的一部分缺失的抗体的试剂
EP4130032A4 (fr) * 2020-03-26 2024-03-13 Denka Company Limited Réactif contenant un anticorps ayant une délétion partielle de région fc
JP7626753B2 (ja) 2020-03-26 2025-02-04 デンカ株式会社 Ｆｃ領域の一部を欠損した抗体を含む試薬
CN115298544B (zh) * 2020-03-26 2026-02-24 电化株式会社 含有Fc区域的一部分缺失的抗体的试剂

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