JP6512214B2 - Modified monomer IgM - Google Patents

Description

  The present invention relates to a modified monomeric IgM, a transformed cell, and a method of measuring a target substance.

  It is known that by combining an antibody with a predetermined protein, an antibody having a function of the protein can be produced. For example, methods for producing labeled antibodies for enzyme immunoassays are generally performed by conjugating the antibodies to an enzyme. It is known that the antibody used for producing a conjugate is preferably so as to have a simple structure, and an F (ab ') fragment obtained by pepsin digestion and reduction of IgG1 through its free cysteine residue Methods for conjugation with enzymes are widely used.

  In addition, in order to produce an antibody to which a function of a protein has been imparted, it has been attempted to fuse the antibody with a protein instead of conjugation of the antibody and the protein (Patent Documents 1 to 3).

JP, 2010-17113, A JP-A-8-70875 Japanese Patent Application Laid-Open No. 10-323187

Multimeric IgM (present as a pentamer or hexamer) is unstable as it tends to aggregate. In addition, when used as an antibody for immunoassay, it is likely to cause a nonspecific reaction with a substance in a sample such as serum, causing a false positive. Thus, the use of multimeric IgM as antibody to be modified by proteins is generally not favored.
Also, 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 method, there are many sites that can be labeled. As a result, as the obtained protein-labeled multimeric IgM (conjugate), non-homogeneous ones can be obtained in which the labeling site of multimeric IgM with protein and the number of labels are various. Therefore, there is a problem that it is difficult to reduce the lot-to-lot difference of the conjugate.
Furthermore, although it is possible to prepare F (ab ') fragments of IgM by pepsin digestion, it is difficult and inefficient to control the reaction for preparing such fragments.
Also, recombinant IgM produced using animal cells as a host often has problems with multimer formation. Therefore, it would be beneficial if it were possible to avoid multimer formation which could cause such problems.
Accordingly, it is an object of the present invention to provide a protein-modified IgM that overcomes the problems unique to multimeric antibody IgM.

  As a result of intensive investigations, the present inventors demonstrate that the modified IgM in which a protein is fused to a predetermined modified constant region exerts the function of an antibody as a monomer despite the fact that the antibody originally functions as a multimer, In addition, they found that the function of the fused protein was well maintained, and completed the present invention.

That is, the present invention is as follows.
[1] an IgM light chain, and a modified IgM heavy chain,
Modified monomeric IgM, wherein the modified IgM heavy chain comprises: A) a heavy chain region comprised 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.
[4] The modified monomeric IgM of [1], wherein the constant region is a constant region having an amino acid residue in a CH3 upstream region as a C-terminal amino acid residue.
[5] The modified monomer IgM of any one of [1] to [4], wherein the protein is an enzyme.
[6] The modified monomeric IgM of any one of [1] to [5], wherein the IgM light chain and the heavy chain region of the modified IgM are derived from birds.
[7] The modified monomeric IgM of [6], wherein the avian is a chicken.
[8] A transformed cell which expresses the modified monomer IgM of any of [1] to [7].
[9] The cell of [8], wherein said cell is a mammalian cell.
[10] A method for measuring a target substance, which comprises measuring a target substance in a sample using the modified monomer IgM of any of [1] to [7].

  The antibody of the present invention is well secreted in the culture supernatant and can retain its ability to bind to an antigen. The antibody of the present invention is useful for greatly simplifying the production process because it can be easily prepared by purification from culture supernatant.

FIG. 1 is a diagram showing Western blotting of various modified antibodies having a fusion protein (modified IgM heavy chain) of an IgM (antibody 1) heavy chain and ALP deleted of C-terminal regions of different lengths. Abbreviations are as follows (Conj: conjugate; ALP: alkaline phosphatase; S: culture supernatant; M: membrane fraction, and so on). FIG. 2 is a diagram showing western blotting of various modified antibodies having a fusion protein (modified IgM heavy chain) of an IgM (antibody 2) heavy chain and ALP deleted of C-terminal regions of different lengths. FIG. 3 shows the association of a modified IgM heavy chain with an IgM light chain. PC: positive control; NC: negative control; BSA: bovine serum albumin. Conjugates for positive controls were made by conjugating ALP with pentameric IgM (an antibody that recognizes the affinity complex of 25OH VD3 and anti-25OH VD3 antibodies) from chicken produced by CHO cells. Conjugates for PC, NC, BSA and positive control are the same as below. FIG. 4 shows the measurement of the affinity complex (indirectly, 25OH VD3) of 25OH VD3 and anti-25OH VD3 antibody by a modified antibody (derived from antibody 2) having a modified IgM heavy chain. pNPP was used as a substrate for ALP. pNPP is converted by an enzymatic reaction into para-nitrophenol which absorbs at a wavelength of 405 nm. In this assay, the absorbance at 405 nm was measured. FIG. 5 shows western blotting of various modified antibodies (derived from antibody 2) having a modified IgM heavy chain. FIG. 6 shows the measurement of the affinity complex (indirectly, 25OH VD3) of 25OH VD3 and anti-25OH VD3 antibody by various modified antibodies (derived from antibody 2) having a modified IgM heavy chain. is there. AMPPD was used as a substrate for ALP. AMPPD is decomposed by an enzyme reaction to generate light. In this assay, the luminescence count was measured. FIG. 7 shows measurement of human IgG with 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 modified monomeric IgM. Hereinafter, the modified monomer IgM of the present invention is abbreviated as the antibody of the present invention, as necessary.

  The antibodies of the invention may be derived from IgM. As IgM, those derived from any animal having the ability to produce IgM can be used. Such animals include, for example, birds (eg, chicken, quail, ostrich), mammals (eg, human, monkey, mouse, rat, hamster, rabbit, cow, horse, sheep, donkey, pig) .

  The antibodies of the invention comprise IgM light chains. IgM light chains are comprised of a variable region (VL) and a constant region (CL). Light chains include, for example, lambda chains and kappa chains.

  Antibodies of the invention also include modified IgM heavy chains. A normal IgM heavy chain is comprised of a variable region (VH) and a constant region (CH). The IgM heavy chain CH is composed of four domains (CH1, CH2, CH3 and CH4). The assignment of the domains (CH1, CH2, CH3 and CH4) in the constant region of the heavy chain of an IgM antibody can be determined based on the amino acid sequence of the heavy chain by common technical knowledge in the art.

  The constant regions of IgM are known to be conserved in certain animal species. That is, the constant region of IgM for one antigen produced by a particular animal species may be identical to the constant region of IgM for another antigen produced by that animal species. This is because IgM has only one subtype without any subtype, and the μ gene encoding the constant region of IgM is identical in a particular animal species. For example, the amino acid sequence of the constant region of chicken-derived IgM is disclosed under GenBank accession number or Swiss-prot accession number of 1) X 01613.1, 2) CAA 25762.1, and 3) P1875.2. The amino acid sequences of the constant regions are identical between these IgMs. The amino acid sequences of constant regions etc. disclosed by these accession numbers are shown as SEQ ID NO: 1. In the amino acid sequence of SEQ ID NO: 1, CH1 (Cμ1 domain) corresponds to an amino acid sequence consisting of amino acid residues 1 to 105, and CH2 (Cμ2 domain) consists of amino acid residues 106 to 209 CH3 (Cμ3 domain) corresponds to an amino acid sequence consisting of amino acid residues at positions 210 to 316, and CH4 (Cμ4 domain) corresponds to an amino acid sequence consisting of amino acid residues at positions 317 to 427. The amino acid sequence consisting of the amino acid residues at positions 428 to 446 in the amino acid sequence of SEQ ID NO: 1 is referred to as a C-terminal region.

  By the way, in the amino acid sequence of the constant region of the IgM antibody, due to the mutation of the μ gene associated with the strain or individual difference of the animal and the step after gene rearrangement, for example, the mutation of the μ gene Mutations of slight amino acid residues may occur. Thus, if the constant region of an IgM directed against one antigen produced by a particular animal species is not completely identical to the constant region of an IgM directed against another antigen produced by that animal species, but is "substantially identical". There is also a possibility. In the present invention, the expression "substantially identical" is intended for the entire constant region of IgM the amino acid sequence of the constant region including the mutation of one or several amino acid residues caused by the mutation as described above . Here, the term "one or several amino acid residues" refers to 1 to 40, preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, with respect to the entire constant region of IgM. It means 10, particularly preferably 1 to 5 amino acid residues. Mutations include, for example, substitutions, insertions, deletions and additions.

  When the mutation of the amino acid residue is a substitution, the substitution of the amino acid residue may be a conservative substitution. The term "conservative substitution" refers to the replacement of a given amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, as such a family, amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having non-charged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acid having nonpolar side chain (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having hydroxyl group (eg, alcoholic or phenolic) -containing side chains (eg, Example, serine, thread Nin, tyrosine), and amino acids (e.g. having sulfur-containing side chains, cysteine, methionine) and the like. Preferably, conservative substitutions of amino acids include substitution between aspartic acid and glutamic acid, substitution between arginine and lysine and histidine, substitution between tryptophan and phenylalanine, phenylalanine and valine. , Leucine, isoleucine and alanine, and glycine and alanine.

In one embodiment, if the IgM is from chicken, then the constant region of the IgM may be:
a) a polypeptide consisting of an amino acid sequence corresponding to amino acid residues 1-427 in the amino acid sequence of SEQ ID NO: 1; or b) corresponding to an amino acid residue 1-47 in the amino acid sequence of SEQ ID NO: 1 A polypeptide consisting of an amino acid sequence comprising a mutation of one or several amino acid residues in the amino acid sequence.

  In another embodiment, when IgM is derived from chicken, the constant region of IgM may be a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1. The percent identity of the amino acid sequence 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. In the present invention, the term "identity" means a value (Identity) obtained using default setting parameters in the NEEDLE program (J MoI Biol 1970; 48: 443-453) search. Such parameters are Gap penalty = 10, Extend penalty = 0.5, Matrix = EBLOSUM62.

The modified IgM heavy chain comprises the following A) B):
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 listed in A) is comprised of a variable region and a C-terminal deleted constant region. The C-terminal deleted constant region is a constant region in which its C-terminal region has been deleted so as to allow formation of monomeric IgM. Deletion of the C-terminal region is carried out by deleting a contiguous unit (region at 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 is a monovalent monomer (ie, Fab-like molecule) or a bivalent monomer (ie, F). (Ab ') like molecule). A cysteine residue (hereinafter sometimes referred to as "CH1 Cys") involved in the crosslinking of heavy chain and light chain by a disulfide bond may be located in CH1. Cysteine residues involved in the cross-linking between heavy chains by disulfide bonds (hereinafter sometimes referred to as "CH2 Cys") may be located in CH2. The cysteine residue (sometimes referred to hereinafter as "CH3 Cys") involved in the cross-linking between monomeric IgM by disulfide bonds (ie, formation of multimeric IgM) may be located in CH3.

  Therefore, from the viewpoint of retention of the antigen binding site, the C-terminal deleted constant region at least includes the essential region from the N-terminal amino acid residue to CH1 Cys as a constant region, and also from the viewpoint of formation of a modified monomer IgM. And at least the region from CH3 Cys to the C-terminal amino acid residue. The modified monomeric IgM having such a C-terminal deleted constant region can have both the function of an antibody (eg, secretory ability and binding ability) and the function of a protein (eg, enzymatic activity). When the C-terminal deleted 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 may be a bivalent monomer. On the other hand, when the C-terminal deleted constant region does not further include the region from CH1 Cys to CH2 Cys in addition to the essential region as a constant region, in other words, the region from CH2 Cys to C-terminal amino acid residue When deleted, the antibodies of the invention may be monovalent monomers.

Specifically, the C-terminal deleted constant region in the antibody of the present invention includes, for example:
i) A region from which a predetermined amino acid residue in CH1 which is C-terminal to CH1 Cys to the C-terminal amino acid residue is deleted (ie, a region composed of a part of CH1);
ii) the region from CH2 to the C-terminal amino acid residue deleted (ie the region consisting entirely of CH1);
iii) a region from which a predetermined amino acid residue in CH2 is deleted to the C-terminal amino acid residue (ie, a region composed of all of CH1 and a part of CH2);
iv) a region that deletes CH3 to the C-terminal amino acid residue (that is, a region composed of all of CH1 and CH2); and v) a region that deletes the CH3 Cys to the C-terminal amino acid residue ( That is, a region composed of the whole of CH1, the whole of CH2, and a part of CH3).

  In a specific embodiment, the C-terminal deleted constant region is a constant region having, as a C-terminal amino acid residue, an amino acid residue in the CH1.CH2 adjacent region, in the CH2.CH3 adjacent region, or in the CH3 upstream region. . An antibody of the present invention having such a C-terminal deleted constant region may be particularly excellent in both the antibody function (eg, secretory and binding ability) and the protein function (eg, enzyme activity).

  In the present invention, the CH1 · CH2 adjacent region means “−1” at the position of the C-terminal amino acid residue of CH1 with respect to the CH1 · CH2 boundary (CH1-CH2), the N-terminal amino acid residue of CH2 Assuming that the position is “+1” (in this case, no amino acid residue at “position 0” exists, but the amino acid residues at “−1 position” and “+1 position” are linked to each other by an amide bond) Refers to a region consisting of 8 consecutive amino acid residues, ranging from the amino acid residue at “position 4” in the CH1 region to the amino acid residue at “+4” in the CH2 region. A C-terminal deleted constant region having an amino acid residue in the CH1.CH2 adjacent region as a C-terminal amino acid residue may correspond to the region of i), ii) or iii) above.

  The CH1 · CH2 flanking region preferably consists of 6 consecutive amino acid residues ranging from the amino acid residue at position "-3" in the CH1 region to the amino acid residue at "+3 position" in the CH2 region. Region b) a region consisting of 4 consecutive amino acid residues ranging from the amino acid residue at position -2 in the CH1 region to the amino acid residue at position +2 in the CH2 region, or c) in the CH1 region It may be a region consisting of two consecutive amino acid residues, ranging from the amino acid residue at "position -1" to the amino acid residue at "position +1" in the CH2 region.

If the C-terminal deleted constant region is a constant region having an amino acid residue in the CH1.CH2 adjacent region as a C-terminal amino acid residue, the C-terminal deleted constant region may be chicken-derived. In this case, the C-terminal deleted constant region may be:
a1) a polypeptide comprising an amino acid sequence corresponding to an amino acid residue at position 1 to X in the amino acid sequence of SEQ ID NO: 1; or b1) corresponding to an amino acid residue at position 1 to X in an amino acid sequence of SEQ ID NO: 1 A polypeptide consisting of an amino acid sequence comprising a mutation of one or several amino acid residues in the amino acid sequence.
X is any integer from 102 to 109, preferably from 103 to 108, more preferably from 104 to 107, and still more preferably 105 or 106. .
In addition, the C-terminal deleted constant region is a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence corresponding to the amino acid residue at position 1 to X in the amino acid sequence of SEQ ID NO: 1 May be X is as described above. The percent identity of the amino acid sequence 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 performed as described above.

  In the present invention, the CH2.CH3 adjacent region refers to the position of the C-terminal amino acid residue of CH2 "-1" with reference to the CH2.CH3 boundary (CH2-CH3), the N-terminal amino acid residue of CH3. Assuming that the position is “+1” (in this case, no amino acid residue at “position 0” exists, but the amino acid residues at “−1 position” and “+1 position” are linked to each other by an amide bond) Refers to a region consisting of 8 consecutive amino acid residues ranging from the amino acid residue at "position 4" in the CH2 region to the amino acid residue at "+4" in the CH3 region. A C-terminal deleted constant region having an amino acid residue in the CH2 · CH3 adjacent region as a C-terminal amino acid residue may correspond to the region of iii), iv) or v) above.

  The CH2 · CH3 flanking region preferably consists of 6 consecutive amino acid residues ranging from the amino acid residue at position "-3" in the CH2 region to the amino acid residue at "+3 position" in the CH3 region. Region b) a region consisting of 4 consecutive amino acid residues ranging from the amino acid residue "position-2" in the CH2 region to the amino acid residue "position 2 + 2" in the CH3 region, or c) in the CH2 region It may be a region consisting of two consecutive amino acid residues, ranging from the amino acid residue at "position -1" to the amino acid residue at "+1" in the CH3 region.

When the C-terminal deleted constant region is a constant region having an amino acid residue in the CH2 · CH3 adjacent region as a C-terminal amino acid residue, the C-terminal deleted constant region may be chicken-derived. In this case, the C-terminal deleted constant region may be:
a2) a polypeptide comprising an amino acid sequence corresponding to an amino acid residue at position 1 to Y in the amino acid sequence of SEQ ID NO: 1; or b2) corresponding to an amino acid residue at position 1 to Y in an amino acid sequence of SEQ ID NO: 1 A polypeptide consisting of an amino acid sequence comprising a mutation of one or several amino acid residues in the amino acid sequence.
Y is any integer of 206 or more and 213 or less, preferably any integer of 207 or more and 212 or less, more preferably any integer of 208 or more and 211 or less, and still more preferably 209 or 210 .
In addition, the C-terminal deleted constant region is a polypeptide consisting of an amino acid sequence having 90% or more identity to an amino acid sequence corresponding to the amino acid residue at position 1 to Y in the amino acid sequence of SEQ ID NO: 1 May be Y is as described above. The percent identity of the amino acid sequence 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 performed as described above.

  In the present invention, when the position of the N-terminal amino acid residue of CH3 is assumed to be “+1”, the CH3 upstream region extends from the “+1 position” amino acid residue to the “+18 position” amino acid residue in the CH3 region. , A region consisting of 18 consecutive amino acid residues. A C-terminal deleted constant region having an amino acid residue in the CH3 upstream region as a C-terminal amino acid residue may correspond to the region v) above.

  The CH3 upstream region is preferably from the amino acid residue at position "+1" in the CH3 region to "position +17", "+16", "+15", "+14", "+13", "+13 , "+12", "+11", "+10", "+9", "+8", "+7", "+6", "+5", "+4", "+3" Or a region consisting of continuous amino acid residues spanning amino acid residues at position "+2".

When the C-terminal deleted constant region is a constant region having an amino acid residue in the CH3 upstream region as a C-terminal amino acid residue, the C-terminal deleted constant region may be chicken-derived. In this case, the C-terminal deleted constant region may be:
a3) a polypeptide comprising an amino acid sequence corresponding to an amino acid residue at position 1 to Z in the amino acid sequence of SEQ ID NO: 1; or b3) corresponding to an amino acid residue at position 1 to Z in an amino acid sequence of SEQ ID NO: 1 A polypeptide consisting of an amino acid sequence comprising a mutation of one or several amino acid residues in the amino acid sequence.
Z is an arbitrary integer of 210 or more and 227 or less. Preferably, 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 proteins mentioned under B) are fused to the heavy chain region. The fusion is carried out 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. In such a fusion protein, the polynucleotide encoding the protein is linked in reading frame downstream of the polynucleotide encoding the heavy chain region, and then the introduction of the linked polynucleotide into an expression vector, and an expression vector The cells can be prepared by introducing them into cells and finally expressing the fusion protein in the cells. The fusion is performed via a linker consisting of one or several (eg, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5) amino acid residues It is also good.

  In the present invention, a protein is an amino acid polymer that imparts some properties to the antibody of the present invention. As a typical protein, for example, a protein (eg, enzyme) capable of producing a substance capable of producing a detectable physical property (eg, absorbance) or capable of generating a detectable signal (eg, luminescence, fluorescence) Or proteins that can have detectable physical properties or can generate detectable signals (eg, fluorescent proteins). Such proteins include, for example, enzymes capable of producing the aforementioned substances (eg, alkaline phosphatase, peroxidase, luciferase, β-galactosidase), proteins capable of generating a signal (eg, green fluorescent protein, red fluorescent protein) Be As protein, soluble protein is preferable. 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. Examples of such antigens include low molecular weight substances, proteins, polysaccharides and allergens.
The term "low molecular weight material" refers to compounds having a molecular weight of less than 1,500. Low molecular weight substances are natural substances or synthetic substances. 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. Examples of low molecular weight substances include ligands, hormones, lipids, fatty acids, vitamins, opioids, neurotransmitters (eg, catecholamines), nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, amino acids, oligopeptides, and drugs. , Poisons, and metabolites. Hormones include, for example, steroid hormones, thyroid hormones and peptide hormones.
Proteins include soluble proteins such as immunoglobulins, and membrane proteins. Immunoglobulins include, for example, IgG, IgA, IgE, IgD, IgY. As proteins such as immunoglobulins, those derived from any animal (preferably, mammals such as humans) as described above can be used.

  The antibodies of the present invention may recognize affinity complexes. The term "affinity complex" refers to a complex formed by association or aggregation (ie non-covalent association) of two or more agents (see, eg, WO 2013/042426). The at least one factor constituting the affinity complex may preferably be a protein. Such proteins include, for example, proteins capable of affinity binding (eg, antibodies) and proteins capable of aggregating. When the protein which is a factor constituting the affinity complex is an antibody, the antibody of the present invention can be used, for example, as a secondary antibody. The at least one factor constituting the affinity complex may also be a small molecule substance.

  In one embodiment, the low molecular weight substance may be a vitamin. As a vitamin, vitamin A, B1, B2, B6, B12, C, D, E, K is mentioned, for example. Preferably, the vitamin is a fat-soluble vitamin (eg, vitamin A, D, E, K), more preferably vitamin D as shown below. The low molecular weight substance may also be a metabolite of vitamin. As a metabolite of a vitamin, for example, a compound in which a hydroxyl group is added to a vitamin as described above, and a conjugate (eg, glucuronide conjugate, sulfate conjugate, glutathione conjugate, acetyl conjugate, amino acid conjugate) Can be mentioned. The low molecular weight substance may further be a vitamin analog therapeutic drug or a metabolite thereof.

  In another embodiment, the low molecular weight substance may be a steroid compound. The steroid compound refers to a compound having a steroid skeleton (cyclopentanoperhydrophenanthrene skeleton). The steroid compounds include steroid hormones and their derivatives (eg, protein anabolic steroids, synthetic steroids such as anti-androgenic agents and anti-oval hormone agents) that retain the steroid skeleton. Examples of steroid hormones include male hormones, follicle hormones, luteinizing hormone, corticoids (eg, glucocorticoids, mineralocorticoids), with preference given to follicle hormones. Follicular hormones include, for example, estrone, estradiol, estriol. The low molecular weight substance may also be a metabolite of a steroid compound. Metabolites of steroid compounds include, for example, compounds in which a hydroxyl group is added to a steroid compound as described above, and conjugates. As a conjugate, for example, a sulfate group is conjugated to a glucuronide conjugate, a sulfate conjugate (eg, a hydroxyl group at either the 3rd or 17th position of estradiol, or both a 3rd and 17th position) Compounds), glutathione conjugates, acetyl conjugates, amino acid conjugates. The low molecular weight substance may further be a steroid compound analog therapeutic drug (eg, estramustine) or a metabolite thereof (eg, estromustine).

In yet another embodiment, 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. Examples of amino acid compounds include α-amino acids (eg, glycine, alanine, asparagine, cysteine, glutamine, isoleucine, leucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, aspartic acid, glutamic acid, arginine, Histidine, lysine, ornithine, citrulline), β-amino acids (eg, β-alanine), γ-amino acids (eg, γ-aminobutyric acid), and derivatives thereof that retain an amino group and a carboxyl group can be mentioned. 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. Metabolites of amino acid compounds include, for example, compounds in which a hydroxyl group is added to an amino acid compound as described above, and a conjugate as described above. The low molecular weight substance may further be an amino acid compound analog therapeutic drug or a metabolite thereof.
Preferably, the amino acid compound may be a tyrosine derivative biosynthesized from tyrosine. Tyrosine derivatives include thyroid hormones (eg, triiodothyronine, thyroxine). The tyrosine derivative may also be a metabolite of thyroid hormone. Metabolic products of thyroid hormone include, for example, 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 analog therapeutic drug or a metabolite thereof.

  The antibody of the present invention may be provided in the form of solution in a solution, but may be provided in the form of being immobilized on a support. The support includes, for example, particles (eg, magnetic particles), membranes (eg, nitrocellulose membranes), glass, plastics, metals, plates (eg, multiwell plates), devices. The antibody of the present invention may also be provided in the form of being impregnated in a medium such as filter paper.

  The antibody of the present invention can be obtained by preparing a transformed cell of the present invention expressing the antibody of the present invention and then culturing the transformed cell of the present invention.

  The host of transformed cells is not particularly limited as long as it can express the antibody of the present invention, for example, prokaryotic cells (eg, E. coli), and eukaryotic cells (eg, single cells such as yeast, and multicellular) Cells of biological origin can be used. Preferably, the host can utilize cells derived from any animal. Such animals include, for example, birds (eg, chicken, quail, ostrich)), mammals (eg, human, monkey, mouse, rat, hamster, rabbit, cow, horse, sheep, donkey, pig) More preferably, the host may be a cell that is widely used for the commercial production of high quality proteins (eg, protein drugs such as antibodies) Such cells include, for example, Chinese hamster ovary ( CHO) cells and their substrains (eg, CHO-K1, DG44), baby hamster kidney (BHK) cells and their substrains.

  The transformed cells of the present invention can be prepared by introducing into the host one or two vectors expressing IgM light chain and modified IgM heavy chain. Specifically, such preparation may be carried out by introducing an expression vector of IgM light chain and an expression vector of modified IgM heavy chain together into a host or expressing IgM light chain and modified IgM heavy chain. It can be carried out by introducing one expression vector into a host. A single expression vector may be a polynucleotide encoding an IgM light chain, and an expression vector in which a polynucleotide encoding a modified IgM heavy chain is linked to different promoters, or an IgM light chain and a modified IgM heavy chain. It may be an expression vector in which the polynucleotide encoding the chain is linked to one promoter. Expression vectors include, for example, plasmids, viral vectors (eg, adenovirus, retrovirus), artificial chromosomes, and integrative 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 of 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 examinations and biological assays.

  The sample is a sample that contains or is suspected of containing the target substance. The origin of the sample is not particularly limited, and may be a biological sample of biological origin or an environmental sample. Examples of 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. Environmental samples include, for example, samples from soil, seawater or fresh water. The sample may be subjected to other processing before being subjected to the measurement method of the present invention. Such treatment includes, for example, 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 immunological techniques. Such immunological techniques include, for example, enzyme-linked immunosorbent assay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Magnetic particle method, immunochromatography method, luminescence immunoassay method, spin immunoassay method, western blotting method, latex agglutination method may be mentioned.

  EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

In the following examples, as the heavy chain of the modified antibody, a complex (affinity complex) of 25OH VD3 and an anti-25OH VD3 antibody (primary antibody) prepared by the method disclosed in WO2013 / 042426 is used. Among two types of chicken-derived IgM (antibody 1 and antibody 2) to be recognized, heavy chains in which C-terminal regions of different lengths were deleted were fused with a protein (ALP, molecular weight: 140 kDa). Alternatively, two chicken-derived IgM (antibody 3, antibody 3, which recognize human IgG prepared using human IgG as an antigen and using ADLib (Autonomously Diversifying Library) system (for example, see WO 2004/011644) In antibody 4), heavy chains in which C-terminal regions of different lengths were deleted were fused with a protein (ALP, molecular weight: 140 kDa), and used as the heavy chain of the modified antibody.
The relationship between the positions of amino acid residues of the antibodies 1 to 4 and the antibody region is as described in Table 1.

The capture description of Table 1 is as follows.
1) The antibodies 1 to 4 differ in the amino acid sequence of the variable region (Vμ), but the amino acid sequences of the constant regions (Cμ1, Cμ2, Cμ3, Cμ4) are completely identical. For example, to explain the relationship between antibodies 1 and 2, the number of amino acid residues constituting the variable regions of antibodies 1 and 2 differs by six. Therefore, based on the positions (n-th position) of the start and end amino acid residues of each constant region of antibody 1, the positions of the start and end amino acid residues of each constant region of antibody 2 are at the “(n + 6) position”. is there.
2) A cysteine residue involved in cross-linking of heavy and light chains by disulfide bond is present in Cμ1 (Antibody 1: cysteine in position 160; cysteine in position 2: 166 in antibody: cysteine in position 3: 158 Antibody 4: cysteine at position 157).
3) The cysteine residue involved in cross-linking between heavy chains by disulfide bonds (ie formation of IgM monomer) is present in Cμ2 (cysteine at antibody 1: 347; cysteine at antibody 2: 353: Antibody 3: cysteine at position 345; antibody 4: cysteine at position 344).
4) The cysteine residue involved in cross-linking between IgM monomers by disulfide bond (ie formation of IgM multimer) is present in Cμ3 (cysteine at antibody 1: 426; cysteine at antibody 2: 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 gene of an antibody heavy chain (H chain) fragment in which C-terminal regions of different lengths were deleted and the gene of alkaline phosphatase (ALP) were linked (5 'End side: H chain fragment: 3' end side: ALP. The same applies to the following expression vectors (six types of expression vector for each of antibodies 1 and 2) and full-length IgM antibody light chain (L chain (λ)) The expression vector was cotransfected into CHO cells to transiently express the modified antibody. The culture supernatant of the obtained CHO cells was analyzed by western blotting (WB). The modified antibody produced is represented by "number + protein (ALP)" for a modified IgM heavy chain. For example, 250 ALP "deleted H chain consisting of amino acid residues 1 to 250 of IgM antibody H chain (in other words, deleted amino acid residue 251 to C terminal amino acid residue It means a modified antibody consisting of “H chain) fused with protein (ALP)” and “full length of IgM antibody L chain”.
As a result, it was found that at least a part of all the engineered antibodies produced was localized in the membrane fraction (M) (FIGS. 1 and 2). This indicates that all the modified antibodies are expressed. On the other hand, the modified antibodies that were found to be secreted into the culture supernatant (S) were the three modified IgM heavy chains (derived from antibody 1) corresponding to 250ALP, 342ALP, and 366ALP, and 256ALP, 348ALP, and 372ALP. Only those with the corresponding three modified IgM heavy chains (derived from antibody 2) (Figures 1, 2).

Example 2 Association of Modified IgM Heavy Chain with IgM Light Chain The culture supernatant of the 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 omitted).
The following assay systems A to D 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 (366 ALP) or Antibody 2 (372 ALP) was used.
(Assay system A)
Anti-L chain antibody (from mouse) was immobilized in the wells of the ELISA plate. A modified antibody followed by an anti-ALP antibody (from rabbit) was added to the wells and allowed to react.
(Assay system B)
Anti-L chain antibody (from mouse) was immobilized in the wells of the ELISA plate. A modified antibody followed by an anti-H chain antibody (from goat) was added to the wells and allowed to react.
(Assay system C)
Anti-H chain antibody (from goat) was immobilized in the wells of the ELISA plate. A modified antibody followed by an anti-L chain antibody (from mouse) was added to the wells and allowed to react.
(Assay system D)
Anti-ALP antibody (from rabbit) was immobilized in the wells of the ELISA plate. A modified antibody followed by an anti-L chain antibody (from mouse) was added to the wells and allowed to react.
After the reaction in assay systems A to D, HRP-labeled anti-rabbit Ig antibody and then TMB (substrate) were added to the wells, and after enzymatic reaction with HRP, the reaction was stopped with sulfuric acid. Finally, the absorbance at 450 nm was measured.
As a result, a signal was detected by assay systems AD (FIG. 3). Thus, it was confirmed that the modified IgM heavy chain was associated with the IgM light chain.

Example 3: Confirmation of Function of Modified Antibody (1)
In order to confirm the function of the antibody of the present invention for a complex (affinity complex) of 25OH VD3 and an anti-25OH VD3 antibody (primary antibody), a method similar to the method disclosed in the example of WO2013 / 042426 is used. Therefore, measurement of 25OH VD3 was performed by ELISA. First, an expression vector (three types of expression vectors for antibody 2) in which genes of antibody heavy chain (H chain) fragments in which C-terminal regions of different lengths are deleted and a gene of alkaline phosphatase (ALP) are linked, and An IgM antibody light chain (L chain (λ)) full-length expression vector was cotransfected into CHO cells to transiently express the modified antibody derived from antibody 2. Then, an assay was performed using the obtained culture supernatant of CHO cells.
As a result, the modified antibodies having two modified IgM heavy chains corresponding to 256 ALP and 372 ALP showed high absorbance values. This indicates that these modified antibodies have both the function (secretion ability and binding ability) of the original antibody (multimer IgM) as a monomer and the function (enzyme activity) of a protein (ALP) ( Figure 4).
From the above, the amino acid residue in the CH1 · CH2 flanking region located N-terminal to heavy chain bridged cysteine (position 353), and the CH3 upstream region located C-terminal to heavy chain bridged cysteine (position 353) It was confirmed that a modified monomeric antibody having a C-terminal deleted constant region having an amino acid residue as a C-terminal amino acid residue may have both the function of the original antibody and the function of a protein.

Example 4: Confirmation of Function of Modified Antibody (2)
An expression vector in which a gene of an antibody heavy chain (H chain) fragment in which C-terminal regions of different lengths are deleted and a gene of alkaline phosphatase (ALP) were linked (11 types of antibody 2 different from Examples 1 to 4) And 6 different expression vectors as in the previous example, and a full-length expression vector of an IgM antibody light chain (L chain (λ)), which are cotransfected into CHO cells and derived from antibody 2 The modified antibody was transiently expressed. 25OH VD3 was measured using the obtained culture supernatant of CHO cells. The measurement of 25OH VD3 was performed by ELISA according to a method similar to the method disclosed in the example of WO2013 / 042426.
As a result, among the modified antibodies having a modified IgM heavy chain, modified antibodies showing high counts in ELISA were 357 ALP, 362 ALP, 367 ALP, 372 ALP, 373 ALP, 374 ALP, and 375 ALP (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 amount and the activity. The low counts were also observed for 250 ALP, confirming the slight retention of function (FIGS. 5 and 6).
From the above, the position of the C-terminal amino acid residue in the heavy chain constant region is determined from the viewpoint of the function (secretion ability and binding ability) of the original antibody and the good retention of the function (activity) of the protein fused to the antibody. It is considered important to be in the CH1 · CH2 adjacent region, or the region C-terminal side to the heavy chain bridged cysteine (in particular, the CH2 · CH3 adjacent region or the CH3 upstream region). In particular, in order to obtain a large amount of modified antibody which may have excellent functions in the culture supernatant, it is in a region C-terminal to heavy chain cross-linked cysteine (in particular, a CH2 · CH3 adjacent region or a CH3 upstream region) It can be important to be.

Example 5: Preparation and evaluation of modified antibodies derived from antibodies capable of binding to different antigens Genes of antibody heavy chain (H chain) fragments deleted of C-terminal regions of different lengths and alkaline phosphatase (ALP) Expression vector (one type of expression vector for each of antibodies 3 and 4), and IgM antibody light chain (L chain (L chain (L chain (5 'end side: H chain fragment: 3' end side: The same applies hereinafter)) λ)) The full-length expression vector was cotransfected into CHO cells to transiently express modified antibodies Antibody 3 (364 ALP) and Antibody 4 (363 ALP). The constant regions of antibody 3 (364 ALP) and antibody 4 (363 ALP) are identical to those of antibody 2 (372 ALP) (see Table 1). ELISA was performed using the obtained culture supernatant of CHO cells.
ELISA was performed according to the following procedure. To the wells of the assay plate, human IgG or buffer alone at a concentration of 5 μg / mL was added and incubated overnight at 4 ° C. 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 wells and allowed to stand for a fixed time, and then washed three times with TBST. AMPPD was added to the wells to cause a luminescence reaction. Finally, the luminescence count was measured.
As a result, since the luminescence count was well measured in the culture supernatant of CHO cells (FIG. 7), the amino acid residue in the CH3 upstream region located C-terminally to the heavy chain cross-linked cysteine remains C-terminal amino acid residue The modified monomeric antibody having a C-terminal deleted constant region as a group has the function (secretion ability and binding ability to human IgG) of the original antibody (multimer IgM) and the function (enzyme activity) of protein (ALP) It was confirmed to have both. This means that regardless of the type of antigen to which the original antibody is capable of binding (ie, the type of amino acid sequence of the variable region of the original antibody), C-terminal amino acid residues in amino acid residues in the same region as above It is shown that the modified monomeric 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 to have the C-terminal deleted constant region as having.
From the above, the present invention is considered to be useful for the production of modified monomeric antibodies against various antigens.

Claims (5)

A chicken-derived IgM light chain, and a chicken-derived modified IgM heavy chain,
Chicken-derived modified IgM heavy chain, A) variable region and a C-terminal deletion constant region heavy chain region composed, and B) seen containing a soluble protein fused to heavy chain regions,
A C-terminal deleted constant region has, as a C-terminal amino acid residue, a) an amino acid residue in a CH2 · CH3 adjacent region, or b) an amino acid residue in a CH3 upstream region, a C-terminal amino acid residue The modified monomer IgM which is a constant region having as Soluble protein is an enzyme, according to claim 1, wherein the modified monomer IgM. A transformed cell which expresses the modified monomer IgM according to claim 1 or 2 . Transformed cell is a mammalian cell, according to claim 3 transformed cell of. A method of measuring a target substance, which comprises measuring a target substance in a sample using the modified monomer IgM according to claim 1 or 2 .