Method for producing polymeric IgA antibodies and multispecific polymeric IgA antibodies

Abstract

Provided is a method for producing trimeric and tetrameric IgA antibodies, the method including mixing dimeric IgA antibodies and monomeric IgA antibodies.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a multimeric IgA antibody, preferably a multispecific multimeric IgA antibody, and to a multispecific multimeric IgA antibody. [Background technology]

[0002] IgA antibodies, the most abundant antibody produced in the body, function as the first line of defense against respiratory infections such as influenza, which target mucosal tissues. This secretory IgA antibody (SIgA) forms dimers and higher multimers, and it has been shown that tetrameric SIgA in particular has higher functional activity than IgG or monomeric IgA.

[0003] In our previous research, we developed a technique for producing monoclonal tetrameric SIgA by artificially converting the antibody into a secretory form and tetramerizing it, by replacing the constant region of a monoclonal IgG antibody other than the variable region sequence with an IgA frame, thereby obtaining an antibody that retains the antigen recognition site of the monoclonal antibody (Patent Document 1). However, the eight Fab regions of the tetrameric SIgA produced by this technique all have the same variable region and are only reactive to a single epitope.

[0004] Currently, multispecific antibodies, in which multiple Fabs each contain a different variable region, are being put to practical use. Multispecific antibodies are a technology that is expected to be applied in a variety of fields, including not only antibody drugs but also the development of research tools and diagnostic agents. However, their production method is complex, and producing a single multispecific antibody requires a great deal of effort, so the types of multispecific antibodies in practical use are limited. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6564777 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for producing multimeric IgA antibodies, particularly multispecific multimeric IgA antibodies, simply and efficiently. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors discovered that bispecific trimeric and tetrameric IgA antibodies can be produced simply and efficiently by mixing in a test tube a dimeric IgA antibody, a monomeric IgA antibody, and a secretory component (hereinafter also referred to as "SC"), each produced in separate cultured cells, and thus completed the present invention. Furthermore, they found that bispecific trimeric and tetrameric IgA antibodies can also be produced without the addition of the secretory component, although with lower efficiency than when the components are mixed. The technology of the present invention can be applied not only to the production of bispecific trimeric and tetrameric antibodies, but also to the production of monospecific trimeric and tetrameric antibodies, and multispecific trimeric and tetrameric antibodies with more than trispecificity.

[0008] That is, the present invention [1] A method for producing trimeric and tetrameric IgA antibodies, comprising mixing dimeric IgA antibodies and monomeric IgA antibodies; [2] The method according to [1], further comprising mixing the secretory fragments. [3] The method according to [1] or [2], wherein at least bispecific trimeric and tetrameric IgA antibodies are produced, wherein the dimeric IgA antibody comprises a first antigen-binding site and the monomeric IgA antibody comprises a second antigen-binding site. [4] The method according to [3], further comprising mixing another monomeric IgA antibody comprising a third antigen-binding site. [5] The method described in [3], wherein the four Fab regions of the dimeric IgA antibody each contain a first antigen-binding site, and the two Fab regions of the monomeric IgA antibody containing the second antigen-binding site each contain a second antigen-binding site. [6] The method described in [4], wherein the four Fab regions of the dimeric IgA antibody each comprise a first antigen-binding site, the two Fab regions of the monomeric IgA antibody comprising the second antigen-binding site each comprise a second antigen-binding site, and the two Fab regions of the other monomeric IgA antibody each comprise a third antigen-binding site. [7] The method according to any one of [2] to [6], wherein the secretory fragment is a wild-type SC or an SC mutant. [8] The method according to any one of claims [1] to [7], further comprising mixing at least one substance selected from the group consisting of molecular chaperone proteins, disulfide bond isomerase, oxidized glutathione, and reduced glutathione. [9] The method according to any one of [1] to [8], wherein the dimeric IgA antibody and the monomeric IgA antibody are recombinant IgA antibodies produced separately in cultured cells.

[10] The method according to any one of [1] to [9], further comprising separating the produced trimeric IgA antibody and tetrameric IgA antibody from other IgA antibodies.

[11] The method described in

[10] , further comprising separating the produced trimeric IgA antibody and tetrameric IgA antibody.

[12] At least a bispecific trimeric or tetrameric IgA antibody comprising a first Fab region comprising a first antigen-binding site and a second Fab region comprising a second antigen-binding site;

[13] The antibody according to

[12] , further comprising a secretory fragment.

[14] The antibody according to

[12] , which is a polymer of one dimeric IgA antibody molecule comprising the first antigen-binding site and one or two monomeric IgA antibody molecules comprising the second antigen-binding site.

[15] The antibody according to

[13] , which is a polymer of one dimeric IgA antibody molecule containing the first antigen-binding site, one or two monomeric IgA antibody molecules containing the second antigen-binding site, and a secretory fragment.

[16] The antibody according to any one of

[12] to

[15] , comprising four first Fab regions and at least two second Fab regions.

[17] The antibody according to any one of

[13] ,

[15] and

[16] , wherein the secretory fragment is a wild-type SC or an SC mutant; and

[18] A pharmaceutical composition comprising the antibody according to any one of

[12] to

[17] . to provide. [Effects of the Invention]

[0009] The multimeric IgA antibodies of the present invention can have one or more types of Fab regions by incorporating four Fab regions derived from one dimeric IgA antibody and two or four Fab regions derived from monomeric IgA antibodies in a single antibody molecule. Therefore, the present invention provides multimeric IgA antibodies, particularly multispecific multimeric IgA antibodies. The multimeric IgA antibodies and multispecific multimeric IgA antibodies of the present invention have high antigen-binding affinity or neutralizing activity for each antigen. Furthermore, since dimeric IgA antibodies and monomeric IgA antibodies cannot polymerize with each other in the methods of the present invention, when a dimeric IgA antibody containing a first antigen-binding site and a monomeric IgA antibody containing a second antigen-binding site are used, the trimeric and tetrameric IgA antibodies produced by the methods of the present invention are multispecific antibodies with a 100% probability. The present invention provides a simple method for producing multimeric antibodies, particularly multispecific multimeric antibodies, particularly multispecific trimeric and tetrameric antibodies, and particularly bispecific trimeric and tetrameric antibodies, by simply mixing at least two proteins, i.e., a dimeric IgA antibody and a monomeric IgA antibody, in vitro. These antibodies are useful as research tools, delivery tools, diagnostic agents, and pharmaceutical development platforms using antibodies. In the present invention, the efficiency of multimeric antibody production is improved by further mixing SC. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing an example of the production of a bispecific tetrameric IgA antibody of the present application. [Figure 2-1] The amino acid sequence of wild-type SC and the amino acid sequence of the SC12 deletion mutant are shown. [Figure 2-2] The nucleotide sequence of wild-type SC is shown. [Figure 2-3] The nucleotide sequence of the SC12 deletion mutant is shown. [Figure 3] This is a graph showing the ability of SC12 to promote IgA antibody multimerization. In the figure, "mA1 + dA1 + SC12" (shown by the solid line) shows the results of mixing a monomeric IgA1 antibody, a dimeric IgA1 antibody, and SC12. "mA1 + SC12" shows the results of mixing a monomeric IgA1 antibody and SC12. "dA1 + SC12" shows the results of mixing a dimeric IgA1 antibody and SC12. "Tri / Tet IgA" shows the trimeric and tetrameric IgA1 antibodies (controls) produced in cells. [Figure 4] This graph shows a comparison of the ability of SC12 and SC-wt to promote IgA antibody tetramer formation. In the figure, "mA1 + mC-dA1 + SC12" shows the results of mixing a monomeric IgA1 antibody, a fluorescently labeled mCherry-fused dimeric IgA1 antibody, and SC12. "mA1 + mC-dA1 + SC-wt" shows the results of mixing a monomeric IgA1 antibody, a fluorescently labeled mCherry-fused dimeric IgA1 antibody, and wild-type SC. "mC-mA1 + dA1 + SC12" shows the results of mixing a fluorescently labeled mCherry-fused monomeric IgA1 antibody, a dimeric IgA1 antibody, and SC12. "mC-mA1 + dA1 + SC-wt" shows the results of mixing a fluorescently labeled mCherry-fused monomeric IgA1 antibody, a dimeric IgA1 antibody, and wild-type SC. [Figure 5A]This graph shows the efficiency of IgA antibody multimer formation in the presence of SC12, with or without an additive (Supple). In the figure, "mA1 + mC-dA1 + SC12" shows the results from mixing a monomeric IgA1 antibody, a fluorescently labeled mCherry-fused dimeric IgA1 antibody, and SC12. "mA1 + mC-dA1 + SC12 + Supple" shows the results from mixing a monomeric IgA1 antibody, a fluorescently labeled mCherry-fused dimeric IgA1 antibody, SC12, and an additive. "mC-mA1 + dA1 + SC12" shows the results from mixing a fluorescently labeled mCherry-fused monomeric IgA1 antibody, a dimeric IgA1 antibody, and SC12. "mC-mA1 + dA1 + SC12 + Supple" shows the results from mixing a fluorescently labeled mCherry-fused monomeric IgA1 antibody, a dimeric IgA1 antibody, SC12, and an additive. [Figure 5B] This graph shows the efficiency of IgA antibody multimer formation in the presence of wild-type SC, with or without an additive (Supple). In the figure, "mA1 + mC-dA1 + SC-wt" shows the results when a monomeric IgA1 antibody, a fluorescently labeled mCherry-fused dimeric IgA1 antibody, and wild-type SC were mixed. "mA1 + mC-dA1 + SC-wt + Supple" shows the results when a monomeric IgA1 antibody, a fluorescently labeled mCherry-fused dimeric IgA1 antibody, wild-type SC, and an additive were mixed. "mC-mA1 + dA1 + SC-wt" shows the results when a fluorescently labeled mCherry-fused monomeric IgA1 antibody, a dimeric IgA1 antibody, and wild-type SC were mixed. "mC-mA1+dA1+SC-wt+Supple" shows the results of mixing fluorescently labeled mCherry-fused monomeric IgA1 antibody, dimeric IgA1 antibody, wild-type SC, and an additive. [Figure 6] 1 is a graph showing the effect of each additive in promoting IgA multimer formation. [Figure 7] 1 is a graph showing the efficiency of multimer formation depending on the difference in the constant region of IgA antibodies (IgA1 and IgA2m2). [Figure 8] 1 is a graph showing the difference in the efficiency of multimer formation depending on the length of reaction time. [Figure 9]1 is a graph showing the difference in the efficiency of trimeric / tetrameric IgA antibody formation with or without SC using monomeric and dimeric IgA antibodies with two different specificities. In the figure, "Hetero" indicates the combination of monomeric IgA2m2 (mA2) and dimeric IgA1 (dA1), which have different specificities, and "Homo" indicates the combination of mA2 and dA1, which have the same specificity. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1. Terminology Terms used herein have meanings commonly used in the art unless otherwise defined.

[0012] An "antibody" is a protein called immunoglobulin (Ig) that has the ability to specifically bind to an antigen. An antibody has a basic unit consisting of four polypeptide chains: two heavy chains (H chains) and two light chains (L chains) arranged in a Y-shape, and one or more basic units combine to form a single antibody molecule. The four polypeptide chains contain constant regions (heavy chain constant regions: CH1, CH2, CH3, and light chain constant region: CL) whose amino acid sequences change relatively little, and variable regions (heavy chain variable region: VH, and light chain variable region: VL) whose amino acid sequences change significantly.

[0013] The term "multimer" refers to an antibody containing two or more of the basic units described above. As described below, the antibodies produced by the production method of the present application are trimeric and tetrameric antibodies. Therefore, as used herein, "multimer" refers to a trimer or tetramer, or a mixture of trimers and tetramers.

[0014] The "Fab region" is a region containing an antigen-binding site, which consists of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain.

[0015] "Multispecific" means that an antibody can specifically bind to two or more different epitopes (antigenic determinants). "Bispecific" means that an antibody can specifically bind to two different epitopes. "Trispecific" means that an antibody can specifically bind to three different epitopes. Similarly, tetraspecific, pentaspecific, hexaspecific, heptaspecific, and octaspecific mean that an antibody can specifically bind to four, five, six, seven, and eight different epitopes, respectively. Generally, a multispecific antibody contains two or more antigen-binding sites (paratopes), each specific for a different epitope. Here, "different epitopes" may be different epitopes on the same antigen or epitopes on different antigens.

[0016] "Monospecific" means that the antibody has one or more antigen-binding sites that bind to the same epitope of the same antigen.

[0017] An "antigen-binding site" refers to a region on an antibody molecule that binds to an epitope. The antigen-binding site comprises the VH of the heavy chain and the VL of the light chain. As used herein, the first, second, third, and fourth antigen-binding sites refer to regions consisting of different amino acid sequences that bind to epitopes on different antigens or different epitopes on the same antigen.

[0018] "IgA" is one of the antibody (immunoglobulin) isotypes. Herein, it is also referred to as "IgA antibody" or "IgA-type antibody." Human IgA is classified into two subclasses, IgA1 and IgA2, based on differences in the constant region. IgA2 is further divided into three allotypes: IgA2m1, IgA2m2, and IgA2(n). IgA exists primarily as monomeric IgA in serum (serotype IgA), with IgA1 being the major component. When secreted into the mucosa, it exists as dimer or higher polymeric IgA (secretory IgA or SIgA), with IgA2 accounting for approximately half. Polymeric IgA is formed by the polymerization of two or more IgA molecules via a J chain (joining chain) or a J chain and SC. Polymeric IgA exists with and without SC. Dimeric IgA generally refers to a molecule with a heavy chain:light chain:J chain ratio of 4:4:1.

[0019] The "SC (or secretory fragment)" is a glycosylated polypeptide with a molecular weight of approximately 70 kDa, derived from the extracellular domain of the polymeric immunoglobulin receptor (pIgR). It has five immunoglobulin-like domains from the N-terminus, designated D1 to D5. Of these, D1 is essential for binding to polymeric IgA, and the structure of D1, which resembles the CDR (complementarity-determining region) of the immunoglobulin variable region, plays a particularly important role in binding to polymeric IgA.

[0020] pIgR is a type I transmembrane protein belonging to the immunoglobulin superfamily that is expressed on the cell membrane of mucosal epithelial cells on the basal membrane side. It consists of an extracellular domain, a transmembrane region, and a cytoplasmic region. pIgR specifically recognizes and binds to polymeric IgA molecules containing J chains produced by plasma cells present in the lamina propria of the mucosal membrane, takes them up into the epithelial cells, and transports them bound to the apical side. Here, pIgR is cleaved between the extracellular domain and the transmembrane region by a protease in the epithelial cells, and polymeric IgA bound to the extracellular domain of pIgR is secreted into the luminal mucosal layer (secretory IgA or SIgA). The extracellular domain of pIgR after cleavage corresponds to the SC. As used herein, IgA antibodies containing an SC in their molecules are referred to as "secretory IgA antibodies."

[0021] The "J chain" is a polypeptide with a molecular weight of approximately 15 kDa that has an N-linked glycan. The J chain is highly conserved among organisms and is essential for the interaction of polymeric IgA with pIgR.

[0022] 2. Method for producing polymeric IgA antibodies In a first aspect of the present invention, a method for producing trimeric and tetrameric IgA antibodies is provided, which comprises mixing a dimeric IgA antibody and a monomeric IgA antibody (hereinafter also referred to as the "production method of the present application"). As a further aspect of the production method of the present application, a method for producing trimeric and tetrameric IgA antibodies is provided, which comprises mixing a dimeric IgA antibody, a monomeric IgA antibody, and an SC. According to the production method of the present application, one tetrameric IgA antibody is produced by polymerizing one dimeric IgA antibody and two monomeric IgA antibodies. Furthermore, according to the production method of the present application, one tetrameric IgA antibody is produced by polymerizing one dimeric IgA antibody, two monomeric IgA antibodies, and an SC ( FIG. 1 ). According to the production method of the present application, one trimeric IgA antibody is produced by polymerizing one dimeric IgA antibody and one monomeric IgA antibody. Furthermore, according to the production method of the present application, one dimeric IgA antibody, one monomeric IgA antibody, and SC are polymerized to produce one trimeric IgA antibody.

[0023] The dimeric and monomeric IgA antibodies to be mixed may contain different variable regions or may contain the same variable regions. When all the variable regions of the dimeric and monomeric IgA antibodies are identical, the resulting trimeric and tetrameric IgA antibodies are monospecific. When the dimeric and monomeric IgA antibodies contain two or more different variable regions, bispecific or octaspecific multispecific antibodies are obtained. For example, bispecific trimeric and tetrameric IgA antibodies may be obtained using a dimeric IgA antibody in which all four Fab regions contain a first antigen-binding site and a monomeric IgA antibody in which both two Fab regions contain a second antigen-binding site. For example, a trispecific tetrameric IgA antibody may be obtained by using a dimeric IgA antibody in which all four Fab regions contain the first antigen-binding site, a first monomeric IgA antibody in which both of two Fab regions contain the second antigen-binding site, and a second monomeric IgA antibody in which both of two Fab regions contain the third antigen-binding site. For example, a trispecific trimeric IgA antibody, and a tria- or tetraspecific tetrameric IgA antibody may be obtained by using a bispecific dimeric IgA antibody in which two of the four Fab regions contain the first antigen-binding site and the remaining two contain the second antigen-binding site, a first monomeric IgA antibody in which both of two Fab regions contain the third antigen-binding site, and a second monomeric IgA antibody in which both of two Fab regions contain the fourth antigen-binding site. In this way, by modifying the variable regions contained in the dimeric and monomeric IgA antibodies to be mixed, desired bispecific to sextuple-specific trimeric IgA antibodies and desired bispecific to octaplex-specific tetrameric IgA antibodies can be obtained. Accordingly, in a preferred embodiment of the present invention, there is provided a method for producing at least bispecific trimeric and tetrameric IgA antibodies, which comprises mixing a dimeric IgA antibody comprising a first antigen-binding site and a monomeric IgA antibody comprising a second antigen-binding site, or which comprises mixing a dimeric IgA antibody comprising a first antigen-binding site, a monomeric IgA antibody comprising a second antigen-binding site, and an SC.

[0024] In the production method of the present application, dimeric IgA antibodies do not polymerize with each other, and monomeric IgA antibodies do not polymerize with each other. Therefore, when a dimeric IgA antibody containing a first antigen-binding site is mixed with a monomeric IgA antibody containing a second antigen-binding site, or when a dimeric IgA antibody containing a first antigen-binding site is mixed with a monomeric IgA antibody containing a second antigen-binding site and SC, at least bispecific trimeric and tetrameric IgA antibodies are obtained with a 100% probability.

[0025] The dimeric IgA antibody and / or monomeric IgA antibody to be mixed preferably has a hydrophobic amino acid at the 458th amino acid residue in its heavy chain constant region. Examples of hydrophobic amino acids include isoleucine, leucine, methionine, tryptophan, and glycine, with isoleucine being a preferred example. The heavy chain constant region of such an IgA antibody may consist of a native sequence, or may be modified by genetic recombination technology to have a hydrophobic amino acid at the 458th amino acid residue. According to the present invention, when an IgA2m2 antibody, in which the 458th amino acid residue in the heavy chain constant region is isoleucine, was used, the efficiency of multimeric IgA antibody formation was improved compared to when only an IgA1 antibody, in which the 458th amino acid residue in the heavy chain constant region is a non-hydrophobic amino acid, was used (Example 5). The amino acid sequence of the IgA1 constant region is shown in SEQ ID NO: 1, and the amino acid sequence of the IgA2m2 constant region is shown in SEQ ID NO: 2.

[0026] The SC may be either a wild-type SC or an SC mutant. Examples of SC mutants include those containing at least domain D1, such as mutants lacking one or more of domains D2 to D5 or a portion of the D2 to D5 region of the SC. Preferred examples include SC mutants containing at least domains D1 and D2, SC mutants lacking domains D4 and D5, and SC mutants lacking all of domains D3 to D5 (referred to herein as "SC12"). More preferred SCs include wild-type SC (SEQ ID NO: 3) or SC12 (SEQ ID NO: 4), or mutants thereof, and even more preferred SC12 or a mutant thereof. Examples of such SC mutants include polypeptides consisting of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. In the production method of the present application, SC may or may not be used, but SC is preferably used. Use of SC improves the efficiency of trimeric and / or tetrameric IgA antibody formation.

[0027] The origin of the IgA antibody used in the production method of the present application is not particularly limited, and examples include human antibodies, non-human mammalian antibodies, rodent-derived antibodies, and avian-derived antibodies.

[0028] The antigen to which an IgA antibody specifically binds is not particularly limited, and an appropriate IgA antibody may be selected to prepare the desired trimeric and / or tetrameric IgA antibody.

[0029] The subclass of the IgA antibody used in the production method of the present application may be either IgA1 or IgA2, or both IgA1 and IgA2 may be used. Preferably, both IgA1 and IgA2 may be used. For example, a dimeric IgA1 antibody and a monomeric IgA2 antibody may be used. Furthermore, any IgA2 allotype may be used.

[0030] The IgA antibody used in the production method of the present application may be a natural IgA antibody or a recombinant IgA antibody. Here, "recombinant antibody" includes antibodies with modified natural antibody sequences and antibodies artificially produced by genetic engineering, regardless of whether the sequence has been modified. For example, a recombinant IgA antibody produced by converting a non-IgA antibody to an IgA type may be used. Examples of non-IgA antibodies include IgG, IgM, IgE, IgD, and IgY antibodies. The origin of the non-IgA antibody is not particularly limited, and examples include human antibodies, non-human mammalian antibodies, rodent-derived antibodies, and avian-derived antibodies. For example, a recombinant IgA antibody produced by grafting the variable region of an IgG antibody onto the backbone framework of an IgA antibody or by grafting only the CDRs of an IgG antibody onto the CDRs of an IgA antibody may be used. Furthermore, recombinant IgA antibodies also include chimeric recombinant IgA antibodies, which combine the variable region of an antibody derived from one animal species with the constant region of an IgA antibody derived from another animal species, and recombinant antibodies in which the complementarity-determining region (CDR) of an antibody derived from one animal species is grafted onto the CDR of an IgA antibody derived from another animal species. Genetic recombination techniques are well known in the art.

[0031] Therefore, as used herein, the term "IgA antibody" refers to an antibody having at least a portion of an amino acid sequence derived from an IgA antibody.

[0032] The dimeric IgA antibody used in the production method of the present application preferably does not contain an SC, and also contains a J chain.

[0033] The dimeric IgA antibody and the monomeric IgA antibody to be mixed may be produced by methods known in the art. For example, the dimeric IgA antibody and the monomeric IgA antibody may each be produced separately in cultured cells. The dimeric IgA antibody may be produced, for example, by coexpressing the heavy chain protein, light chain protein, and J chain that constitute the IgA antibody in a single host cell. The monomeric IgA antibody may be produced, for example, by coexpressing the heavy chain protein and light chain protein that constitute the IgA antibody in a single host cell.

[0034] Host cells include mammalian cells, insect cells, bacteria, yeast, etc. Mammalian cells include, but are not limited to, 293F cells, CHO cells, etc. Insect cells include, but are not limited to, Sf9 cell line, Sf21 cell line, etc.

[0035] The step of co-expressing the above proteins (heavy chain protein, light chain protein, and J chain in the case of a dimeric IgA antibody; heavy chain protein and light chain protein in the case of a monomeric IgA antibody) in a single host cell can be carried out, for example, by introducing into the cell an expression vector containing nucleic acids encoding these proteins. Expression vectors and introduction methods are well known in the art. Phage vectors, viral vectors, plasmid vectors, etc. can be used as expression vectors, and a vector appropriate for the host can be selected as appropriate by those skilled in the art. After introducing the expression vector, the cells are cultured to express the target protein. Cell culture conditions can be selected as appropriate by those skilled in the art.

[0036] In the production method of the present application, in addition to the dimeric IgA antibody and the monomeric IgA antibody, or the dimeric IgA antibody, the monomeric IgA antibody, and the SC, at least one substance selected from the group consisting of a molecular chaperone protein, a disulfide bond isomerase, oxidized glutathione, and reduced glutathione may be further mixed. Molecular chaperone proteins are proteins that contribute to the folding of protein molecules. Examples of molecular chaperone proteins include the Hsp60 family, Hsp70 family, Hsp90 family, Hsp100 family, low-molecular-weight Hsp family, isomerases, and their cofactors, such as Dnak, DnaJ, GrpE, GroE, GroEL, and GroES. Two or more molecular chaperone proteins may also be used. Disulfide bond isomerases are enzymes that contribute to the formation of disulfide bonds between amino acid residues. Oxidized glutathione creates the oxidative conditions necessary for disulfide bond formation. The use of at least one of these substances promotes tetramer formation of IgA antibodies. In the production method of the present application, preferably, at least two substances selected from the group consisting of a molecular chaperone protein, a disulfide bond isomerase, oxidized glutathione, and reduced glutathione may be further mixed, and more preferably, a molecular chaperone protein, a disulfide bond isomerase, and oxidized glutathione are further mixed. Furthermore, in the production method of the present application, preferably, at least one substance selected from the group consisting of a molecular chaperone protein and a disulfide bond isomerase may be further mixed.

[0037] In the production method of the present application, the mixing of a dimeric IgA antibody and a monomeric IgA antibody, or a dimeric IgA antibody, a monomeric IgA antibody, and an SC, or a dimeric IgA antibody, a monomeric IgA antibody, and at least one substance selected from the group consisting of a molecular chaperone protein, disulfide bond isomerase, oxidized glutathione, and reduced glutathione, or a dimeric IgA antibody, a monomeric IgA antibody, an SC, and at least one substance selected from the group consisting of a molecular chaperone protein, disulfide bond isomerase, oxidized glutathione, and reduced glutathione may be carried out in a buffer adjusted to a pH of 6 to 10, preferably a pH of 7 to 8. Examples of buffers include, but are not limited to, phosphate buffer, Tris-HCl buffer, Good's buffer, etc. The appropriate temperature during mixing can be determined appropriately by those skilled in the art, but is, for example, 25° C. to 45° C., preferably 30° C. to 43° C., more preferably 35° C. to 40° C., for example, around 37° C. The mixing time is not particularly limited and can be determined appropriately by those skilled in the art based on other conditions such as the reaction volume, but may be, for example, 6 hours or more, 12 hours or more, 24 hours or more, or 48 hours or more, and preferably about 24 to 72 hours. The mixing ratio of dimeric IgA antibody and monomeric IgA antibody, or the mixing ratio of dimeric IgA antibody, monomeric IgA antibody, and SC, or the mixing ratio of dimeric IgA antibody, monomeric IgA antibody, and at least one substance selected from the group consisting of molecular chaperone protein, disulfide bond isomerase, oxidized glutathione, and reduced glutathione, or the mixing ratio of dimeric IgA antibody, monomeric IgA antibody, SC, and at least one substance selected from the group consisting of molecular chaperone protein, disulfide bond isomerase, oxidized glutathione, and reduced glutathione is not particularly limited and can be determined appropriately by one skilled in the art.

[0038] The trimeric and tetrameric IgA antibodies thus produced can be separated from other IgA antibodies (non-multimerized dimeric IgA antibodies and monomeric IgA antibodies) based on their characteristics, such as molecular size, and trimeric and tetrameric IgA antibodies can also be separated from each other. For example, size exclusion chromatography, ultrafiltration, ion exchange chromatography, mass spectrometry, etc. can be used to separate trimeric and tetrameric IgA antibodies from other IgA antibodies. Therefore, the production method of the present application may further comprise a step of separating trimeric and tetrameric IgA antibodies from other IgA antibodies.

[0039] Furthermore, the specificity of the trimeric and tetrameric IgA antibodies produced by the production method of the present application can be confirmed by known methods such as ELISA, size exclusion chromatography, and affinity chromatography. Most of the multimeric IgA antibodies produced by the production method of the present application are tetrameric IgA antibodies. Therefore, the production method of the present application produces a mixture of trimeric and tetrameric IgA antibodies that contains a higher proportion of tetrameric IgA antibodies than trimeric IgA antibodies.

[0040] 3. Multispecific multimeric IgA antibodies In a second aspect, the present invention provides at least a bispecific trimeric or tetrameric IgA antibody comprising a first Fab region comprising a first antigen-binding site, and a second Fab region comprising a second antigen-binding site (hereinafter also referred to as the "multispecific multimeric IgA antibody of the present application"). As a further aspect of the multispecific multimeric IgA antibody of the present application, there is provided at least a bispecific trimeric or tetrameric IgA antibody comprising a first Fab region comprising a first antigen-binding site, a second Fab region comprising a second antigen-binding site, and an SC. The multispecific multimeric IgA antibody of the present application is preferably obtained by the production method of the present application described above.

[0041] The multispecific multimeric IgA antibody is preferably a polymer of one dimeric IgA antibody and one or two monomeric IgA antibodies. The multispecific multimeric IgA antibody is more preferably a polymer of one dimeric IgA antibody, one or two monomeric IgA antibodies, and an SC. For example, one dimeric IgA antibody and one or two monomeric IgA antibodies are trimerized or tetramerized via the SC. For example, the dimeric IgA antibody comprises a first antigen-binding site, and the monomeric IgA antibody comprises a second antigen-binding site. In a preferred example, the multispecific multimeric IgA antibody of the present application comprises four first Fab regions and at least two second Fab regions. In a more preferred example, the multispecific multimeric IgA antibody of the present application comprises four first Fab regions, at least two second Fab regions, and an SC. In an even more preferred example, the multispecific multimeric IgA antibody of the present application may be a bispecific trimeric IgA antibody comprising four first Fab regions and two second Fab regions, a bispecific tetrameric IgA antibody comprising four first Fab regions and four second Fab regions, or a trispecific tetrameric IgA antibody comprising four first Fab regions, two second Fab regions, and two third Fab regions. In an even more preferred example, the multispecific multimeric IgA antibody of the present application may be a bispecific trimeric IgA antibody comprising four first Fab regions, two second Fab regions, and an SC, a bispecific tetrameric IgA antibody comprising four first Fab regions, four second Fab regions, and an SC, or a trispecific tetrameric IgA antibody comprising four first Fab regions, two second Fab regions, two third Fab regions, and an SC. In an even more preferred embodiment, the four first Fab regions are derived from a dimeric IgA antibody, and the two or four second or third Fab regions are derived from a monomeric IgA antibody.

[0042] The multispecific multimeric IgA antibody of the present application may be either IgA1 or IgA2, or may contain both. Preferably, the multispecific multimeric IgA antibody of the present application may contain both IgA1 and IgA2. More preferably, the multispecific multimeric IgA antibody may contain four Fab regions derived from dimeric IgA1 and two or four Fab regions derived from monomeric IgA2. Furthermore, the IgA2 contained in the multispecific multimeric IgA antibody of the present application may be IgA2m1, IgA2m2, or IgA2(n).

[0043] The 458th amino acid residue in the heavy chain constant region of the multispecific multimeric IgA antibody of the present application may be a hydrophobic amino acid. Examples of hydrophobic amino acids include isoleucine, leucine, methionine, tryptophan, and glycine, with isoleucine being a preferred example.

[0044] The multispecific multimeric IgA antibody of the present application may be a recombinant IgA antibody. For example, when the multispecific multimeric IgA antibody of the present application is a polymer of one dimeric IgA antibody and one or two monomeric IgA antibodies, or a polymer of one dimeric IgA antibody and one or two monomeric IgA antibodies and SC, either or all of the dimeric IgA antibody and the monomeric IgA antibody may be recombinant IgA antibodies.

[0045] The IgA antibodies and SCs constituting the multispecific multimeric IgA antibodies of the present application are as described above in "2. Method for producing multimeric IgA antibodies."

[0046] 4. Pharmaceutical Compositions In a third aspect, the present invention provides a pharmaceutical composition comprising the multispecific multimeric IgA antibody of the present application (hereinafter also referred to as the "pharmaceutical composition of the present application"). The pharmaceutical composition of the present application preferably comprises the multispecific multimeric IgA antibody of the present application or a mixture of such multispecific multimeric IgA antibodies as an active ingredient.

[0047] The pharmaceutical composition of the present application can be used, for example, for the treatment, prevention, or diagnosis of a disease. The target disease for which the pharmaceutical composition of the present application is used is not particularly limited, and examples include infectious diseases, such as infections caused by pathogens such as parasites, bacteria, fungi, viruses, and abnormal prions, as well as malignant tumors. Further examples of infectious diseases include mucosal infections such as influenza virus infection, respiratory syncytial virus infection, Ebola virus infection, severe fever with thrombocytopenia syndrome (SFTS), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and acquired immunodeficiency syndrome (AIDS).

[0048] The multispecific multimeric IgA antibody contained in the pharmaceutical composition of the present application has an antigen-binding site appropriate for the intended use of the pharmaceutical composition. For example, when used for the diagnosis, prevention, treatment, etc. of a disease, a multispecific multimeric IgA antibody that specifically binds to two different proteins (e.g., a causative protein and a marker protein) associated with a single target disease can be used. For example, the multispecific multimeric IgA antibody contained in the pharmaceutical composition of the present application may specifically bind to the HA protein and the NA protein of influenza virus, respectively. Furthermore, the multispecific multimeric IgA antibody contained in the pharmaceutical composition of the present application may specifically bind to a protein specific to a target cell and a protein effective in treatment, respectively. Examples of antigens or epitopes that can be targeted by the multispecific multimeric IgA antibody contained in the pharmaceutical composition of the present application include, but are not limited to, multiple epitopes of the glycoprotein of Ebola virus, the glycoproteins Gn and Gc of SFTS virus, lymphocyte marker proteins such as CD3 and CD8, and cancer-specific antigens.

[0049] The pharmaceutical composition of the present application can be administered by formulating it into a dosage form such as a powder or a liquid. The pharmaceutical composition of the present application may further contain excipients, additives, thickeners, etc. known in the pharmaceutical field. For example, the pharmaceutical composition of the present application may be administered by spraying onto the nasal mucosa, or by inhalation into the lower respiratory tract using a nebulizer.

[0050] The pharmaceutical composition of the present application may be intended for humans, or may be intended for non-human mammals, for example, livestock animals such as horses, cows, goats, sheep, and pigs, pet animals such as dogs and cats, primates such as chimpanzees, gorillas, and cynomolgus monkeys, and rodents such as mice, rats, and guinea pigs.

[0051] Preferably, any of the heavy chain, light chain, SC, and J chain of the multispecific multimeric IgA antibody contained in the pharmaceutical composition of the present application comprises an amino acid sequence derived from the target animal of the pharmaceutical composition (the target animal type). More preferably, the heavy chain, light chain, SC, and J chain of the multispecific multimeric IgA antibody contained in the pharmaceutical composition of the present application all comprise an amino acid sequence derived from the target animal of the pharmaceutical composition (the target animal type). Here, with respect to the heavy chain and light chain, "of the target animal type" means that the constant regions of the heavy chain and light chain of the multispecific multimeric IgA antibody have the amino acid sequences of the constant regions of the IgA heavy chain and IgA light chain of the target animal. With respect to the SC and J chain, "of the target animal type" means that the SC and J chain of the multispecific multimeric IgA antibody have the amino acid sequences of the SC and J chain of the target animal. The amino acid sequences of the IgA heavy chain, IgA light chain, SC, and J chain may contain mutations as long as they have the desired antigen-binding activity. [Example]

[0052] The present invention will be further described below using examples, but the present invention is not limited to these examples.

[0053] Materials and Preparation 1. Construction of IgA expression plasmid vector An expression vector for the α1 heavy chain (HC), which is the constant region of human IgA1, was constructed as follows. PCR was performed using PrimeSTAR TMPCR was performed using ®MAX DNA Polymerase (Takara Bio, Kusatsu, Japan). Briefly, the human IgA1 antibody constant region gene was amplified by PCR using pFUSE-CHIg-hA1 (InvivoGen) as a template and an appropriate primer set for amplifying the IgA1 antibody constant region, including restriction enzyme sites recognized by XhoI and HindIII. PCR conditions included 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 10 seconds, for 30 cycles. Next, PCR was performed using the human γ1HC expression vector reported by T. Tiller et al. (J Immunol Methods, 329, 112-24, 2008) as a template, with an appropriate primer set for removing the IgG1 antibody constant region γ1HC, excluding the signal sequence, including XhoI and HindIII sites. PCR conditions included 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 30 seconds, for 30 cycles. The PCR product was purified using the MonoFas DNA Purification Kit I (GL Sciences Inc., Tokyo, Japan). The purified expression vector from T. Tiller et al., from which α1HC and γ1HC had been removed, was digested with restriction enzymes XhoI (New England Biolabs) and HindIII-HF (New England Biolabs) at 37°C. The restriction enzyme products were purified using the MonoFas DNA Purification Kit I. The digested DNA was ligated using the DNA Ligation Kit.<Mighty Mix> (Thermo Fisher Scientific), and the entire amount was transformed into Competent Quick DH5α at 42°C. Plasmid extraction was performed using PureYield TM The procedure was carried out using the Plasmid Miniprep System (Promega). The extracted plasmid was sequenced using an Applied Biosystems 3130 Genetic Analyzer. The sequencing reaction was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit, and the plasmid was purified using the BigDye XTerminator. TMAll of the above procedures were carried out according to the attached instructions.

[0054] The human IgA2 allotype, α2m2 HC, was artificially synthesized (GeneArt Strings DNA Fragments) by codon-optimizing the sequence registered in IMGT / GENE-DB (accession numbers for α2m2 HC are M60192 and AJ012264) for human use. To facilitate cloning of the variable region, the codons for the last amino acid, alanine, in the variable region and the first amino acid, serine, in the constant region were altered to create an NheI cleavage site. The synthetic sequence, treated with NheI-HF (New England Biolabs) and HindIII-HF, and α1 HC treated with the same restriction enzymes, were purified using the MonoFas DNA Purification Kit I. The restriction enzyme-treated DNA was ligated using the DNA Ligation Kit<Mighty Mix> (Thermo Fisher Scientific), and a portion was transformed into Competent Quick DH5α at 42°C. Plasmid extraction was performed using PureYield TM The procedure was carried out using the Plasmid Miniprep System (Promega). The extracted plasmid was sequenced using an Applied Biosystems 3130 Genetic Analyzer. The sequencing reaction was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit, and the plasmid was purified using the BigDye XTerminator. TM All of the above procedures were carried out according to the attached instructions.

[0055] Since the human light chain (LC) is common to each antibody isotype, the λ LC expression vector reported by T. Tiller et al. was used.

[0056] 2. Cloning of antibody variable region genes into α1, α2m2 HC, and λ LC expression vectors F045-092, a known antibody clone that binds broadly to influenza virus (H3N2) hemagglutinin (HA), was used. F045-092 was artificially synthesized (GeneArt Strings DNA Fragments) by optimizing the codons for human use from the sequence registered with Nucleotide (accession numbers AB649270 for the heavy chain and AB649271 for the light chain) so that it could be incorporated into expression vectors for α1HC (heavy chain), α2m2HC (heavy chain), and λLC (λ chain). The synthetic sequences were digested with AgeI-HF (all chains), NheI-HF (heavy chain), and XhoI (λ chain) (New England Biolabs), and the expression vectors for α1HC, α2m2HC, and λLC were digested with the same restriction enzymes and purified using the MonoFas DNA Purification Kit I. The restriction enzyme-digested DNA was ligated using the DNA Ligation Kit.<Mighty Mix> (Thermo Fisher Scientific), and a portion was transformed into Competent Quick DH5α at 42°C. Plasmid extraction was performed using PureYield TM The procedure was carried out using the Plasmid Miniprep System (Promega). The extracted plasmid was sequenced using an Applied Biosystems 3130 Genetic Analyzer. The sequencing reaction was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit, and the plasmid was purified using the BigDye XTerminator. TM All of the above procedures were carried out according to the attached instructions.

[0057] 3. Preparation of expression plasmid DNA for fluorescently labeled IgA1 (mC-A1) and IgA2m2 antibody (mC-A2) IgA1Δ and IgA2m2Δ mutants, consisting of the CH2 and CH3 heavy chains of the α1 and α2m2 IgA antibody subclasses, lacking cysteine ​​up to position 241, were generated by PCR using a specific primer set containing restriction enzyme sites and PrimeSTAR Max DNA Polymerase (Takara Bio, Kusatsu, Japan). PCR was also performed using a primer set containing AgeI recognition sequences at the 5' and 3' ends and PrimeSTAR Max DNA Polymerase (Takara Bio) to specifically amplify the fluorescent protein mCherry sequence. Each IgAΔ mutant and mCherry were then digested with AgeI (New England Biolabs) under optimal conditions, followed by agarose gel electrophoresis to isolate the desired product bands. The collected target product was purified using the MonoFas DNA Purification Kit I (GL Sciences Inc., Tokyo, Japan). The purified DNA fragment encoding the IgAΔ mutant and the DNA fragment encoding mCherry were mixed at a ratio of 1:3 (= IgA1Δ:mCherry) and ligated with equal amounts of DNA Ligation Kit<Mighty Mix> Ligation was performed by adding 100% ribonucleotides (Thermo Fisher Scientific) and incubating at 16°C for 30 minutes. The circularized IgAΔ and mCherry were transformed into E. coli DH5α Competent Cells (Takara Bio, Kusatsu, Japan) at 42°C. Plasmid DNA was extracted using the PureYield Plasmid Miniprep System (Promega), and sequence analysis was performed using the Sanger method. mCherry was added to the 5' end of the coding sequences for IgA1Δ and IgA2m2Δ. The IgAΔ mutants fused with the fluorescent protein mCherry were designated mC-A1 and mC-A2 antibodies, respectively. All procedures were performed according to the accompanying instructions.

[0058] 4. Cloning of the J chain and secretory fragment (SC) J chain (GenBank accession no. NM_144646) was artificially synthesized by an artificial gene synthesis service (Eurofins Genomics) with an XhoI recognition sequence and a Kozak sequence added to the 5' end of the coding sequence and a NotI recognition sequence added to the 3' end. Then, restriction enzyme digestion was carried out under optimal conditions using XhoI (New England Biolabs) and NotI-HF (New England Biolabs). The pCXSN vector, an expression vector for mammalian cultured cells, was also digested under the same conditions. The restriction enzyme-digested DNA was ligated using a DNA Ligation Kit.<Mighty Mix> (Thermo Fisher Scientific), and a portion was transformed into Competent Quick DH5α at 42°C. Plasmid extraction was performed using PureYield TM The procedure was carried out using the Plasmid Miniprep System (Promega). The extracted plasmid was sequenced using an Applied Biosystems 3130 Genetic Analyzer. The sequencing reaction was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit, and the plasmid was purified using the BigDye XTerminator. TM All of the above procedures were carried out according to the attached instructions.

[0059] Wild-type SC (SC-wt) was synthesized using 1809 bp (including the signal sequence) from the 5' end of pIgR (GenBank accession no. NM_002644). A DNA fragment (SEQ ID NO: 7) containing an XhoI recognition sequence and a Kozak sequence on the 5' side of the SC sequence, and a HindIII recognition sequence, a Thrombin cleavage sequence, and a 6x His tag on the 3' side was artificially synthesized using GeneArt® Strings DNA Fragments (Life Technologies). The synthesized DNA fragment was subjected to PCR using PrimeSTAR Max DNA Polymerase (Takara Bio, Kusatsu, Japan), and DNA purification was performed. Subsequently, restriction enzyme digestion with XhoI and HindIII was performed under optimal conditions. The pCXSN vector, an expression vector for mammalian cultured cells, was also digested with these enzymes under the same conditions. The digested DNA was ligated using a DNA Ligation Kit.<Mighty Mix> (Thermo Fisher Scientific), and a portion was transformed into Competent Quick DH5α at 42°C. Plasmid extraction was performed using PureYield TM The procedure was carried out using the Plasmid Miniprep System (Promega). The extracted plasmid was sequenced using an Applied Biosystems 3130 Genetic Analyzer. The sequencing reaction was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit, and the plasmid was purified using the BigDye XTerminator. TM All of the above procedures were carried out in accordance with the attached instructions. The gene sequence encoding SC-wt is shown in Figure 2-2.

[0060] 5. Preparation of expression plasmid DNA for SC12 deletion mutants Using the five-domain SC as a template, PCR was performed using specific primers and PrimeSTAR Max DNA Polymerase (Takara Bio, Kusatsu, Japan) under optimal conditions, taking into account the optimal primer set and base length. The SC domains were determined according to Beth et al.'s previous study (eLife, 5, e10640, 2016). After PCR, specific amplification of the desired base length was confirmed by agarose gel electrophoresis. The PCR product was purified using the MonoFas DNA Purification Kit I (GL Sciences Inc., Tokyo, Japan). The purified DNA fragment was phosphorylated with T4 polynucleotide kinase (Takara Bio, Kusatsu, Japan) and self-ligated into a circular form using T4 DNA ligase (Takara Bio, Kusatsu, Japan). After the ligation procedure was completed, the circularized plasmid DNA (SEQ ID NO: 8) encoding the deletion mutant was transformed into E. coli DH5α Competent Cells (Takara Bio, Kusatsu, Japan) at 42°C. Plasmid DNA was extracted using the PureYield Plasmid Miniprep System. The extracted plasmid was sequenced using an Applied Biosystems 3130 Genetic Analyzer. The sequencing reaction was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit, and purification was performed using the BigDye XTerminator. TM All of the above procedures were carried out in accordance with the attached instructions. The gene sequence encoding the SC12 deletion mutant is shown in Figure 2-3.

[0061] 6. Expression of Monomeric and Dimeric IgA Antibodies Monomeric IgA antibodies are composed of heavy and light chains, while dimeric IgA antibodies are composed of heavy, light, and J chains. Plasmid DNA expressing the molecules that make up monomeric and dimeric IgA antibodies was transfected into 2.9 x 10 Expi293F human cells (Thermo Fisher Scientific), which are mammalian cultured cells derived from human kidney cells. 6 The cells / mL were co-transfected with each gene using the Expi293 Expression System Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). After incubation at 37°C, 8% CO2, and 120 rpm with shaking, the cell culture medium was harvested after one week.

[0062] 7. Expression of mC-A1 and mC-A2 antibodies Since the mC-A1 and mC-A2 antibodies do not have light chains, the plasmid DNA encoding each heavy chain and J chain was transfected into 2.9 x 10 Expi293F human cells (Thermo Fisher Scientific), which are mammalian cultured cells derived from human kidney cells. 6 Cells / mL were co-transfected using the Expi293 Expression System Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). After incubation at 37°C, 8% CO2, and 120 rpm with shaking, the cell culture medium was harvested after one week.

[0063] 8. Purification of recombinant IgA antibodies and mC-A1 / 2 The harvested cell culture medium was centrifuged at 3000 rpm for 20 minutes to remove debris, such as cells, and the culture supernatant was collected. This centrifugation was performed twice. The supernatant was then filtered using a glass fiber filter and a Stericup (Merck KGaA, Darmstadt, Germany). Purification of the recombinant IgA antibody and mC-A1 / 2 was performed using CaptureSelect IgA Affinity Matrix (Thermo Fisher Scientific), which specifically recognizes the constant region of human IgA antibodies. Purification was performed according to the manufacturer's instructions. Briefly, the purification method is described below. The column was equilibrated with 10 CV of PBS, and the filtered culture supernatant was loaded onto the column. The column was washed with 10 CV of PBS, and the antibody was eluted with 5 CV of 0.1 M glycine-HCl (pH 3.0). The eluate was neutralized with 1 M Tris-HCl (pH 8.0). Antibody concentration was performed using Amicon® Ultra Centrifugal Filter Devices (Millipore) according to the manufacturer's instructions.

[0064] 9. Separation of Monomeric and Dimeric IgA Antibodies by Gel Filtration Chromatography After concentration, the monomeric and dimeric IgA antibodies were fractionated by gel filtration chromatography. Gel filtration chromatography was performed using Superose 6 10 / 300 GL (GE Healthcare) for fractionation of recombinant IgA antibodies, and Superose 12 10 / 300 GL for fractionation of mC-A1 / 2, according to the manufacturer's instructions. The chromatography conditions were PBS at a flow rate of 0.5 mL / min, column equilibration of 1.5 CV, and elution of 0.2 mL / fraction (total 1.5 CV). Fractions containing monomeric and dimeric IgA antibodies were concentrated using Amicon® Ultra Centrifugal Filter Devices. Concentrations were measured using a NanoDrop.

[0065] 10. Expression and Purification of Recombinant SC12 Plasmid DNA encoding SC12 was transfected into 2.9 x 10 Expi293F human cells (Thermo Fisher Scientific), which are mammalian cells derived from human kidney cells. 6 Transfection was performed using the Expi293 Expression System Kit (Thermo Fisher Scientific) at 1000 cells / mL. The cells were then cultured at 37°C, 8% CO2, and 120 rpm with shaking for one week. The cell culture medium was then harvested. The harvested cell culture medium was then centrifuged at 3000 rpm for 20 minutes to remove debris, and the culture supernatant was collected. This centrifugation step was repeated twice. SC12 in the culture supernatant was purified using an affinity column packed with Ni Sepharose excel (GE Healthcare), which recognizes the C-terminal His tag of each protein. Briefly, the equilibration solution was 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4; the wash solution was 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4; and the elution solution was 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4. After equilibrating the column with 5 CV (column volume) of equilibration solution, the culture supernatant was passed through. Washing was performed with 20 CV of washing solution, elution was performed with 5 CV of elution solution, and re-equilibration was performed with 5 CV of equilibration solution. SC12 was concentrated using Amicon® Ultra Centrifugal Filter Devices (Millipore) according to the manufacturer's instructions.

[0066] Example 1: Extracellular formation of polymeric IgA antibodies Our previous study (Patent Document 1) demonstrated that coexpression of the heavy chain (HC), light chain (LC), J chain (JC), and secretory fragment (SC) constituting trimeric and tetrameric secretory IgA antibodies (tSIgA) in cultured mammalian cells resulted in efficient production of tSIgA. Furthermore, the difference in tSIgA formation efficiency depending on the presence or absence of SC suggested that SC functions as a promoter of IgA antibody trimer and tetramer formation in cultured cells. The ability of SC to promote IgA antibody trimer and tetramer formation was evaluated by measuring trimeric and tetrameric IgA antibodies (Tri / Tet IgA) formed by the interaction of four types of proteins coexisting in the cells. Therefore, we evaluated whether SC functions as a promoter of multimeric IgA antibody formation from isolated proteins produced from separate cells under extracellular conditions, rather than in the limited intracellular environment. In addition, SC12, which is composed of domains 1 and 2 of SC, has been shown to have a higher ability to promote trimer and tetramer formation in cells than SC-wt, so in this example, SC12 was used as the SC.

[0067] The SC12, monomeric IgA1 antibody (mA1), and dimeric IgA1 antibody (dA1) prepared as described above were mixed in phosphate buffer (pH 7.4) to a final concentration of 1.25 mg / mL, and incubated at 37°C for one week using a rotator. The antibody sample was then pretreated using a Cosmos Spin Filter H (Nacalai Tesques). The Tri / Tet IgA produced in the reaction mixture was separated by size exclusion chromatography (SEC) and then detected by UV (280 nm) to assess the degree of antibody polymerization. An Agilent 1260 Infinity HPLC system (Agilent Technologies) was used, and a KW404-4F (Shodex) column was used. Phosphate buffer (pH 7.4) was used as the eluent, and the flow rate was 0.2 mL / min. 9 μL of each IgA-antibody mixture was used. Chromatograms were analyzed using OpenLAB CDS ChemStation Edition (Agilent Technologies).

[0068] The results are shown in Figure 3. In the figure, "mA1 + dA1 + SC12" shows the results when a monomeric IgA antibody, a dimeric IgA antibody, and SC12 were mixed. "mA1 + SC12" shows the results when a monomeric IgA antibody and SC12 were mixed. "dA1 + SC12" shows the results when a dimeric IgA antibody and SC12 were mixed. As controls, trimeric and tetrameric IgA antibodies (Tri / Tet IgA) produced in cells using the method described in Patent Document 1 were used.

[0069] As a result, no trimeric / tetrameric IgA antibody peaks were detected with the combinations of "mA1 + SC12" and "dA1 + SC12." On the other hand, when the three proteins were mixed ("mA1 + dA1 + SC12"), a polymeric IgA antibody peak was detected, which has a larger molecular weight than dA1 and at a detection time equivalent to that of "Tri / Tet IgA" produced under intracellular conditions. These results indicate that the coexistence of mA1 and dA1, as well as the presence of SC12, is necessary for trimeric / tetrameric IgA antibody formation. Furthermore, it was demonstrated that trimeric / tetrameric IgA antibodies can be formed extracellularly, just as they are intracellularly. Furthermore, it was shown that polymerization between monomers and dimers does not occur. Therefore, trimeric / tetrameric IgA antibodies are formed by polymerization of dimeric and monomeric IgA antibodies.

[0070] Example 2: Comparison of polymeric IgA antibody formation in the presence of SC12 and SC-wt As shown in Example 1, multimeric IgA antibodies were formed extracellularly by mixing SC with monomeric and dimeric IgA antibodies, demonstrating that SC contributes to the formation of multimeric IgA antibodies. Furthermore, previous research by the inventors showed that SC12 has a higher ability to promote trimer / tetramer formation than SC-wt under intracellular conditions. Therefore, to explore a method for efficiently producing trimeric / tetrameric IgA antibodies under extracellular conditions, the tetramer formation-promoting abilities of SC12 and SC-wt were compared.

[0071] The SC12, SC-wt, mA1, and dA1 prepared as described above, as well as the mCherry-fused IgA antibody (mC-A1) in which the fluorescent protein mCherry was substituted in the region corresponding to the Fab region of the IgA antibody, were mixed in phosphate buffer (pH 7.4) to a final concentration of 0.5 mg / mL, respectively, as a monomer (mC-mA1) and a dimer (mC-dA1). The mixture was incubated at 37°C for 6 hours using a rotator, and the trimeric / tetrameric IgA produced in the reaction mixture was detected by ELISA.

[0072] The ELISA was performed as follows. 50 μl of recombinant HA (derived from A / Sydney / 5 / 1997 (H3N2), 5 μg HA / mL) was immobilized overnight at 4°C on a 96-well half plate, followed by blocking with 1% BSA-PBS at room temperature for 1 hour. A 3-fold serial dilution of a 200-fold diluted antibody sample was then prepared and added to each well for 2 hours at 37°C. After washing with PBST, the antibody captured on the plate via the HA antigen was reacted with 5 ng / mL Direct-Blot HRP anti-mCherry (BioLegend) for 1 hour at 37°C. After washing with PBST, the color reaction was developed using Immobilon Western Chemilum HRP Substrate (MERCK), and the absorbance at 450 nm was measured. A coefficient was calculated for each plate to ensure that the control OD value was 1.5, and this coefficient was multiplied by each sample to correct for differences between plates.

[0073] The results are shown in Figure 4. When mA1 and mC-dA1 were reacted in the presence of SC12 or SC-wt, the OD values ​​were higher than when mC-mA1 and dA1 were reacted. Furthermore, when comparing SC12 and SC-wt, there was no difference when mC-mA1 was used, but when mC-dA1 was used, the OD values ​​were higher in the presence of SC12. These results demonstrate that SC12 has a high ability to promote IgA antibody multimerization. Furthermore, the IgA antibodies used in this experiment were an IgA antibody with a Fab region containing the variable region of F045-092 and mC-A1. These antibodies have the same constant region but different variable regions. In other words, the method for producing multimeric IgA antibodies of the present application is variable region-independent and capable of producing IgA antibodies with dual or more specificities, preferably bispecific or trispecific, with different specificities.Furthermore, it has been shown that multimeric IgA antibodies can be formed even if the dimeric IgA antibodies and the monomeric IgA antibodies have different variable regions.

[0074] [Example 3: Identification of factors involved in promoting multimer formation 1] Example 2 revealed that SC12 has a higher ability to promote IgA antibody multimer formation than SC-wt. Therefore, in order to further enhance multimer formation, the addition of protein components other than SC and IgA antibody was investigated.

[0075] SC12, SC-wt, mA1, and dA1, as well as mC-mA1 and mC-dA1, prepared as described above, were mixed at a final concentration of 0.5 mg / mL with the additive (Supple) in phosphate buffer (pH 7.4) and incubated for 6 hours at 37°C using a rotator. The additive contained the chaperone proteins DnaK Mix (5 μM DnaK, 1 μM DnaJ, 1 μM GrpE) and GroE Mix (0.5 μM GroEL, 1 μM GroES), disulfide bond isomerase DsbC (375 μg / mL), and oxidized glutathione GSSG (30 mM). Trimeric / tetrameric IgA produced in the reaction mixture was detected by ELISA as described in Example 2.

[0076] The results are shown in Figures 5A and 5B. In the presence of SC12, the addition of the supplement to both the "mA1 + mC-dA1" and "mC-mA1 + dA1" mixtures increased the OD value, an indicator of trimeric / tetrameric IgA formation (Figure 5A). Furthermore, the OD value increased more than twofold in both mixtures. Furthermore, in the presence of SC-wt, the addition of the supplement also significantly increased the OD value, comparable to that observed with SC12 (Figure 5B).

[0077] [Example 4: Identification of factors involved in promoting multimer formation 2] The contribution of each of the four major components of Supplement to multimer formation was assessed by reacting mC-mA1 and dA1 alone in the presence of SC12. The reaction and detection were performed as in Example 3, except that DnaK Mix, GroE Mix, GSSG, or DsbC was added alone.

[0078] The results are shown in Figure 6. Similar OD values ​​were observed when DnaK Mix or GroE Mix, which act as molecular chaperones, were added. Meanwhile, DsbC, a disulfide bond isomerase, showed the highest increase in OD value among the four components. However, when oxidized glutathione GSSG, which creates the oxidative conditions necessary for disulfide bond formation, was added, it showed the lowest efficiency of promoting trimer / tetramer IgA formation among the four components. These results demonstrate that the supplement used in this experiment is useful for promoting trimerization / tetramerization of IgA antibodies.

[0079] [Example 5: Identification of factors involved in promoting multimer formation 3] We focused on the constant region of IgA antibodies as a factor contributing to IgA antibody multimerization. IgA antibodies are classified into two subclasses, IgA1 and IgA2, based on differences in their constant regions. IgA2 is further divided into three allotypes: IgA2m1, IgA2m2, and IgA2(n). In our previous study (Patent Document 1), differences in these constant regions resulted in different trimer / tetramer IgA antibody formation efficiencies under intracellular conditions. Therefore, we evaluated whether differences in multimer formation efficiency existed between IgA antibody subclasses under extracellular conditions as well. Specifically, we compared the multimer formation efficiencies due to differences in the constant regions of IgA antibodies using two different subclasses, IgA1 and IgA2m2.

[0080] As in Example 3, mC-A1 and mC-A2 prepared as described above were reacted with a combination of monomeric and dimeric IgA antibodies, SC12, and Supplement, and then the formation of trimeric / tetrameric IgA was evaluated.

[0081] The results are shown in Figure 7. Compared to the combination of IgA1 antibody monomers and dimers, the combination of IgA2m2 antibody and IgA1 antibody showed an increased OD value. These results demonstrate that IgA2m2 has a high ability to form tetramers.

[0082] [Example 6: Identification of factors involved in promoting multimer formation 4] We investigated the effect of reaction time on IgA antibody multimerization. mC-mA1 and dA1 were reacted in phosphate buffer (pH 7.4) at 37°C in the presence of SC12 and Supplement. Samples were collected 6, 12, and 24 hours after the start of the reaction, and trimeric / tetrameric IgA formation was evaluated by ELISA.

[0083] The results are shown in Figure 8. Although no significant changes were observed, an increase in the OD value was observed by extending the reaction time. Based on these results, it is expected that the efficiency of polymeric IgA antibody formation will be further improved by exploring the optimal reaction time.

[0084] Example 7: Evaluation of bispecific IgA antibodies using HA of different subtypes In Example 2, we demonstrated that mixing mCherry-fused IgA antibody monomers and dimers in the presence of SC-wt and SC12 resulted in the formation of trimeric / tetrameric IgA antibodies with different variable regions. Furthermore, in Example 5, we also demonstrated that the efficiency of multimer formation differs depending on the subclass of the IgA antibody. Therefore, in this Example, we verified the bispecificity of the trimeric / tetrameric IgA antibodies formed by the method of the present application using two influenza virus protein HAs of different subtypes to which detection tags were added, and IgA antibody clones specific for each HA.

[0085] The antibody clones used were 18-18K (H1) and 15-19L (H3), which are specific for influenza A H1 and H3 subtypes. These clones were isolated from subjects in a clinical trial of a nasal influenza vaccine. The variable regions from these antibodies were cloned into the α1 HC, α2m2 HC, and λ LC expression vectors using the same method as described above to generate dimeric IgA1 antibodies from antibody clone 18-18K, which is specific for influenza virus A / California / 7 / 2009 (H1N1) (H1 / HA), and monomeric IgA2m2 and dimeric IgA1 antibodies from antibody clone 15-19L, which is specific for influenza virus A / New York / 39 / 2012 (H3N2) (H3 / HA). The gene sequence of the heavy chain variable region of 18-18K(H1) is shown in SEQ ID NO: 9, the amino acid sequence is shown in SEQ ID NO: 10, the gene sequence of the light chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12. The gene sequence of the heavy chain variable region of 15-19L(H3) is shown in SEQ ID NO: 13, the amino acid sequence is shown in SEQ ID NO: 14, the gene sequence of the light chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16. SC and SC12 were also prepared as described above.

[0086] The 18-18K dimeric IgA1 antibody, the 15-19L monomeric IgA2m2 antibody, the dimeric IgA1 antibody, and SC or SC12 were mixed in phosphate buffer (pH 7.4) to a final concentration of 0.3 mg / mL. Specifically, the hetero group (18-18K dimeric IgA1 antibody and the 15-19L monomeric IgA2m2 antibody) and the homo group (15-19L monomeric IgA2m2 antibody and the dimeric IgA1 antibody) were mixed with SC-wt or SC12, or with neither SC-wt nor SC12. Reduced glutathione (Fujifilm Wako Pure Chemical Corporation) was added to a final concentration of 0.03 mM, and the mixture was incubated at 37°C for 12 hours in a thermal cycler. Trimeric / tetrameric IgA produced in the reaction mixture was detected by ELISA.

[0087] ELISA was performed as follows. 40 μl of strept-tagged recombinant H3 / HA-Strept (derived from A / NewYork / 39 / 2012 (H3N2) virus, 5 μg HA / mL) was immobilized overnight at 4°C in a 384-well plate, followed by blocking with 1% BSA-PBS at room temperature for 1 hour. A 2-fold serial dilution of a 100-fold diluted antibody sample was then prepared and added to each well. The plate was incubated at 37°C for 2 hours. After washing with PBST, 40 μl of His-tagged recombinant H1 / HA-His (derived from A / California / 7 / 2009 (H1N1) virus, 1 μg HA / mL) was added to each well and incubated at 37°C for 1 hour. After washing with PBST, 40 μl of 6000-fold diluted mAb-HRP-Direct (MBL) was added to each well and incubated at 37°C for 1 hour. After washing with PBST, a color reaction was carried out using Immobilon Western Chemilum HRP Substrate (MERCK), and the absorbance at a wavelength of 450 nm was measured.

[0088] The results are shown in Figure 9. In the hetero group, which combined antibody clones with different specificities, an increase in OD value was observed in the presence of SC compared to the absence of SC. Furthermore, as in previous results, a further increase in OD value was observed in the presence of SC12 compared to SC-wt. On the other hand, in the homo group, which combined monomeric IgA2m2 (mA2) and dimeric IgA1 (dA1) with the same specificity, no OD value comparable to that of the hetero group in the absence of SC was detected, even in the presence of SC-wt or SC12.

[0089] These results indicate that the acquisition of H1 / HA-binding ability is not due to the formation of trimeric / tetrameric IgA antibodies per se, but rather due to the formation of a single trimeric / tetrameric IgA antibody molecule by two IgA antibody molecules: dA1 from clone 15-19L, which is specific for H3 / HA, and mA2 from clone 18-18K, which is specific for H1 / HA. Furthermore, trimeric / tetrameric IgA antibodies were also formed by the combination of IgA allotypes IgA1 and IgA2m2 in the absence of SC. Furthermore, it was revealed that bispecific trimeric / tetrameric IgA antibodies could be more efficiently generated by reacting IgA antibodies in the presence of SC-wt or SC12. [Sequence List Free Text]

[0090] SEQ ID NO:1; Amino acid sequence of IgA1 constant region SEQ ID NO:2; Amino acid sequence of IgA2m2 constant region SEQ ID NO:3; Amino acid sequence of wild-type SC SEQ ID NO:4; Amino acid sequence of SC12 SEQ ID NO:5; Amino acid sequence of wild-type SC containing signal sequence, thrombin site, His tag SEQ ID NO:6; Amino acid sequence of SC12 containing signal sequence, thrombin site, His tag SEQ ID NO:7; Nucleotide sequence of wild-type SC containing signal sequence, thrombin site, His tag SEQ ID NO:8; Nucleotide sequence of SC12 containing signal sequence, thrombin site, His tag SEQ ID NO:9; Nucleotide sequence of heavy chain variable region of 18-18K(H1 specific) SEQ ID NO:10; Amino acid sequence of heavy chain variable region of 18-18K(H1 specific) SEQ ID NO:11; Nucleotide sequence of light chain variable region of 18-18K(H1 specific) SEQ ID NO:12; Amino acid sequence of light chain variable region of 18-18K(H1 specific) SEQ ID NO:13; Nucleotide sequence of heavy chain variable region of 15-19L(H3 specific) SEQ ID NO:14; Amino acid sequence of heavy chain variable region of 15-19L(H3 specific) SEQ ID NO:15; Nucleotide sequence of light chain variable region of 15-19L(H3 specific) SEQ ID NO:16; Amino acid sequence of light chain variable region of 15-19L(H3 specific)

Claims

1. A method for producing trimeric and tetrameric IgA antibodies comprising mixing dimeric and monomeric IgA antibodies.

2. The method of claim 1, further comprising mixing secreted fragments, wherein the secreted fragments are wild-type SC or SC mutants containing domain D1.

3. 3. The method of claim 1 or 2, wherein at least bispecific trimeric and tetrameric IgA antibodies are produced, wherein the dimeric IgA antibody comprises a first antigen-binding site and the monomeric IgA antibody comprises a second antigen-binding site.

4. The method of claim 3, further comprising mixing another monomeric IgA antibody comprising a third antigen-binding site.

5. The method of claim 3, wherein the four Fab regions of the dimeric IgA antibody each contain a first antigen-binding site, and the two Fab regions of the monomeric IgA antibody containing the second antigen-binding site each contain a second antigen-binding site.

6. The method of claim 4, wherein the four Fab regions of the dimeric IgA antibody each comprise a first antigen-binding site, the two Fab regions of the monomeric IgA antibody comprising the second antigen-binding site each comprise a second antigen-binding site, and the two Fab regions of the other monomeric IgA antibody each comprise a third antigen-binding site.

7. The method according to any one of claims 1 to 6, further comprising mixing at least one substance selected from the group consisting of a molecular chaperone protein, a disulfide bond isomerase, oxidized glutathione, and reduced glutathione.

8. The method according to any one of claims 1 to 7, wherein the dimeric IgA antibody and the monomeric IgA antibody are recombinant IgA antibodies produced separately in cultured cells.

9. The method according to any one of claims 1 to 8, further comprising separating the produced trimeric and tetrameric IgA antibodies from other IgA antibodies.

10. 10. The method of claim 9, further comprising separating the trimeric and tetrameric IgA antibodies produced.

11. At least a bispecific trimeric or tetrameric IgA antibody comprising a first Fab region comprising a first antigen binding site and a second Fab region comprising a second antigen binding site.

12. The antibody of claim 11, further comprising a secretory piece, wherein the secretory piece is a wild-type SC or an SC mutant comprising domain D1.

13. The antibody according to claim 11, which is a polymer of one dimeric IgA antibody molecule comprising the first antigen-binding site and one or two monomeric IgA antibody molecules comprising the second antigen-binding site.

14. The antibody of claim 12, which is a polymer of one dimeric IgA antibody molecule containing the first antigen-binding site, one or two monomeric IgA antibody molecules containing the second antigen-binding site, and a secretory fragment.

15. The antibody of any one of claims 12 to 14, comprising four first Fab regions and at least two second Fab regions.

16. A pharmaceutical composition comprising the antibody according to any one of claims 11 to 15.

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