Immunoglobulin

Abstract

The present disclosure provides an immunoglobulin multimer, a method for producing the same, and a modified immunoglobulin capable of forming an immunoglobulin multimer. In another aspect, the present disclosure has an effect of providing: a method for utilizing an immunoglobulin multimer or a modified immunoglobulin capable of forming an immunoglobulin multimer; and a composition containing an immunoglobulin multimer or a modified immunoglobulin capable of forming an immunoglobulin multimer.

Description

[Technical Field]

[0001] The present disclosure relates to methods for producing immunoglobulin multimers, as well as immunoglobulin multimers, immunoglobulin variants capable of forming immunoglobulin multimers, compositions containing them, and uses thereof. [Background technology]

[0002] Immunoglobulins exist in various isotypes, such as IgG, IgM, IgA, IgD, and IgE, each of which has a different function in the body. Immunoglobulins such as IgA and IgM are known to function in the body by forming multimers, such as dimers and pentamers. Because immunoglobulins have more antigen-binding sites in the multimer state, they exhibit higher physiological activity than monomeric immunoglobulins, and it is believed that the greater the number of immunoglobulins contained in a multimer, the greater the physiological activity.

[0003] Various methods for producing immunoglobulin multimers have been investigated to date (for example, Patent Document 1: WO2021 / 193553). However, there is a need for a method that can stably produce immunoglobulin multimers with high quality. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2021 / 193553A1 Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides immunoglobulin multimers, methods for producing the same, and immunoglobulin variants that can be used in the methods. In another aspect, the present disclosure provides methods and compositions that utilize the immunoglobulin multimers and immunoglobulin variants. [Means for solving the problem]

[0006] The present inventors have explored methods for artificially producing immunoglobulin multimers and have unexpectedly found that high-quality immunoglobulin multimers can be efficiently obtained by using immunoglobulin variants having a deletion at the C-terminus of at least one heavy chain. In particular, they have found that high-quality immunoglobulin multimers can be efficiently obtained by adding a heterologous amino acid sequence to the C-terminus of an immunoglobulin variant having a deletion at the C-terminus of at least one heavy chain to promote multimerization.

[0007] Based on the above findings, in one aspect, the present disclosure provides a method for producing an immunoglobulin multimer.

[0008] In another aspect, the present disclosure provides immunoglobulin multimers.

[0009] In another aspect, the present disclosure provides immunoglobulin variants capable of forming immunoglobulin multimers.

[0010] In another aspect, the present disclosure provides a pharmaceutical composition, food composition, feed composition, or reagent composition containing the immunoglobulin multimer or immunoglobulin variant.

[0011] In another aspect, the present disclosure provides methods for treating or preventing a disease, disorder, or condition using immunoglobulin multimers or immunoglobulin variants.

[0012] In another aspect, the present disclosure provides test, testing or diagnostic methods using immunoglobulin multimers or immunoglobulin variants.

[0013] More specifically, the present disclosure provides: [Item 1] contacting two or more immunoglobulins, including at least one immunoglobulin variant having a deletion in the C-terminal multimerization-promoting region of at least one heavy chain; and conjugating the two or more immunoglobulins A method for producing an immunoglobulin multimer, comprising: [Item 2] Item 10. The method of item 1, wherein the deletion is a deletion of one or more amino acids in the multimerization-promoting region. [Item 3] 2. The method according to Item 1, wherein a heterologous amino acid sequence that promotes multimerization is added to the C-terminus of the immunoglobulin variant that has a deletion in the C-terminal multimerization-promoting region. [Item 4] 2. The method of claim 1, wherein each of the plurality of immunoglobulins is IgA or IgM. [Item 5] An immunoglobulin multimer produced by the method described in item 1. [Item 6] An immunoglobulin multimer comprising two or more immunoglobulins, wherein at least one of the immunoglobulins constituting the two or more immunoglobulins is an immunoglobulin variant having a deletion in the C-terminal multimer formation ability-promoting region of at least one heavy chain. [Item 7] 7. The immunoglobulin multimer according to item 5 or 6, which has a bacterial growth inhibitory effect. [Item 8] 7. The immunoglobulin multimer according to item 5 or 6, which has bacterial agglutination activity. [Item 9] A modified immunoglobulin having a deletion in the C-terminal multimerization promoting region and having the ability to form immunoglobulin multimers. [Item 10] A food composition, feed composition, pharmaceutical composition, or reagent composition comprising the immunoglobulin multimer according to Item 5 or 6 or the immunoglobulin variant according to Item 9. [Item 11] A nucleic acid comprising a nucleic acid sequence encoding the immunoglobulin variant of item 9. [Item 12] A cell containing the nucleic acid according to item 11. [Effects of the Invention]

[0014] The present disclosure provides immunoglobulin multimers, methods for producing the same, and immunoglobulin variants capable of forming immunoglobulin multimers. In another aspect, the present disclosure has the effect of providing methods for using immunoglobulin multimers or immunoglobulin variants capable of forming immunoglobulin multimers, and compositions comprising immunoglobulin multimers or immunoglobulin variants capable of forming immunoglobulin multimers. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the heavy chain amino acid sequence (signal sequence not shown) of antibody CHO W27HAACOMP. [Figure 2] FIG. 2 shows the heavy chain amino acid sequence (signal sequence not shown) of antibody CHO W27HKKCOMP. [Figure 3] FIG. 3 shows the heavy chain amino acid sequence (signal sequence not shown) of antibody CHO W27HdirectCOMP. [Figure 4] FIG. 4 shows the heavy chain amino acid sequence (signal sequence not shown) of antibody CHO W27notailCOMP. [Figure 5] FIG. 5 shows the results of examining the influence of the structure of the C-terminal region of the IgA H chain and the structure of the linker on multimer formation. [Figure 6] FIG. 6 shows the binding ability of W27 IgA multimers to E. coli. [Figure 7] FIG. 7 shows the results confirming that immunoglobulin multimers have excellent in vitro bacterial growth inhibitory effects. [Figure 8] FIG. 8 shows the results confirming that immunoglobulin multimers have an excellent in vivo inhibitory effect on bacteria-associated enteritis. DETAILED DESCRIPTION OF THE INVENTION

[0016] The inventors of the present disclosure attempted to produce immunoglobulin multimers, but found it particularly difficult to efficiently produce immunoglobulin multimers that retain physiological activity. Furthermore, after independent trial and error, they surprisingly and unexpectedly found that high-quality immunoglobulin multimers can be efficiently obtained by using immunoglobulin variants with deletions in the C-terminal multimerization-promoting region of their heavy chains. In particular, they unexpectedly found that high-quality immunoglobulin multimers can be efficiently obtained by adding a heterologous amino acid sequence to the C-terminus of the heavy chain of an immunoglobulin variant to promote multimerization.

[0017] (definition) In this specification, when multiple ranges of numerical values ​​are shown, the same applies to ranges formed by combining any lower limit value and upper limit value of those multiple ranges.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0019] As used herein, the term "about" in reference to a numerical value x means that the value can vary within, for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01%.

[0020] As used herein, the term "comprise" has the same meaning as commonly understood by a person skilled in the art to which the present disclosure belongs, and includes, for example, "comprises" and "consists of." Specifically, a composition "comprising" A may contain another component, B, in addition to containing only A.

[0021] As used herein, "separate" has the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains, and may refer, for example, to selectively obtaining a specific component from a mixed composition and excluding other components. As used herein, in cases such as separating immunoglobulins from a culture medium, "separate" may have the same meaning as "isolate," "purify," "enrich," or "concentrate."

[0022] As used herein, the term "protein" refers to a substance composed of a polypeptide in which amino acids are linked by peptide bonds. When referring to a protein in this specification, there is no particular limitation on its molecular weight. Therefore, when referring to a protein in this specification, it also encompasses the meaning of polypeptides and peptides.

[0023] As used herein, the term "deficient" has the same meaning as commonly understood by those skilled in the art to which this disclosure pertains, and means, for example, that the function or property originally possessed by the target molecule, region, etc. is reduced or completely lost.

[0024] As used herein, the term "variant" has the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains, but when the subject is a protein, the variant refers to a mutation in the amino acid sequence, such as substitution, deletion, or addition of one or more amino acids. In another embodiment, the variant refers to a variant to which a heterologous molecule is attached.

[0025] (Method for producing immunoglobulin multimers) In one aspect, the present disclosure provides a method for producing an immunoglobulin multimer. Typically, the method for producing an immunoglobulin multimer of the present disclosure has the following configuration. contacting two or more immunoglobulins, including at least one immunoglobulin variant having a deletion in the C-terminal multimerization-promoting region of at least one heavy chain; and conjugating the two or more immunoglobulins; A method for producing an immunoglobulin multimer, comprising:

[0026] The method of the present disclosure may further comprise the step of separating the immunoglobulin multimers produced by said binding.

[0027] In the methods of the present disclosure, the environment in which the step of contacting two or more immunoglobulins is carried out is not particularly limited, as long as it allows binding of the two or more immunoglobulins. For example, the step can be carried out in an environment such as inside a cell, in a cell suspension, or in a test tube containing cellular components. In one embodiment, the step of contacting two or more immunoglobulins can be achieved by expressing nucleic acids encoding the immunoglobulins in the cells, thereby contacting the two or more immunoglobulins within the cells.

[0028] In the method of the present disclosure, the environment in which the step of binding two or more immunoglobulins is performed is not particularly limited as long as it allows binding of the two or more immunoglobulins. For example, the step can be performed in an environment such as inside a cell, a cell suspension, or a test tube containing cellular components. In one embodiment, the step of binding two or more immunoglobulins can be achieved by contacting two or more immunoglobulins inside a cell, resulting in interaction between globulin molecules. The binding mode of the two or more immunoglobulins includes any mode, including, for example, a covalent bond, an ionic bond, and a hydrophobic bond. The binding of the two or more immunoglobulins may be via a heterologous molecule bound to the immunoglobulin.

[0029] In the method of the present disclosure, the step of separating the immunoglobulin multimers produced by the binding is not particularly limited and can be carried out using various methods commonly used for separating proteins in the technical field of the present disclosure, such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, ethanol precipitation, reverse-phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, which can be used alone or in combination.

[0030] In the method for producing immunoglobulin multimers of the present disclosure, in the step of separating immunoglobulin multimers in the culture medium from other culture medium components, the produced immunoglobulin multimers can be separated according to a standard method. In the method for producing immunoglobulin multimers of the present disclosure, a process such as cell disruption is not required for the isolation and purification of immunoglobulin multimers. In a preferred embodiment, the method for producing immunoglobulin multimers of the present disclosure does not include a process for cell disruption. In one embodiment, the cells used in the method for producing immunoglobulin multimers of the present disclosure may produce immunoglobulins intracellularly. Cells containing immunoglobulin multimers intracellularly can be incorporated into a composition as an active ingredient. For example, when yeast or filamentous fungi are used as cells producing immunoglobulin multimers, the cells themselves that produce immunoglobulin multimers can be orally administered. When such cells containing immunoglobulin multimers are orally administered, the cells may or may not be disrupted prior to administration.

[0031] When cells are used in the methods of the present disclosure, the cells may express two or more immunoglobulin variants having different types of deletions as immunoglobulin variants having a deletion in at least one C-terminal multimerization-promoting region of their heavy chains. In one embodiment, the cells used in the methods of the present disclosure may express an immunoglobulin that does not have a deletion in its C-terminal multimerization-promoting region, in addition to an immunoglobulin having a deletion in at least one C-terminal multimerization-promoting region of its heavy chain.

[0032] In the method of the present disclosure, the type of immunoglobulin multimer produced is not particularly limited and can be selected from, for example, IgG, IgA, IgM, IgD, or IgE. In a preferred embodiment, the immunoglobulin is IgA or IgM. In a preferred embodiment, the resulting immunoglobulin multimer has physiological activity, for example, antigen-binding activity.

[0033] The immunoglobulin multimers produced by the methods of the present disclosure may contain immunoglobulins from various organisms, such as humans, non-human primates, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, horses, deer, wild boars, sheep, goats, camels, etc. In a preferred embodiment, the immunoglobulin is a human immunoglobulin.

[0034] In addition to the deletion of the C-terminal multimerization-promoting region, the immunoglobulins produced by the methods of the present disclosure may have a native amino acid sequence in the heavy or light chain, or may have mutations relative to the native sequence. Such mutations include addition, deletion, or substitution of amino acids, and include truncation of the N-terminus or C-terminus. Such mutations can adjust the antigen-binding ability, stability, and other properties of the produced immunoglobulins.

[0035] The immunoglobulins contained in the immunoglobulin multimers produced by the methods of the present disclosure can include immunoglobulins with a deletion in the C-terminal multimerization-promoting region as well as immunoglobulins without such a deletion. Immunoglobulins with a deletion in the C-terminal multimerization-promoting region and immunoglobulins without such a deletion may have a native amino acid sequence in the heavy or light chain, or may have a mutation relative to the native sequence, in addition to the deletion in the C-terminal multimerization-promoting region. Such mutations include addition, deletion, or substitution of amino acids, and include truncation of the N-terminus or C-terminus. Such mutations can adjust the antigen-binding ability, stability, and other properties of the produced immunoglobulin.

[0036] In one embodiment, the immunoglobulins constituting the immunoglobulin multimers produced by the method of the present disclosure may be a mixture of IgG, IgA, IgM, IgD, and IgE. For example, they may be IgG, IgA, IgM, IgD, or IgE to which the multimerization-promoting region or other region of IgA or IgM has been added by recombinant technology or other means, or in which a natural region has been partially replaced with such a region. Alternatively, the immunoglobulins constituting the immunoglobulin multimers produced by the method of the present disclosure may be IgA or IgM in which portions other than the multimerization-promoting region have been replaced with immunoglobulins of other classes. In the present disclosure, when an immunoglobulin is referred to as IgG, IgA, IgM, IgD, or IgE, it encompasses IgG, IgA, IgM, IgD, or IgE that contain portions of other types of immunoglobulins and / or have been replaced with portions of other types of immunoglobulins. When the immunoglobulins making up the immunoglobulin multimers produced by the methods of the present disclosure are referred to as IgG, IgA, IgM, IgD, or IgE, this encompasses IgG, IgA, IgM, IgD, or IgE that contain and / or are substituted with portions of other types of immunoglobulins, respectively.

[0037] The immunoglobulin multimers produced by the methods of the present disclosure comprise two or more immunoglobulins. In preferred embodiments, the immunoglobulin multimers produced by the methods of the present disclosure comprise 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, or 2 to 9 immunoglobulins.

[0038] The immunoglobulin variants having a deletion in the C-terminal multimerization-promoting region of at least one heavy chain used in the methods of the present disclosure can be the immunoglobulin variants described below (Immunoglobulin variants having a deletion in the C-terminal multimerization-promoting region).

[0039] Cells expressing immunoglobulin variants having deletions in at least one heavy chain C-terminal multimerization-promoting region are not particularly limited and can be appropriately selected by those skilled in the art. In one embodiment, it is preferable to use cells capable of producing various proteins derived from mammals, etc., that function by forming higher-order structures. Such cells can be appropriately selected from known cells, such as, but not limited to, mammalian-derived cells such as COS cells, HEK cells, HELA cells, and CHO cells; insect-derived cells such as Sf9 cells; yeast; and filamentous fungi.

[0040] Cells expressing the immunoglobulins or immunoglobulin variants can be obtained by standard genetic engineering techniques, for example, by transducing cells capable of producing immunoglobulins with nucleic acids encoding the immunoglobulins or immunoglobulin variants.

[0041] When transducing cells used in the methods of the present disclosure with nucleic acids encoding immunoglobulins or the like, those skilled in the art can employ specific nucleic acid introduction methods based on publicly known information. For example, the nucleic acid may be introduced separately from the host cell genome in the form of a plasmid, artificial chromosome, or the like, or may be integrated into the host cell genome using genome editing techniques. The genome modification techniques (genome editing techniques) used in the methods of the present disclosure can utilize various methods commonly used in the technical field of the present disclosure. For example, CRISPR-Cas systems, meganucleases (MN), zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN) can be used. In one embodiment, it is preferable to use a modification technique that does not retain the genome modification plasmid in the modified host cells. For example, when using filamentous fungi as host cells, genome-modified cells in which the genome modification plasmid does not remain in the modified host cells can be obtained by using the genome editing plasmid for creating multiple mutant strains through multiple steps, as disclosed in Japanese Patent Publication No. 2018-191551, in single-step or multi-step mutation procedures.

[0042] The vectors or nucleic acid constructs used in the genome modification procedures included in the methods for producing host cells of the present disclosure can be any of various vectors or nucleic acid constructs commonly used in the technical field of the present disclosure. For example, vectors having a selectable marker gene, cloning site, and control region (promoter and terminator) as disclosed in Japanese Patent Application Publication No. 2012-179011 can be used. In one embodiment, when the CRISPR-Cas system is used in the genome modification procedures, vectors containing the A. nidulans-derived DNA fragment AMA1, which enables autonomous replication of the plasmid, and a DNA fragment designed to enable high expression of the Aoace2 gene, whose high expression significantly impairs growth, under specific conditions, as disclosed in Japanese Patent Application Publication No. 2018-191551, can be preferably used.

[0043] Those skilled in the art can appropriately adjust the culture conditions, recovery method, purification method, etc. of the cells into which the nucleic acid has been introduced depending on the type of cells, etc., and can produce the immunoglobulin multimers according to the present disclosure.

[0044] In the above-described cell culturing step, cell culture conditions, such as medium composition, culture temperature, medium pH, and shaking conditions, can be adjusted and optimized as desired by those skilled in the art based on known techniques. Optimizing these conditions can improve the yield, yield, and quality of immunoglobulins, as well as produce immunoglobulin multimers with desired properties, such as stability.

[0045] In one embodiment, the pH of the medium in which the cells are cultured can be adjusted to any pH between 4 and 11. Preferably, the pH of the medium is selected from the ranges of 4.0 to 11.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.5, or 6.0 to 8.0. In one embodiment, the pH of the medium can be set to 5.0 or higher, 5.5 or higher, 6.0 or higher, 6.5 or higher, 7.0 or higher, 7.5 or higher, 8.0 or higher, 8.5 or higher, 9.0 or higher, and 9.5 or higher, as well as 11.0 or lower, 10.5 or lower, 10.0 or lower, 9.5 or lower, 9.0 or lower, 8.5 or lower, 8.0 or lower, 7.5 or lower, 7.0 or lower, 6.5 or lower, 6.0 or lower, 5.5 or lower, and 5.0 or lower, or any combination of these upper and lower limits.

[0046] In the method of the present disclosure, when a cell suspension or an in vitro environment containing cell components is used, the cells and cell components used are not particularly limited, and can be carried out using various configurations used in the technical field of the present disclosure for protein expression, reaction, etc.

[0047] The polymeric form of the isolated immunoglobulin can be confirmed by various methods commonly used in the technical field of the present disclosure, for example, by analyzing the immunoglobulin isolated from the culture medium by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0048] (immunoglobulin multimers) In one aspect, the present disclosure provides immunoglobulin multimers. Typically, the immunoglobulin multimers provided by the present disclosure have the following structure: An immunoglobulin multimer comprising two or more immunoglobulins, wherein at least one of the immunoglobulins constituting the two or more immunoglobulins is an immunoglobulin variant having a deletion in the C-terminal multimer formation ability-promoting region of at least one heavy chain.

[0049] In one embodiment, the immunoglobulin multimers of the present disclosure are produced by the production method described above (Method for producing immunoglobulin multimers).

[0050] Thus, the immunoglobulin multimers of the present disclosure can include immunoglobulin variants having a deletion in the C-terminal multimerization-promoting region of at least one heavy chain, and can include two or more immunoglobulin variants having different types of deletions.

[0051] Furthermore, the type of immunoglobulin constituting the immunoglobulin multimer of the present disclosure is not particularly limited and can be selected from, for example, IgG, IgA, IgM, IgD, or IgE. In a preferred embodiment, the immunoglobulin is IgA or IgM. In a preferred embodiment, the immunoglobulin multimer of the present disclosure has physiological activity, for example, antigen-binding activity.

[0052] The immunoglobulin multimers of the present disclosure may comprise immunoglobulins from various organisms, such as humans, non-human primates, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, horses, deer, wild boars, sheep, goats, camels, etc. In a preferred embodiment, the immunoglobulin is a human immunoglobulin.

[0053] The immunoglobulin multimers of the present disclosure may include immunoglobulins that have a deletion in the C-terminal multimerization-promoting region as well as immunoglobulins that do not have this deletion. Immunoglobulins that have a deletion in the C-terminal multimerization-promoting region and immunoglobulins that do not have this deletion may have a native amino acid sequence in the heavy or light chain, or may have a mutation relative to the native sequence, in addition to the deletion in the C-terminal multimerization-promoting region. Such mutations include addition, deletion, or substitution of amino acids, and include truncation of the N-terminus or C-terminus. Such mutations can adjust the antigen-binding ability, stability, and other properties of the immunoglobulins produced.

[0054] In one embodiment, the immunoglobulins constituting the immunoglobulin multimers of the present disclosure may be a mixture of IgG, IgA, IgM, IgD, and IgE. For example, they may be IgG, IgA, IgM, IgD, or IgE to which the multimerization-promoting region or other region of IgA or IgM has been added by recombinant technology or the like, or in which a natural region has been partially replaced with such a region. Alternatively, the immunoglobulins constituting the immunoglobulin multimers of the present disclosure may be IgA or IgM in which portions other than the multimerization-promoting region have been replaced with immunoglobulins of other classes. In the present disclosure, when an immunoglobulin is referred to as IgG, IgA, IgM, IgD, or IgE, it encompasses IgG, IgA, IgM, IgD, or IgE that contain portions of other types of immunoglobulins and / or have been replaced with portions of other types of immunoglobulins.

[0055] The immunoglobulin multimer of the present disclosure comprises two or more immunoglobulins. In preferred embodiments, the immunoglobulin multimer of the present disclosure comprises 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, or 2 to 9 immunoglobulins.

[0056] In one embodiment, the immunoglobulin variant contained in the immunoglobulin multimer of the present disclosure is an immunoglobulin variant described below (Immunoglobulin variant having a deletion in the C-terminal multimer formation-promoting region).

[0057] (Immunoglobulin variants with deletions in the C-terminal multimerization-promoting domain) In one aspect, the present disclosure provides immunoglobulin variants having deletions in their C-terminal multimerization-promoting regions. Typically, immunoglobulins having deletions in their C-terminal multimerization-promoting regions of the present disclosure have the following configuration: An immunoglobulin variant having a deletion in a C-terminal multimerization-promoting region, wherein the immunoglobulin variant has the ability to form immunoglobulin multimers.

[0058] In one embodiment, the immunoglobulin variant having a deletion in the C-terminal multimerization-promoting region of the present disclosure is used in the above-mentioned (method for producing immunoglobulin multimers).

[0059] Thus, the immunoglobulin variants disclosed herein having a deletion in the C-terminal multimerization-promoting region, and immunoglobulin variants having a deletion in the C-terminal multimerization-promoting region of at least one heavy chain, can include two or more types of immunoglobulin variants having different types of deletions.

[0060] Furthermore, the type of immunoglobulin of the immunoglobulin variants of the present disclosure having deletions in the C-terminal multimerization-promoting region is not particularly limited and can be selected from, for example, IgG, IgA, IgM, IgD, or IgE. In a preferred embodiment, the immunoglobulin variants of the present disclosure are IgA or IgM. In a preferred embodiment, the immunoglobulin variants of the present disclosure have physiological activity, such as antigen-binding activity.

[0061] The immunoglobulin variants of the present disclosure may include immunoglobulins from various organisms, such as humans, non-human primates, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, pigs, horses, deer, wild boars, sheep, goats, camels, etc. In a preferred embodiment, the immunoglobulin variant is a human immunoglobulin.

[0062] In one embodiment, immunoglobulin variants of the present disclosure having a deletion in the C-terminal multimerization-promoting region may have a mixture of IgG, IgA, IgM, IgD, and IgE structures. For example, they may be IgG, IgA, IgM, IgD, or IgE to which the multimerization-promoting region of IgA or IgM or other region has been added, or a native region has been partially replaced with such a region, by recombinant technology or the like. Alternatively, immunoglobulin variants of the present disclosure having a deletion in the C-terminal multimerization-promoting region may be IgA or IgM in which portions other than the multimerization-promoting region have been replaced with immunoglobulins of other classes. In the present disclosure, when an immunoglobulin variant is referred to as IgG, IgA, IgM, IgD, or IgE, it encompasses IgG, IgA, IgM, IgD, or IgE that contain and / or have been replaced with portions of other types of immunoglobulins, respectively.

[0063] The heavy chain C-terminal multimerization-promoting region of an immunoglobulin variant used in the methods of the present disclosure encompasses a region that functions to promote the multimerization process when an immunoglobulin forms multimers of dimers or higher, or a region that stabilizes the formed multimers. This region may be contained in a naturally occurring immunoglobulin, or may contain an amino acid mutation or a heterologous molecule. Preferably, this region is contained in a naturally occurring immunoglobulin.

[0064] The deletion in the heavy chain C-terminal multimerization-promoting region of the immunoglobulin variant described above includes any embodiment that reduces or eliminates the multimerization-promoting function. In one embodiment, the deletion results in the deletion of the beta sheet structure of the immunoglobulin heavy chain C-terminal region, which contributes to the multimerization-promoting function (Int J Mol Sci. 2021 Dec; 22(23): 12776.). In one embodiment, the deletion may be the deletion, substitution, or insertion of one or more amino acid sequences. In one embodiment, the deletion may be the binding of a heterologous molecule to the region. The deletion may be located anywhere in the multimerization-promoting region. In a preferred embodiment, the deletion is the deletion of an amino acid sequence, and in a more preferred embodiment, the deletion is the deletion of one or more amino acids from the C-terminus. For example, when the immunoglobulin is mouse IgA, the deletion includes the amino acid sequence PTNVSVSVIMSEGDGICY (SEQ ID NO: 7) located at the C-terminus of the heavy chain.

[0065] The immunoglobulin variants used in the methods of the present disclosure may contain a heterologous amino acid sequence to promote multimerization. Examples of such heterologous amino acid sequences include amino acid sequences that form coiled-coil domains. In one embodiment, the heterologous amino acid sequence may be derived from cartilage oligomer matrix protein (COMP), mannose-binding protein A, coiled-coil serine-rich protein 1, polypeptide-releasing factor 2, SNAP-25, SNARE, Lac repressor, or apolipoprotein E. In a preferred embodiment, the heterologous amino acid sequence for promoting multimerization may be, for example, the coiled-coil assembly domain of cartilage oligomer matrix protein (COMP) described in JP-A-2016-512309. The heterologous amino acid sequence may be fused to the N-terminus or C-terminus of the immunoglobulin protein, preferably the C-terminus. Various linker sequences may be used for the fusion, as long as they do not impair the desired physiological function of the multimeric immunoglobulin, but they are not required. In one embodiment, a linker consisting of the amino acid sequence SSADDAKKDAAKKDDAKKDDAKKDAS (SEQ ID NO: 4) or a linker consisting of the amino acid sequence SSADDAAADAAAADDAAADDAAADAS (SEQ ID NO: 5) can be used. In one embodiment, the heterologous sequence linker or linkers to promote multimerization can be fused to the shortened N- or C-terminus of the immunoglobulin.

[0066] The class, amino acid sequence, etc. of immunoglobulins having deletions in the C-terminal multimerization-promoting region of the present disclosure are not particularly limited as described above. However, as an example, the W27 antibody described in WO 2014 / 142084 and the CHO cell-produced recombinant IgA of the W27 antibody, RS_H000_L001, in which a mutation for protein L binding has been introduced into the light chain, can be used (see WO 2023 / 277142. Hereinafter, RS_H000_L001 will be referred to as "W27 IgA"). More specifically, as an immunoglobulin derived from the W27 IgA antibody, an immunoglobulin having an amino acid deletion in the C-terminal region of the heavy chain and an added COMP sequence (CHO W27notailCOMP IgA) can be used.

[0067] The heavy chain amino acid sequence of CHO W27notailCOMP IgA expressed in mouse cells is shown below. SEQ ID NO: 1: CHO W27notailCOMP IgA H amino acid sequence (without signal sequence) TIFF0007760200000001.tif57154

[0068] When expressing the immunoglobulin in mouse cells (for example, CHO), a mouse IgV signal sequence, for example, a signal sequence containing the amino acid sequence MKCSWIIFFLMAVVTGVNS (SEQ ID NO: 6), can be added to the N-terminus.

[0069] Furthermore, the light chain of CHO W27notailCOMP IgA has the same amino acid sequence as the light chain of a modified CHO cell-produced recombinant IgA of the W27 antibody (RS_H000_L001), and the amino acid sequence of its light chain variable region (RS_LV001) contains a mutation for binding to Protein L. The amino acid sequence of the light chain variable region RS_LV001 is shown below. >SEQ ID NO: 3: RS_LV001 light chain variable region amino acid sequence TIFF0007760200000002.tif19155

[0070] Nucleic acids encoding immunoglobulin variants In one aspect, the disclosure provides nucleic acids encoding the immunoglobulin variants described above (immunoglobulin variants having deletions in the C-terminal multimerization-promoting region).

[0071] The nucleic acid may be a ribonucleotide or a deoxynucleotide, and may be in the form of a single strand or a double strand, without any particular limitation.

[0072] The base sequence encoding the amino acid sequence described above is not limited to nucleic acids consisting of one type of base sequence per amino acid sequence, but may be determined by appropriately selecting various codons encoding the amino acids depending on the intended use of the nucleic acid. Examples of intended use include using the base sequence to express and produce an immunoglobulin having a deletion in the C-terminal multimerization-promoting region according to the present disclosure. In particular, various codons may be selected taking into consideration the codon frequency corresponding to the type of host cell used during production.

[0073] Such a nucleotide sequence can be easily determined in silico, for example, by using a known program. The nucleic acid according to the present disclosure may have a nucleotide sequence encoding an amino acid sequence obtained by subjecting SEQ ID NOS: 1 to 50 to the mutations described above in (Immunoglobulins having deletions in the C-terminal multimerization-promoting region).

[0074] Nucleic acids encoding the immunoglobulins of the present disclosure can be prepared, for example, by preparing nucleic acids having nucleotide sequences encoding heavy chain variable regions and light chain variable regions, and nucleotide sequences encoding heterologous heavy chain constant regions and light chain constant regions, and then binding the prepared nucleotide sequences encoding the heavy chain variable regions to nucleic acids having nucleotide sequences encoding the heavy chain constant regions, and also by binding the prepared nucleotide sequences encoding the light chain variable regions to nucleic acids having nucleotide sequences encoding the light chain constant regions.

[0075] In one embodiment, the immunoglobulin of the present disclosure is an antibody in which heavy and / or light chain CDRs 1 to 3 have amino acid sequences derived from mouse, and other regions have amino acid sequences derived from human (this may also be referred to as a humanized antibody). Such a humanized antibody can be produced by preparing nucleic acids having nucleotide sequences encoding heavy and / or light chain CDRs 1 to 3 and nucleotide sequences encoding other regions, and recombining the nucleic acids to form heavy and light chains. In one embodiment, some bases may be substituted with other bases to maintain the binding ability of the antibody.

[0076] The base sequence of the nucleic acid of the present disclosure is not particularly limited, but an example thereof is the base sequence SEQ ID NO: 2, which corresponds to the amino acid sequence SEQ ID NO: 1 described above (immunoglobulin variant having a deletion in the C-terminal multimer formation ability-promoting region). SEQ ID NO: 2: Nucleotide sequence of CHO W27HnotailCOMP (including the portion encoding the N-terminal mouse IgV signal sequence "MKCSWIIFFLMAVVTGVNS") TIFF0007760200000003.tif154153

[0077] (vector) In one aspect, the present disclosure provides a vector comprising a nucleic acid encoding an immunoglobulin variant. The vector can be produced by incorporating the nucleic acid encoding the immunoglobulin variant into a vector known in the art. The production method is not particularly limited, and various methods known to those skilled in the art can be used.

[0078] The vectors of the present disclosure can be used to express immunoglobulin variants by introducing them into host cells, and can also be used for genetic engineering applications in vitro, etc.

[0079] In one aspect, when the vector of the present disclosure is used as an expression vector, a phage vector, a viral vector, a plasmid vector, or the like can be used.

[0080] (cell) In one aspect, the present disclosure provides a cell comprising a nucleic acid encoding an immunoglobulin variant. The cell may be any cultured cell known in the art or a cell obtained from an in vivo source.

[0081] In one embodiment, the cell is a cell used in the above (method for producing immunoglobulin multimers) and expresses the immunoglobulin variant encoded by the nucleic acid. The various cells described above (method for producing immunoglobulin multimers) can be used as the cell.

[0082] In one embodiment, the cell does not necessarily express the immunoglobulin variant encoded by the nucleic acid. For example, the cell may contain a nucleic acid encoding an immunoglobulin variant for the purpose of performing intracellular genetic engineering or the like. The specific type of the cell is not particularly limited, and various cells known in the art can be used. In one embodiment, the cell may be, for example, but is not limited to, a mammalian cell, an avian cell, a reptilian cell, an amphibian cell, a fish cell, a fungal cell, a bacterial cell, an archaeal cell, or the like.

[0083] (composition) In one aspect, the disclosure provides compositions comprising immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimerization-promoting region. The compositions of the present disclosure can be provided as compositions containing purified immunoglobulin multimers or immunoglobulin variants, as well as compositions containing host cells expressing immunoglobulin multimers or immunoglobulin variants, compositions containing part or all of the culture medium of host cells expressing immunoglobulin multimers or immunoglobulin variants, or compositions containing host cells and part or all of the culture medium. The compositions of the present disclosure are preferably provided in the form of pharmaceutical compositions, food compositions, feed compositions, reagent compositions, etc., but are not limited to these. In one aspect, the pharmaceutical compositions, food compositions, feed compositions, and reagent compositions of the present disclosure each comprise a material composition used to produce a final product.

[0084] (Pharmaceutical composition) The pharmaceutical compositions of the present disclosure are used to treat diseases, disorders, or conditions by utilizing the effects of immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimerization-promoting region.

[0085] In one aspect, the immunoglobulin multimers or immunoglobulin variants disclosed herein bind to an in vivo pathogen or an in vivo molecule exhibiting an abnormal function associated with a disease, disorder, condition, etc., and exert an effect of inhibiting the proliferation of the pathogen in the body and / or an effect of promoting the excretion of the pathogen or the in vivo molecule exhibiting an abnormal function from the body. Pharmaceutical compositions containing such immunoglobulin multimers or immunoglobulin variants are used for the prevention or treatment of diseases, disorders, conditions, etc. associated with the pathogen or the in vivo molecule exhibiting an abnormal function.

[0086] In the present disclosure, "treatment" of a disease, disorder, or condition includes any treatment that improves or inhibits the progression of the disease, disorder, or condition. Also, in the present disclosure, "prevention" of a disease, disorder, or condition includes any treatment that prevents, delays, or alleviates the "disease, disorder, or condition." In one embodiment, the "prevention" includes treating a subject who has a predisposition to developing the "disease, disorder, or condition."

[0087] The pathogens mentioned above include any pathogens associated with diseases, disorders, conditions, etc. in living organisms, including, but not limited to, viruses, bacteria, fungi, and parasites. The biological molecules that exhibit abnormal functions mentioned above include any biological molecules that exhibit abnormal functions associated with diseases, disorders, conditions, etc. The biological molecules include molecules that are originally contained in the target organism but exhibit abnormal functions due to changes in expression level, structural mutations, etc., as well as molecules that are introduced into the living organism by pathogens, etc.

[0088] In one aspect, the immunoglobulin multimers or immunoglobulin variants disclosed herein that have deletions in the C-terminal multimer formation-promoting region include W27 IgA and variants thereof, which bind to C. difficile bacteria and have the effect of preventing or treating diseases, disorders, conditions, etc.

[0089] Diseases associated with C. difficile bacteria include, but are not limited to, inflammatory bowel disease, ulcerative colitis, Crohn's disease, allergies, asthma, obesity, autoimmune diseases, neonatal necrotizing enterocolitis, etc., with inflammatory bowel disease being preferred.

[0090] Such pharmaceutical compositions according to the present disclosure may contain an effective amount of an immunoglobulin multimer or an immunoglobulin variant having a deletion in a C-terminal multimer formation-promoting region according to the present disclosure, and can be appropriately determined, for example, so that the content of the antibody according to the present disclosure in 100% by weight of the pharmaceutical composition is in the range of 0.001 to 99.99% by weight, taking into consideration the type of disease to be treated, the dosage form, the method of administration, the recipient, the severity of the symptoms in the recipient, and the degree of effect exerted by administration.

[0091] As used herein, the term "effective amount" refers to the amount of the immunoglobulin multimer or immunoglobulin variant having a deletion in the C-terminal multimer formation-promoting region according to the present disclosure that exerts a desired physiological effect in a living body.

[0092] Pharmaceutical compositions according to the present disclosure may contain pharmaceutically acceptable carriers or additives in combination with the immunoglobulin multimers or immunoglobulin variants having deletions in their C-terminal multimerization-enhancing regions of the present disclosure. The pharmaceutically acceptable carriers or additives refer to any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener, and any known pharmaceutically acceptable carriers or additives may be used.

[0093] Such subjects for administration are not particularly limited, but include mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.

[0094] The dosage and administration method of the pharmaceutical composition vary depending on the type of disease, sex, species, age, general condition, severity of disease, desired effect, etc. The dosage is usually set appropriately within the range of 0.001 to 100 mg / kg / day.

[0095] The administration method is not particularly limited, but administration to mucosal tissue is preferred, and examples of such administration methods include oral administration, nasal administration, and enteral administration.

[0096] Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy. The location into which the digestive tract is inserted is not limited to the intestine, but may include the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc.

[0097] The pharmaceutical composition according to the present disclosure may be administered in the above-mentioned amount once a day or in divided doses. The administration interval may be daily, every other day, weekly, biweekly, every 2-3 weeks, monthly, bimonthly, or every 2-3 months, as long as it has a therapeutic effect against the above-mentioned diseases.

[0098] (Food and feed compositions) Food or feed compositions according to the present disclosure contain immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimer formation-promoting region according to the present disclosure. Food compositions according to the present disclosure encompass all forms of food intake, including general foods, foods for specified health uses (including conditional foods for specified health uses), nutritional supplements, functional foods, and foods for medical conditions. By using such food or feed compositions, the physiological activities of immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimer formation-promoting region according to the present disclosure can be utilized. Food compositions can be provided as food compositions labeled for intestinal regulation, intestinal environment improvement, intestinal environment optimization, intestinal putrefaction prevention, etc.

[0099] The proportion of immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimer formation-promoting region in such food or feed compositions is not particularly limited and may be appropriately adjusted depending on the form, intended use, etc. of the food or feed composition, and is usually about 0.001 to 99% by weight of the total amount of the composition.

[0100] The intake amount of the food composition or feed composition according to the present disclosure is not particularly limited and can be set depending on the desired effect, the desired degree of effect, and other conditions, etc. For example, the amount converted into the amount of immunoglobulin multimer according to the present disclosure is usually about 0.001 to 100 mg / kg / day, which may be taken once a day or in divided doses several times a day.

[0101] The specific form of the food composition is not particularly limited, and examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and dairy drinks; frozen desserts such as ice cream, ice sherbet, and shaved ice; sweets such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jellies, jams, cream, and baked goods; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; processed seafood and livestock foods such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and processed oil foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressing; condiments such as sauces and dressings; soups, stews, salads, side dishes, furikake (seasoning), pickles, bread, and cereals. In the case of foods for specified health uses, dietary supplements, and functional foods, the food may be in the form of powders, granules, capsules, lozenges, tablets, syrups, and the like.

[0102] The food or feed compositions of the present disclosure can be provided as compositions containing immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimerization-promoting region. They can also be provided as compositions containing host cells expressing immunoglobulins or multimeric immunoglobulins, compositions containing part or all of the culture medium of host cells expressing immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimerization-promoting region, or compositions containing the host cells and part or all of the culture medium. In one aspect, the food or feed compositions of the present disclosure are provided as compositions produced through a host cell culture process, e.g., a fermentation process. In such cases, the culture or brewing fermentation product may be used as a food composition directly, or a purified portion of the brewing fermentation product may be used as a food composition. For example, solid components containing host cells can be filtered from the culture or brewing fermentation product and provided as a liquid composition. Such compositions can include the host cells themselves, parts of the host cells, extracts of the host cells, components of the host cell culture medium, or mixtures thereof. Non-limiting examples of such food compositions using filamentous fungi as host cells include mirin, sake, amazake, miso, soy sauce, shio koji, rice koji, soy sauce koji, barley koji, soybean koji, koji natto, sake lees fish koji: fish sauce base, ragi (Indonesia), ketchup (Indonesian miso, no tomatoes whatsoever), soy sauce: hishi sake brewing, moromi lees, mochi koji, maki koji, cheonggukjang, bonito flakes and other miscellaneous dried fish.

[0103] The feed composition of the present disclosure can be provided as a feed composition labeled for intestinal regulation, intestinal environment improvement, intestinal environment optimization, intestinal putrefaction prevention, etc. for various animals.

[0104] The specific form of the feed composition is not particularly limited, and for example, as long as the effects of the feed composition according to the present disclosure are not impaired, the feed composition may be prepared by mixing it with a normal feed or, if necessary, by mixing it with other ingredients that can be added to a normal feed, or the feed composition itself may be used as feed. For example, the feed may be prepared by mixing the feed with pomace used in the production of a fermented food obtained by host cells.

[0105] (Reagent composition) The reagent compositions of the present disclosure are provided as compositions that utilize the physiological activity of immunoglobulin multimers or immunoglobulin variants having deletions in their C-terminal multimerization-promoting regions. For example, they are provided as reagents that utilize the specific antigen-binding activity or agglutination activity of immunoglobulin multimers or immunoglobulin variants having deletions in their C-terminal multimerization-promoting regions. Such reagent compositions can be used for purposes such as detection, quantification, recovery, and removal of target molecules.

[0106] (Preventive or therapeutic methods for diseases, etc.) In one aspect, the present disclosure provides a method for preventing or treating a disease, disorder, or condition. Typically, the method for preventing or treating a disease, disorder, or condition of the present disclosure utilizes the physiological activity of an immunoglobulin multimer or an immunoglobulin variant having a deletion in a C-terminal multimerization-promoting region according to the present disclosure. The immunoglobulin multimer or an immunoglobulin variant having a deletion in a C-terminal multimerization-promoting region according to the present disclosure binds to an in vivo pathogen or an in vivo molecule exhibiting an abnormal function associated with a disease, disorder, or condition, and exhibits an effect of inhibiting the growth of the pathogen in the body and / or an effect of promoting the excretion of the pathogen or the in vivo molecule exhibiting an abnormal function. By utilizing the physiological activity of such an immunoglobulin multimer or an immunoglobulin having a deletion in a C-terminal multimerization-promoting region, a method for preventing or treating a disease, disorder, condition, or the like associated with the pathogen or the in vivo molecule exhibiting an abnormal function is provided for a subject in need of such prevention or treatment.

[0107] In one aspect, the method for preventing or treating a disease, disorder, condition, etc. of the present disclosure has the following features. For a subject in need of prevention or treatment of a disease, disorder or condition associated with a pathogen or a biological molecule exhibiting abnormal function, A method for preventing or treating a disease, disorder, or condition associated with a pathogen or a biological molecule that expresses an abnormal function, comprising the step of administering a composition containing an immunoglobulin multimer that binds to the pathogen or the biological molecule that expresses an abnormal function, or an immunoglobulin that has a deletion in the C-terminal multimer formation ability-promoting region.

[0108] In one aspect, the disclosed method for preventing or treating a disease, disorder, or condition includes W27 IgA and its variants as an immunoglobulin multimer or immunoglobulin variant, and utilizes the ability of the immunoglobulin multimer or immunoglobulin variant to bind to C. difficile bacteria, thereby providing a method for preventing or treating a disease, disorder, condition, etc. associated with C. difficile bacteria.

[0109] The method for preventing or treating a disease, disorder, or condition disclosed herein comprises the step of administering to a subject in need of prevention or treatment of the disease, disorder, or condition an immunoglobulin multimer or an immunoglobulin variant having a deletion in the C-terminal multimer formation-promoting region disclosed herein.

[0110] The administration mode, administration target, and disease may be the same as those explained above in (Pharmaceutical composition).

[0111] (Tests, Inspections and Diagnostic Methods) In one aspect, the present disclosure provides test, testing, and diagnostic methods. Typically, the test, testing, and diagnostic methods of the present disclosure utilize the specific binding activity of immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimerization-promoting region of the present disclosure. The immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimerization-promoting region of the present disclosure can specifically bind to target molecules in test samples, test samples, or in vivo tissues. Using the specific binding activity of such immunoglobulin multimers or immunoglobulin variants having deletions in the C-terminal multimerization-promoting region, test and testing methods are provided for determining the presence or absence, or quantifying the amount of target molecules in test samples, test samples, or in vivo tissues. Furthermore, in one aspect, test or diagnostic methods are provided for determining a subject's condition, disease, prognosis, disease risk, etc. based on the results of these tests and tests.

[0112] In one aspect, the test, examination and diagnostic methods of the present disclosure comprise the following: contacting a test sample, a specimen, or an in vivo tissue with an immunoglobulin multimer or an immunoglobulin variant having a deletion in the C-terminal multimerization-promoting region; and A testing, examination and diagnostic method comprising a step of detecting or quantifying molecules that bind to the immunoglobulin multimer or immunoglobulin variant having a deletion in the C-terminal multimer formation ability-promoting region in the test sample, examination sample or in vivo tissue. [Example]

[0113] Hereinafter, the present disclosure will be described in more detail with reference to examples, but these are merely illustrative and do not limit the present disclosure.

[0114] Methods and Materials 1.DNA manipulation method E. coli DH5α was used for DNA manipulation. PrimeSTARHS DNA polymerase (TaKaRa) was used for PCR. The In-Fusion HD Cloning Kit (TaKaRa) was used for plasmid construction. Plasmids were extracted using the alkaline SDS method. Nucleotide sequences were analyzed by a contract with FASMAC Corporation.

[0115] 2. Immunoglobulin Quantification and Non-reducing SDS-PAGE Purified immunoglobulins were quantified by ELISA. The immunoglobulin quantification method is detailed below. First, anti-goat-mouse IgA (Southern Biotech: 1040-01) was diluted to 2 μg / ml with 0.05 M NaCO3. 50 μl of this solution was added to each well of a C96 MaxiSorp Nunc-Immuno Plate (Thermo Fisher) (hereafter referred to as the ELISA plate) at 4°C overnight. The plate was washed three times with PBS using a Vacuum-Pette / 96 multiwell pipetter (Sigma-Aldrich) (hereafter referred to as the plate washer). Then, 150 μl of PBS containing 1% bovine serum albumin (BSA) (Wako) (PBS-BSA) was added to each well and allowed to stand overnight at 4°C for blocking. A series of antibody solutions was prepared in a 96-well plate. The antibody solution was serially diluted using PBS-BSA at the following dilutions: 1x (x1; x1 / 100 and x1 / 1000 of the purified antibody are considered x1), 3x (x1 / 3), 10x (x1 / 10), 30x (x1 / 30), 100x (x1 / 100), 300x (x1 / 300), 1000x (x1 / 1000), and 3000x (x1 / 3000). After completely removing the PBS-BSA from the plate, 50 μl of the serial dilutions were added to each well and the plate was left to stand at room temperature for 1 hour. After washing the plate three times with PBS-T containing 0.05% Tween 20 (Chem Cruz), 50 μl of secondary antibody, alkaline phosphatase (ALP)-conjugated anti-goat mouse IgA (Southern Biotech: 1040-04), diluted in PBS-BSA to a final concentration of 0.5 μg / ml, was added to each well. The plate was then washed three times with PBS-T and 50 μl of substrate solution (5 ml carbonate buffer (6.5 mM Na2CO3, 18.5 mM NaHCO3), 10 μl of 1 M MgCl2, and one tablet of Substrate for Alkaline Phosphatase (Sigma-Aldrich) was added to each well. After leaving the mixture at room temperature in the dark, sufficient color development was confirmed visually, and the absorbance at optical density (OD) 405 nm was measured using a TriStar2 LB942 (BERTHOLD TECHNOLOGIES).

[0116] Separately, the production of immunoglobulin multimers was confirmed by non-reducing SDS-PAGE. Sample preparation consisted of adding 3x Native buffer (0.2 M Tris-HCl pH 6.8, 30% glycerol, 0.015% bromophenol blue) to immunoglobulin (5 μg). After electrophoresis on a 6% SDS-PAGE gel using Wide Range Gel Preparation Buffer (4x) for PAGE (Nacalai Tesque), the gel was stained with Coomassie Brilliant Blue (CBB). For reducing SDS-PAGE, a buffer containing 2-ME was added to the sample, which was then denatured at 95°C for 10 minutes. The gel was then electrophoresed on the same 6% SDS-PAGE gel as for the non-reducing SDS-PAGE, followed by Coomassie Brilliant Blue (CBB) staining.

[0117] 3. Analysis of the binding strength of immunoglobulin multimers to E. coli To compare the binding avidity of immunoglobulin multimers, an ELISA was performed on E. coli. E. coli (DH5α or BW38092 and their mutants, both K12 strains) were cultured aerobically at 37°C overnight in LB medium and collected by centrifugation. After washing with PBS, the bacteria were suspended in 0.05 M Na2CO3 buffer and coated onto ELISA plates (4°C overnight). After blocking with 1% BSA in PBS, as in the above-described purified IgA antibody quantification ELISA, serial dilutions of each antibody were prepared in a 96-well plate and added to the wells. The plate was then washed with 0.05% Tween 20 in PBS. The secondary antibody, alkaline phosphatase (ALP)-conjugated anti-goat mouse IgA (Southern Biotech: 1040-04), was diluted in PBS-BSA to a final concentration of 0.5 μg / ml and added at 50 μl / well. The plate was then incubated at room temperature for 1 hour. After washing the plate three times with PBS-T using a plate washer, the color reaction was carried out as described above using Alkali Phosphatase tablets (Sigma). To ensure sufficient reaction, the ELISA plate was incubated overnight at 4°C, and the OD at 405 nm was measured using a TriStar2 LB942 (Berthold Technologies).

[0118] 4. In vitro growth inhibition test of immunoglobulin multimers against Clostridioides difficile (C. difficile) bacteria C. difficile cultured overnight anaerobically was diluted to 10,000 cfu per tube, and test antibodies were added at 2.4 mg / ml. Anaerobically culture was then performed. After 6 hours, the bacterial solution was diluted and plated. The number of colonies was counted after further anaerobically culture. The test antibodies used were the rW27 pentameric IgA antibody (a pentameric IgA antibody consisting of an IgA heavy chain having SEQ ID NO: 4: CHO W27notailCOMP and the light chain of antibody RS_H000_L001 (see WO 2014 / 142084)), the rW27 dimeric IgA antibody (a dimeric IgA antibody consisting of an IgA and J chain having the heavy and light chains of antibody RS_H000_L001 (see WO 2014 / 142084)), and a control dimeric IgA antibody.

[0119] 5. Suppressive effect of immunoglobulin polymers against C. difficile enteritis Using a method similar to that described in WO 2023 / 277166, the test antibody was orally administered to mice to test its effect of suppressing enteritis. Specifically, a spore suspension of a C. difficile strain (VPI10483) was prepared and stored at -80°C in small aliquots. The spore suspension was thawed, cultured, and then plated onto C. difficile selective medium plates (TCCFA plates). The number of viable bacteria obtained was then calculated. The method for producing spores is as follows. C. difficile was plated on SMC medium and cultured anaerobically at 37°C for 7 days. 3 ml of ice-cold sterile water was added to the plate, and colonies were scraped off with a cell scraper and transferred to a 50 ml tube. The plate was washed with 3 ml of ice-cold sterile water, and the wash was transferred to the same tube. After centrifugation at 8,000 g for 10 minutes at 4°C, the supernatant was discarded and the cells were suspended in 20 ml of ice-cold sterile water. After centrifugation at 8,000 g for 10 minutes at 4°C, the supernatant was discarded and the cells were suspended in 10 ml of ice-cold sterile water. After suspension, 10 ml of ethanol was added, the mixture was vortexed, and the mixture was left at room temperature for 1 hour. After centrifugation at 8,000 g for 10 minutes at 4°C, the supernatant was discarded and the cells were suspended in 20 ml of ice-cold sterile water. This procedure was repeated twice, the supernatant was discarded, and the cells were suspended in 5 ml of ice-cold sterile water. The mixture was dispensed in 100 μl aliquots into 1.5 ml tubes and stored at −80° C. The composition of the SMC medium is shown below. SMC medium composition The above reagents were dissolved in 200 ml of sterile distilled water and sterilized in an autoclave. After cooling to approximately 60°C, 600 μl of 10% (w / v) L-cysteine ​​was added, and the mixture was dispensed into 10 cm plates.

[0120] C. difficile selective medium plates were prepared by dissolving the reagents listed in Table 1 below in 400 ml of sterile distilled water and sterilizing the mixture in an autoclave. After allowing the mixture to cool to 60°C, 2 ml of cyclocloserine (Sigma-Aldrich) (50 mg / ml) and 2 ml of cefoxitin (Sigma-Aldrich) (1.6 mg / ml) were added, and the mixture was dispensed into 10 cm plates.

[0121] (Table 1) Composition of C. difficile selective medium TIFF0007760200000005.tif78154

[0122] C57BL / 6 mice (8 weeks old) were purchased from CLEA Japan and acclimated for one week in a sterile isolator for infection experiments. After a week of acclimation, a triple antibiotic mixture (gentamicin: 500 mg / kg, kanamycin: 150 mg / kg, metronidazole: 50 mg / kg) was orally administered using a probe for four days. After four days, each mouse was weighed, and those that lost weight due to antibiotic administration were excluded. The remaining mice were randomly assigned to groups. The day after antibiotic administration ended, they were inoculated with C. difficile spores (10x10 3 Each mouse was orally infected with 100 μg of 100 μg of the test antibody via a probe. Six hours later, 300 μg of each test antibody was orally administered via a probe (n=6 mice for each test antibody). The test antibodies used were the rW27 pentameric IgA antibody (a pentameric IgA antibody consisting of an IgA heavy chain having SEQ ID NO: 4: CHO W27notailCOMP and the light chain of antibody RS_H000_L001 (see WO 2014 / 142084)), the rW27 dimeric IgA antibody (a dimeric IgA antibody consisting of an IgA and J chain having the heavy and light chains of antibody RS_H000_L001 (see WO 2014 / 142084)), and a control dimeric IgA antibody. Subsequently, 300 μg of each antibody was administered once daily for a total of 7 days. From day 8 onward, the mice were observed without antibody administration.

[0123] Example 1: Construction of immunoglobulins with deletions in the C-terminal multimerization-promoting region We investigated the effect of deletion of the C-terminal oligomerization-promoting region of immunoglobulins on oligomerization.

[0124] Genes encoding immunoglobulins and vectors containing them were constructed and introduced into CHO cells according to standard methods, similar to the method described in Example 8 of WO2023 / 277166.

[0125] As exemplary immunoglobulins, the W27 antibody described in International Publication No. 2014 / 142084 and RS_H000_L001 (W27 IgA), a CHO cell-produced recombinant IgA of the W27 antibody, in which a mutation for protein L binding has been introduced into the light chain, were used (see Patent Application No. 2021-110421). Using the W27 IgA antibody, genome-modified CHO cells were prepared by standard methods using a nucleic acid construct encoding IgA with a native L chain and a fusion protein with the following structure as the H chain, and the multimerization state of the secreted IgA was examined. TIFF0007760200000006.tif88153

[0126] The amino acid sequences of the IgA heavy chains with each structure and the nucleic acid sequences encoding them are shown below. When expressing them in CHO cells, the mouse IgV signal sequence "MKCSWIIFFLMAVVTGVNS" was added to the N-terminus. SEQ ID NO: 8: CHO W27 HAACOMP IgA H chain amino acid sequence (without signal sequence) TIFF0007760200000007.tif51152>SEQ ID NO: 9: CHO W27 HKKCOMP IgA H chain amino acid sequence (without signal sequence) TIFF0007760200000008.tif50156>SEQ ID NO: 10: CHO W27HdirectCOMP IgA H amino acid sequence (without signal sequence) TIFF0007760200000009.tif45158>SEQ ID NO: 1 (supra): CHO W27notailCOMP IgA H amino acid sequence (without signal sequence) TIFF0007760200000010.tif59154>SEQ ID NO: 11: Nucleotide sequence of CHO W27HAACOMP (including N-terminal signal sequence) TIFF0007760200000011.tif165156> SEQ ID NO: 12: Nucleotide sequence of CHO W27HKKCOMP (including N-terminal signal sequence) TIFF0007760200000012.tif166152> SEQ ID NO: 13: Nucleotide sequence of CHO W27HdirectCOMP (including N-terminal signal sequence) TIFF0007760200000013.tif159152>SEQ ID NO: 2: Nucleotide sequence of CHO W27HnotailCOMP (including N-terminal signal sequence) TIFF0007760200000014.tif153154

[0127] The light chain of the W27 IgA antibody has the same amino acid sequence as the light chain of a modified CHO cell-produced recombinant IgA of the W27 antibody (RS_H000_L001) (see International Publication No. 2023 / 277142), and the amino acid sequence of its light chain variable region (RS_LV001) has a mutation for protein L binding. >RS_LV001 (SEQ ID NO: 3) (supra) TIFF0007760200000015.tif16152

[0128] As shown in Figure 5, it was confirmed that the structure of the IgA H chain C-terminal region and the structure of the linker can affect IgA multimer formation. In particular, it was observed that a higher proportion of IgA was produced as multimers when an H chain to which the COMP sequence was attached without a linker was used, and especially when an H chain to which the COMP sequence was attached after a partial deletion of the IgA C-terminal region was used.

[0129] Example 2: Binding properties of immunoglobulin multimers The binding ability of multimerized W27 IgA as an immunoglobulin multimer to E. coli was examined. As described in International Publication WO 2014 / 142084, it is known that W27 IgA antibodies expressed in mammalian cells bind to E. coli. Multimerized W27 IgA secreted from CHO cells was purified, and the E. coli-binding ability of these IgA multimers was tested. For comparison, W27 IgA monomer (W27monomer) and W27 IgA dimer (W27dimer), obtained by expressing unmodified W27 in CHO cells, were also tested. As a result of measuring the binding affinity to E. coli, all W27IgA antibody multimers secreted from CHO cells exhibited significantly higher binding affinity than the W27 IgA monomer (W27monomer) and W27 IgA dimer (W27dimer), as shown in Figure 6. Furthermore, among the W27 IgA antibody multimers secreted from CHO cells, W27notailCOMP, which lacks the C-terminal region and is linked to COMP without a linker, exhibited the highest binding affinity to E. coli.

[0130] Example 3: Inhibitory effect of immunoglobulin multimers on C. difficile growth (in vitro) As an immunoglobulin multimer, the multimerized W27 IgA was tested for its growth inhibitory effect on C. difficile bacteria. As a result, both the rW27 pentameric IgA antibody and the rW27 dimeric IgA antibody showed superior growth inhibitory effects against C. difficile bacteria compared to the control dimeric IgA antibody. These results demonstrate that the immunoglobulin polymers rW27 pentameric IgA antibody and rW27 dimeric IgA antibody have excellent inhibitory effects on the growth of C. difficile in vitro.

[0131] Example 4: Inhibitory effect of immunoglobulin multimers on C. difficile-associated enteritis (in vivo) The inhibitory effects of rW27 pentameric IgA antibody and rW27 dimeric IgA antibody on C. difficile-associated enteritis were confirmed in vivo. The results showed that the rW27 dimeric IgA antibody slightly improved survival rate compared to the control dimeric IgA antibody, whereas the rW27 pentameric IgA antibody significantly improved survival rate compared to both the rW27 dimeric IgA antibody and the control dimeric IgA antibody (p=0.0295). As a result, it was found that the rW27 pentameric IgA antibody in particular had an excellent inhibitory effect on C. difficile-associated enteritis in vivo.

[0132] The sequence information set forth in this disclosure is reproduced below. SEQ ID NO: 1: CHO W27notailCOMP IgA H amino acid sequence (without signal sequence) TIFF0007760200000016.tif51152> SEQ ID NO: 2: Nucleotide sequence of CHO W27HnotailCOMP (including the portion encoding the N-terminal mouse IgV signal sequence "MKCSWIIFFLMAVVTGVNS") TIFF0007760200000017.tif153154>SEQ ID NO: 3RS_LV001 Light chain variable region amino acid sequence TIFF0007760200000018.tif21155>SEQ ID NO: 4: Linker (KK) TIFF0007760200000019.tif6154>SEQ ID NO: 5: Linker (AA) TIFF0007760200000020.tif7149>SEQ ID NO: 6: Mouse IgV signal sequence TIFF0007760200000021.tif6152>SEQ ID NO: 7: notail deletion sequence TIFF0007760200000022.tif6148>SEQ ID NO: 8: CHO W27 HAACOMP IgA H chain amino acid sequence (without signal sequence) TIFF0007760200000023.tif40154>SEQ ID NO: 9: CHO W27 HKKCOMP IgA H chain amino acid sequence (without signal sequence) TIFF0007760200000024.tif39153>SEQ ID NO: 10: CHO W27HdirectCOMP IgA H amino acid sequence (without signal sequence) TIFF0007760200000025.tif37156>SEQ ID NO: 11: Nucleotide sequence of CHO W27HAACOMP (including N-terminal signal sequence) TIFF0007760200000026.tif167151> SEQ ID NO: 12: Nucleotide sequence of CHO W27HKKCOMP (including N-terminal signal sequence) TIFF0007760200000027.tif165154>SEQ ID NO: 13: Nucleotide sequence of CHO W27HdirectCOMP (including N-terminal signal sequence) TIFF0007760200000028.tif160152> SEQ ID NO: 14: Nucleotide sequence of CHO W27HnotailCOMP (including N-terminal signal sequence) TIFF0007760200000029.tif152153

Claims

1. contacting two or more immunoglobulins, including at least one immunoglobulin variant having a deletion in the C-terminal multimerization-promoting region of at least one heavy chain; and conjugating the two or more immunoglobulins Including, an immunoglobulin having a deletion in a C-terminal multimerization-promoting region, wherein a heterologous amino acid sequence that promotes multimerization is added to the C-terminus thereof, the heterologous amino acid sequence comprising an amino acid sequence that forms a coiled-coil domain; each of the plurality of immunoglobulin molecules is IgA or IgM; A method for producing immunoglobulin multimers.

2. The method of claim 1, wherein the deletion is a deletion of one or more amino acids in the multimerization-promoting region.

3. An immunoglobulin multimer comprising two or more immunoglobulins, wherein at least one of the immunoglobulins constituting the two or more immunoglobulins has a deletion in the C-terminal multimerization-promoting region of at least one heavy chain, a heterologous amino acid sequence that promotes multimerization is added to the C-terminus of the immunoglobulin having the deletion, the heterologous amino acid sequence comprising an amino acid sequence that forms a coiled-coil domain; Immunoglobulin multimers that are immunoglobulin variants that are IgA or IgM.

4. The immunoglobulin multimer according to claim 3, which has a bacterial growth inhibitory effect.

5. The immunoglobulin multimer according to claim 3, which has bacterial agglutination activity.

6. An immunoglobulin variant that is an IgA or IgM having a deletion in a C-terminal multimerization-promoting region and has the ability to form immunoglobulin multimers, a heterologous amino acid sequence that promotes multimerization is added to the C-terminus thereof, the heterologous amino acid sequence including an amino acid sequence that forms a coiled-coil domain; The immunoglobulin variant.

7. A food composition, feed composition, pharmaceutical composition, or reagent composition comprising the immunoglobulin multimer of claim 3 or the immunoglobulin variant of claim 6.

8. A nucleic acid comprising a nucleic acid sequence encoding the immunoglobulin variant of claim 6.

9. A cell comprising the nucleic acid of claim 8.

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