WO2024248127A1 - Drug for preventing or treating allergic diseases - Google Patents

Abstract

The present invention addresses the problem of developing a substance capable of preventing or treating allergic diseases, and a pharmaceutical composition containing said substance, on the basis of the previous finding that allergic disease reactions occurring in a living body can be prevented by a substance that binds to an Ibp protein of bacteria belonging to the family Lachnospiraceae.　The inventors of the present invention have found that an antibody or antibody derivative is created as a substance which, instead of direct binding to IgE, binds to a region important for binding to IgE in an Ibp protein produced by bacteria belonging to the family Lachnospiraceae, which are intestinal bacteria associated with allergic diseases, and a pharmaceutical composition for preventing or treating allergic diseases, containing said antibody or antibody derivative, can be provided.

Description

Drugs for preventing and treating allergic diseases

The present invention relates to the development of pharmaceuticals for preventing or treating allergic diseases. More specifically, the present invention relates to the development of antibody pharmaceuticals for preventing or treating allergic diseases.

　Allergies occur when allergens (antigens) - substances that cause allergies, such as those derived from food, pollen, or dust mites - enter the body, causing the production of IgE antibodies (sensitization). If an allergen is then re-entered into the body, the IgE antibodies react, causing the mast cells to release chemical messengers such as histamine, which leads to the symptoms of the allergic disease.

Although the mechanism of allergy onset remains largely unknown, in recent years attention has been focused on its connection with the intestinal microflora. Studies of pediatric allergy patients have reported an increase in Ruminococcus gnavus (Mediterraneibacter gnavus) (hereafter referred to as "R. gnavus") in the intestines, and experiments in which R. gnavus was administered to mice have reported that administration of this bacterium is associated with and promotes the onset of allergic diseases, particularly respiratory allergies (Non-Patent Document 1).

　Antihistamines are generally used to treat allergies, but they have side effects such as drowsiness, and their effectiveness can decrease with continued use. The anti-IgE antibody omalizumab (trade name: Xolair) is also available commercially as an antibody-based allergy treatment. Xolair binds to human IgE and inhibits the binding between human IgE and IgE receptors, thereby suppressing allergic reactions. Xolair is an antibody that targets the human IgE antibody itself, and there is concern that it may cause serious side effects such as anaphylactic reactions, and that it may affect the host's immune defense mechanism mediated by IgE, such as in the case of parasitic infections, because it reduces the amount of IgE in the blood.

Under these circumstances, it was discovered that the Ibp protein of bacteria of the Lachnospiraceae family, which includes the enterobacterium R. gnavus, is involved in the onset of allergic diseases, and the pathogenesis mechanism of allergic diseases involving the Ibp protein of bacteria of the Lachnospiraceae family was elucidated (Patent Document 1). It was shown that allergic disease reactions occurring in the body can be prevented by administering to the body or producing in the body a substance that binds to the Ibp protein of bacteria of the Lachnospiraceae family (Patent Document 1).

The binding ability of R. gnavus to IgA was originally studied, and it was reported that the immunoglobulin-binding proteins (Ibp) IbpA and IbpB contained in R. gnavus bind to human and mouse IgA as superantigens, and that these superantigens act on B cells to increase IgA production (Non-Patent Document 2).

Patent application 2021-195941

Chua, et al., Gastroenterology. 154: 154-167, 2018 Bunker, et al., Sci Transl Med, 11, eaau9356, 2019

The present invention aims to develop a substance capable of preventing or treating allergic diseases and a pharmaceutical composition containing such a substance, based on the knowledge that a substance that binds to the Ibp protein of bacteria of the Lachnospiraceae family can prevent allergic disease reactions occurring in the body.

The inventors of the present invention have demonstrated that they can create antibodies or antibody derivatives that do not directly bind to IgE but bind to a region important for binding to IgE in the Ibp protein produced by Lachnospiraceae bacteria, an enterobacteria associated with allergic diseases, and provide pharmaceutical compositions for preventing or treating allergic diseases that contain such antibodies or antibody derivatives.

More specifically, in order to solve the above-mentioned problems, the present application provides the following aspects:
[1]: (1) heavy chain complementarity determining regions, CDR1 (GFTFSNYGMA, SEQ ID No.: 1), CDR2 (TISYDGSSTYYRDSVKG, SEQ ID No.: 2), and CDR3 (ESFGTTVVPHYFDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (QASQDIGNWLS, SEQ ID No.: 4), CDR2 (GATSLAD, SEQ ID No.: 5), and CDR3 (LQAYSAPWT, SEQ ID No.: 6);
an antibody or antibody derivative having binding activity against an Ibp antigen, comprising the heavy and light chain complementarity determining regions of
[2]: The antibody or antibody derivative according to [1], wherein the antibody or antibody derivative is a human antibody;
[3]: The antibody or antibody derivative according to [2], wherein the human antibody derivative is a human antibody variant or a functional fragment thereof selected from a humanized antibody, a chimeric antibody, a multivalent antibody, and a multispecific antibody;
[4]: the antibody or antibody derivative according to [3], wherein the functional fragment is a Fab, a Fab', a F(ab')2, a single-chain variable region fragment (scFv) or a disulfide-stabilized (dsFv) antibody;
[5]: The amino acid sequence of the heavy chain variable region VH domain of an antibody or human antibody derivative having binding ability to an Ibp antigen is
(VH-1-1) EVMLVESGGG LVQPGRSMKL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNAKSTLY LQMNSLRSED TATYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 7) or an amino acid sequence of SEQ ID NO: 7 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-2) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 9) amino acid sequence, or an amino acid sequence of SEQ ID NO: 9 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-3) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRSED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 10) amino acid sequence, or an amino acid sequence of SEQ ID NO: 10 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-4) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 11) amino acid sequence, or an amino acid sequence containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the amino acid sequence of SEQ ID NO: 11 other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
The antibody or antibody derivative according to [1] or [2], selected from the group consisting of:
[6]: The amino acid sequence of the light chain variable region VL domain of an antibody or human antibody derivative having binding activity to an Ibp antigen is
(VL-1-1) DIQMTQSPAS LSASLEEIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YSLKISRLQV EDIGIYYCLQ AYSAPWTFGG GTKLELK (SEQ ID NO: 8), or an amino acid sequence of SEQ ID NO: 8 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-2) DIQMTQSPSS LSASVGDRVT ITCQASQDIG NWLSWYQQKP GKSPKLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 12) amino acid sequence, or an amino acid sequence of SEQ ID NO: 12 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-3) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 13) amino acid sequence, or an amino acid sequence containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the amino acid sequence of SEQ ID NO: 13 other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-4) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YTLTISSLQP EDIATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 14) amino acid sequence, or an amino acid sequence of SEQ ID NO: 14 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
The antibody or antibody derivative according to [1] or [2], selected from the group consisting of:
[7]: The antibody or antibody derivative according to any one of [1] or [2], which binds to Ibp and inhibits the binding of Ibp to IgE in vivo;
[8]: the antibody or antibody derivative according to [7] for preventing or treating an allergic disease;
[9]: the antibody or antibody derivative according to [8], wherein the allergic disease is atopic dermatitis;
[10]: (1) heavy chain complementarity determining regions, CDR1 (GFTFSNYGMA, SEQ ID No.: 1), CDR2 (TISYDGSSTYYRDSVKG, SEQ ID No.: 2), and CDR3 (ESFGTTVVPHYFDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (QASQDIGNWLS, SEQ ID No.: 4), CDR2 (GATSLAD, SEQ ID No.: 5), and CDR3 (LQAYSAPWT, SEQ ID No.: 6);
a pharmaceutical composition for preventing or treating an allergic disease, comprising an antibody or an antibody derivative having binding affinity to an Ibp antigen and comprising the heavy and light chain complementarity determining regions of said antibody;
[11]: The pharmaceutical composition for preventing or treating an allergic disease according to [10], wherein the antibody or antibody derivative is a human antibody;
[12]: A pharmaceutical composition for preventing or treating an allergic disease according to [11], wherein the allergic disease is atopic dermatitis.

　Based on the findings that allergic reactions occurring in the body can be prevented by substances that bind not directly to IgE but to a region important for IgE binding in the Ibp protein of Lachnospiraceae bacteria (e.g., Ruminococcus bacteria), an enterobacteria associated with allergic diseases, the present invention provides an antibody or antibody derivative that binds to the Ibp protein of Lachnospiraceae bacteria or a modified version thereof and can prevent or treat allergic reactions occurring in the body. This antibody or antibody derivative can suppress allergic reactions by exerting an inhibitory effect on allergens. Therefore, it does not bind to human IgE and can suppress allergic reactions without inhibiting the host's immune system, thereby reducing the problem of side effects.

FIG. 1 shows schematic diagrams of various fragments of Ibp protein that were prepared to investigate the binding mode between Ibp protein and IgE. Figure 2 shows the in vitro activity of Clone A antibody and Clone B antibody selected in the present invention in inhibiting the binding of Ibp protein to IgE. Figure 3 shows an outline of the experimental protocol used to investigate the effect of anti-Ibp antibodies in inhibiting the binding of human IgE to Ibp in vivo. Figure 4 shows the results of an investigation into whether Clone A antibodies exhibit an allergy-suppressing effect in vivo in SPF mice.

　Based on the previous knowledge that allergic reactions occurring in the body can be prevented by substances that bind to the Ibp protein of Lachnospiraceae bacteria, the present invention aims to eliminate the side effects of antihistamines that are commonly used in the treatment of allergic diseases and the anti-IgE antibody omalizumab (trade name: Xolair) used to treat allergic diseases, and has demonstrated that, based on the previous knowledge, it is possible to create an antibody or antibody derivative as a substance that binds to a region important for binding to IgE in the Ibp protein produced by Lachnospiraceae bacteria, an intestinal bacterium associated with allergic diseases, rather than directly binding to IgE, as a new treatment tool for allergic diseases, and to provide a pharmaceutical composition for preventing or treating allergic diseases that contains such an antibody or antibody derivative.

Specifically, in one aspect, the present invention can provide an antibody or antibody derivative that has binding affinity to an Ibp antigen and includes heavy and light chain complementarity determining regions (CDR1 to CDR3) represented by specific amino acid sequences.

　In previous studies, the inventors of the present invention have shown that the Ibp protein of bacteria of the genus Ruminococcus binds to IgE, causing allergic diseases (Patent Document 1). Specifically, they have shown that the Ibp protein of bacteria of the family Lachnospiraceae, more specifically bacteria of the genus Ruminococcus among bacteria of the family Lachnospiraceae, and even more specifically, the Ibp protein derived from Ruminococcus gnavus (R. gnavus) among bacteria of the genus Ruminococcus, binds to IgE in the body and induces histamine production from mast cells, resulting in allergies.

On the other hand, the inventors of the present invention have also shown that by inhibiting the non-specific binding of the Ibp protein or a modified form thereof of Lachnospiraceae bacteria to IgE in the body, it is possible to inhibit histamine production from mast cells, thereby inhibiting the onset of allergic diseases (Patent Document 1).

Ibp protein is an immunoglobulin binding protein found in R. gnavus, and R. gnavus Ibp protein has two homologues, IbpA and IbpB. R. gnavus IbpA has 607 amino acids (WP_105084811.1) and IbpB has 625 amino acids (WP_105084812.1). Both proteins have a signal peptide (SP), variable region (VR), conserved repeat sequence, and Ig binding domain arranged from the N-terminus (see Figure 1). It was known that the Ig binding domain in the C-terminal region of this Ibp protein gives it the ability to nonspecifically bind to mouse IgG, IgM, and IgA.

The inventors previously created ibp gene fragments of various lengths using R. gnavus genomic DNA (NCBI Reference Sequence: NZ_CP027002.1) as a template, created various truncated Ibp proteins based on each fragment, and examined the portions involved in binding to IgE. As a result, it was revealed that the repeat region protein with the amino acid sequence of amino acid numbers 111 to 378 of the IbpA protein (WP_105084811.1) or amino acid numbers 126 to 393 of the IbpB protein (WP_105084812.1), and the C-terminal region protein with the amino acid sequence of amino acid numbers 379 to 574 (ibpA C200) of the IbpA protein (WP_105084811.1) or amino acid numbers 394 to 592 (ibpB C200) of the IbpB protein (WP_105084812.1) are involved in binding to IgE.

From these results, it is preferable that the antibody or antibody derivative of the present invention binds to a structural region on the Ibp protein of a bacterium of the family Lachnospiraceae that is involved in the binding between the Ibp protein and IgE. An example of such a structure on the Ibp protein is the C-terminal region of the IbpA protein (WP_105084811.1) or the IbpB protein (WP_105084812.1). An example of the C-terminal region of the IbpA protein (WP_105084811.1) or the IbpB protein (WP_105084812.1) is, for example, a C-terminal region protein having an amino acid sequence of amino acid numbers 379 to 574 of the IbpA protein (WP_105084811.1) or amino acid numbers 394 to 592 of the IbpB protein (WP_105084812.1).

The antibody or antibody derivative of the present invention is intended to inhibit the binding of Ibp proteins to IgE in vivo, and therefore preferably binds to the C-terminal region of these Ibp proteins, thereby inhibiting the binding of Ibp proteins to IgE in vivo.

As the antibody or antibody derivative of the present invention having the above-mentioned characteristics, it is possible to provide an antibody or antibody derivative comprising heavy and light chain complementarity determining regions (CDR1 to CDR3) represented by specific amino acid sequences. Examples of the heavy and light chain complementarity determining regions (CDR1 to CDR3) include the following:
(1) heavy chain complementarity determining regions, CDR1 (GFTFSNYGMA, SEQ ID No.: 1), CDR2 (TISYDGSSTYYRDSVKG, SEQ ID No.: 2), and CDR3 (ESFGTTVVPHYFDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (QASQDIGNWLS, SEQ ID No.: 4), CDR2 (GATSLAD, SEQ ID No.: 5), and CDR3 (LQAYSAPWT, SEQ ID No.: 6).

Antibodies or antibody derivatives containing these heavy and light chain variable regions bind to the C-terminal region of the Ibp protein, and as a result have the activity of inhibiting the binding of the Ibp protein to IgE in vivo. By possessing such activity, the antibodies or antibody derivatives of the present invention can be used to prevent or treat allergic diseases.

Among the antibodies or antibody derivatives of the present invention, the monoclonal antibody obtained as Clone A having binding activity to the Ibp antigen has an amino acid sequence of the heavy chain variable region VH domain:
It was revealed that the amino acid sequence of (VH-1-1) EVMLVESGGG LVQPGRSMKL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNAKSTLY LQMNSLRSED TATYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 7).

It is common knowledge regarding the amino acid sequence structure of antibodies that the amino acid sequence of the heavy chain variable region VH domain other than the sequences of CDR1 to CDR3 is allowed to be variable. Therefore, the amino acid sequence of the above-mentioned (VH-1-1) other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3) is allowed to contain one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions.

That is, the present invention relates to an antibody or human antibody derivative having binding ability to an Ibp antigen, the heavy chain variable region VH domain of which has an amino acid sequence of:
(VH-1-1) EVMLVESGGG LVQPGRSMKL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNAKSTLY LQMNSLRSED TATYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 7) or an amino acid sequence of SEQ ID NO: 7 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
It is possible to provide an antibody or antibody derivative, wherein

The antibody or antibody derivative of the present invention may be a heavy chain variable region VH domain having the same complementarity determining region (CDR) as the heavy chain variable region VH domain of Clone A having the above-mentioned ability to bind to the Ibp antigen but having a different framework region (FR), and the amino acid sequence of the heavy chain variable region VH domain may be
(VH-1-2) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 9);
(VH-1-3) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRSED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 10);
(VH-1-4) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 11);
The amino acid sequence may be any of the following:

As mentioned above, variability is permitted in the amino acid sequence of the heavy chain variable region VH domain other than the sequences of CDR1 to CDR3, and the amino acid sequences of the above-mentioned (VH-1-2) to (VH-1-4) other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3) are permitted to contain one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions.

That is, the present invention relates to an antibody or human antibody derivative having binding ability to an Ibp antigen, the heavy chain variable region VH domain of which has an amino acid sequence of:
(VH-1-2) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 9) amino acid sequence, or an amino acid sequence of SEQ ID NO: 9 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-3) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRSED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 10) amino acid sequence, or an amino acid sequence of SEQ ID NO: 10 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-4) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 11) amino acid sequence, or an amino acid sequence containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the amino acid sequence of SEQ ID NO: 11 other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
Also provided is an antibody or antibody derivative selected from the group consisting of:

Among the antibodies or antibody derivatives of the present invention, the monoclonal antibody obtained as Clone A having binding activity to the Ibp antigen has an amino acid sequence of the light chain variable region VL domain:
It was revealed that the amino acid sequence of (VL-1-1) DIQMTQSPAS LSASLEEIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YSLKISRLQV EDIGIYYCLQ AYSAPWTFGG GTKLELK (SEQ ID NO: 8).

It is common knowledge regarding the amino acid sequence structure of antibodies that the amino acid sequence of the light chain variable region VL domain other than the sequences of CDR1 to CDR3 is allowed to be variable. Therefore, the amino acid sequence of the above-mentioned (VL-1-1) other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6) is allowed to contain one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions.

That is, the present invention relates to an antibody or human antibody derivative having binding ability to an Ibp antigen, the amino acid sequence of the light chain variable region VL domain of which is
(VL-1-1) DIQMTQSPAS LSASLEEIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YSLKISRLQV EDIGIYYCLQ AYSAPWTFGG GTKLELK (SEQ ID NO: 8), or an amino acid sequence of SEQ ID NO: 8 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
It is possible to provide an antibody or antibody derivative, wherein

The antibody or antibody derivative of the present invention may be a light chain variable region VL domain having the same complementarity determining region (CDR) as the light chain variable region VL domain of Clone A having the above-mentioned binding ability to the Ibp antigen but having a different framework region (FR), and the amino acid sequence of the light chain variable region VL domain is
(VL-1-2) DIQMTQSPSS LSASVGDRVT ITCQASQDIG NWLSWYQQKP GKSPKLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 12);
(VL-1-3) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 13);
(VL-1-4) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YTLTISSLQP EDIATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 14);
The amino acid sequence may be any of the following:

As mentioned above, variability is permitted in the amino acid sequence of the light chain variable region VL domain other than the sequences of CDR1 to CDR3, and the amino acid sequences of (VL-1-2) to (VL-1-4) described above other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6) are permitted to contain one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions.

That is, the present invention relates to an antibody or human antibody derivative having binding ability to an Ibp antigen, the amino acid sequence of the light chain variable region VL domain of which is
(VL-1-2) DIQMTQSPSS LSASVGDRVT ITCQASQDIG NWLSWYQQKP GKSPKLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 12) amino acid sequence, or an amino acid sequence of SEQ ID NO: 12 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-3) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 13) amino acid sequence, or an amino acid sequence containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the amino acid sequence of SEQ ID NO: 13 other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-4) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YTLTISSLQP EDIATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 14) amino acid sequence, or an amino acid sequence of SEQ ID NO: 14 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
Also provided is an antibody or antibody derivative selected from the group consisting of:

The antibody or human antibody derivative having binding ability to the Ibp antigen that can be used in the present invention may be an antibody or human antibody derivative formed from any combination of the heavy chain variable region VH domain and the light chain variable region VL domain described above. That is, it can be formed by combining any of the VH domains (VH-1-1) to (VH-1-4) with any of the VL domains (VL-1-1) to (VL-1-4). More specifically,
- A combination of the VH domain (VH-1-1) and the VL domain (VL-1-1): a rat antibody; - A combination of the VH domain (VH-1-1) and any of the VL domains (VL-1-2) to (VL-1-4), or a combination of the VH domain (VH-1-1) and any of the VL domains (VH-1-2) to (VH-1-4) and the VL domain (VL-1-1): a chimeric antibody; - A combination of the VH domain (VH-1-2) to (VH-1-4) and any of the VL domains (VL-1-2) to (VL-1-4): a humanized antibody.

The antibody or antibody derivative of the present invention can be made into a human antibody derivative while maintaining the above-mentioned sequences of CDR1-3 of the heavy and light chains based on known techniques generally relating to antibodies or antibody derivatives. Such human antibody derivatives can be selected from human antibody variants or functional fragments thereof selected from humanized antibodies, chimeric antibodies, multivalent antibodies, and multispecific antibodies. Here, functional fragments of antibodies or antibody derivatives can include Fab, Fab', F(ab')2, single chain variable region fragments (scFv), disulfide stabilized (dsFv) antibodies, etc.

In the present invention, it has been shown that the antibody or antibody derivative described above can inhibit the binding of Ibp protein to IgE in the body, thereby suppressing the secretion of histamine from mast cells. This indicates that the occurrence of allergic diseases in the body can be prevented or treated. Therefore, in another aspect of the present invention, an invention can be provided relating to the medicinal use of an antibody or antibody derivative containing heavy and light chain complementarity determining regions (CDR1 to CDR3) represented by specific amino acid sequences as the antibody or antibody derivative of the present invention having the characteristics described above.

Specifically, the present invention relates to
(1) heavy chain complementarity determining regions, CDR1 (GFTFSNYGMA, SEQ ID No.: 1), CDR2 (TISYDGSSTYYRDSVKG, SEQ ID No.: 2), and CDR3 (ESFGTTVVPHYFDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (QASQDIGNWLS, SEQ ID No.: 4), CDR2 (GATSLAD, SEQ ID No.: 5), and CDR3 (LQAYSAPWT, SEQ ID No.: 6);
It is possible to provide a pharmaceutical composition for preventing or treating allergic diseases, which comprises an antibody or an antibody derivative having binding affinity to an Ibp antigen and which comprises the heavy and light chain complementarity determining regions of Ibp.

Alternatively, the present invention relates to a medical use of the antibody or antibody derivative,
- a method for preventing or treating allergic diseases by administering the above-mentioned antibody or antibody derivative;
- an antibody or an antibody derivative as defined above for use in preventing or treating an allergic disease;
- use of an antibody or antibody derivative as described above in the manufacture of a medicament for the prevention or treatment of an allergic disease;
It can also be written as:

The antibody or antibody derivative of the present invention binds to the IgE-binding region on the Ibp protein of Lachnospiraceae bacteria, competing with IgE to prevent IgE from binding to the Ibp protein, thereby inducing IgE-induced stimulation of mast cells and inhibition of histamine production and release by mast cells. Due to this mechanism of action, the antibody or antibody derivative of the present invention can be used for preventive purposes in addition to therapeutic purposes for allergic diseases. Allergic diseases often develop symptoms due to sensitization to allergens at unintended times, and in the medical field, it is desirable to use them not only for symptomatic treatment but also for preventive purposes.

The antibody or antibody derivative used in the medical applications of the present invention may have the characteristics already described above, and in particular when administered to humans, it is preferable that the antibody or antibody derivative be a human antibody or antibody derivative.

Allergic diseases that can be prevented or treated with the antibody or antibody derivative of the present invention include bronchial asthma, atopic dermatitis, allergic rhinitis, hay fever, food allergies, and allergic conjunctivitis. Of these, atopic dermatitis is in demand as an allergic disease that can be prevented or treated with the antibody or antibody derivative of the present invention.

When the antibody or antibody derivative of the present invention is used for medical purposes, it can be administered by common routes such as intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration, or intraperitoneal administration. In addition, since the Lachnospiraceae bacteria that produce the target Ibp protein are enterobacteria, it can also be administered by routes such as rectal administration. By applying an appropriate drug delivery system (DDS), it can also be administered by other routes.

The present invention will be specifically illustrated below with examples. The examples shown below are not intended to limit the present invention in any way.

Example 1: Obtaining anti-Ibp antibodies Previous studies have revealed that the C-terminal region of Ibp plays an important role in the binding of Ibp to IgE (Patent Document 1). Therefore, we thought that creating an antibody targeting the C-terminus of Ibp would lead to the development of a new treatment for allergic diseases, and produced a monoclonal antibody. In this example, we first produced an anti-Ibp monoclonal antibody.

Rats were immunized with a truncated protein of the region common to IbpA and IbpB (IbpA C200, amino acid numbers 375-574 of WP_105084811.1) as an antigen to produce monoclonal antibodies. Two female Wistar rats were immunized twice with 0.1 mg of antigen via the subcutaneous route, followed by a final booster dose of 0.04 mg of antigen via the subcutaneous route. After three days, spleen cells were obtained from each of the three rats, and B cells were harvested. Cell fusion was then performed between these B cells and mouse myeloma cells (P3U1) to obtain hybridomas.

The obtained hybridomas were cultured in 2 mL (1 mL per clone per well, for 2 wells) of culture medium (culture medium composition: RPMI1640, penicillin-streptomycin-glutamine solution, sodium pyruvate, 10% FBS) in a 24-well cell culture plate for 4 to 7 days, after which the culture supernatant was collected and positive wells were identified by screening with ELISA using IbpA C200. The cells from the obtained positive wells were cloned by limiting dilution, and this process was repeated twice to obtain 180 hybridoma clones.

For the monoclonal antibodies against IbpA C200, 180 clones showing a reaction to IbpA C200 were obtained in the primary screening. To select useful clones from the 180 clones, (1-1) binding to wild-type Ruminococcus gnavus (WT Rg) and R. gnavus lacking ΔIbpAB (ΔIbpAB R.gnavus:ΔIbpAB Rg) was evaluated by FACS, (1-2) binding to IbpA C200, IbpA N200, and IbpA CR was evaluated by ELISA, and (1-3) inhibition of histamine production by LAD2 cells was verified. These tests were performed using hybridoma supernatants derived from the 180 clones obtained.

(1-1) Evaluation of binding to wild-type Ruminococcus gnavus (WT Rg) and R. gnavus lacking ΔIbpAB (ΔibpAB R.gnavus: ΔibpAB Rg) by FACS 50 μl of glycerol stock solution of wild-type Ruminococcus gnavus (JCM6515T) or ΔibpAB Ruminococcus gnavus (described in Patent Document 1) was suspended in 5 ml of GAM medium (Nissui) and cultured in an Anaeropack or anaerobic chamber (Bactron300) at 37°C under anaerobic conditions for 16 to 24 hours. After centrifugation at 8000 rpm and 4°C for 5 minutes to collect the cells, the cells were washed three times with PBS. After the third centrifugation, the cells were suspended in PBS, adjusted to OD 1.0/mL, and dispensed. The tubes containing the aliquots were centrifuged at 8000 rpm at 4°C for 5 minutes, the supernatant was removed, and the pellet was stored at -80°C until use.

The bacterial pellets prepared by the above bacterial culture were suspended in 1 ml of PBS, and the OD was measured (Yamato Picoexplorer). OD = 1.0 = 8 x 10 ⁸ bacteria, and 1 x 10 ⁸ suspension was transferred to a new 1.5 ml tube. 2% BSA (Jacson Immuno Research Bovine Serum Albumin (IgG-Free, Protease-Free))/PBS solution was added to prepare a total of 1 ml of bacterial suspension (1 x 10 ⁸ /ml), and the mixture was blocked at room temperature for 15 minutes.

100 μl (1×10 ⁷ ) was dispensed into 1.5 ml tubes for measurement according to the number of samples to be measured, and centrifuged at 14,000 rpm and 4° C. for 5 minutes. The supernatant was removed, and the following antibodies were added:
Either of the following was added to the plate: hybridoma supernatant, isotype control (negative control) (Invitrogen Rat IgG Isotype Control (2.5 mg/ml)) diluted 1:250 with 2% BSA PBS solution, or isotype control (negative control) (BD Pharmingen Purified Rat IgM, k Isotype Control (0.5 ml/ml)) diluted 1:250 with 2% BSA PBS solution, and then incubated at 4°C for 30 minutes. 1 ml of PBS was added, and the plate was centrifuged at 14,000 rpm at 4°C for 5 minutes, and the supernatant was removed. This step was repeated two more times for a total of three washes.

Secondary antibodies as follows:
Either Invitrogen Alexa Fluor 488 goat anti-rat IgG (H+L) (2 mg/ml) diluted 1:500 in 2% BSA PBS solution or Biolegend Alexa Fluor 488 anti-rat IgM (0.5 mg/ml) diluted 1:250 in 2% BSA PBS solution was added and incubated at 4°C for 30 minutes in the dark. 1 ml of PBS was added, centrifuged at 14000 rpm at 4°C for 5 minutes, and the supernatant was removed. This step was repeated two more times for a total of three washes.

After adding 400 μl of PBS and vortexing, FACS analysis was performed using a BD Accuri C6 Plus Flow Cytometer to screen for antibodies that bound to WT R.g but not to ΔIbpAB R.g. The data obtained was analyzed using FlowJo (BD Japan).

The 10 antibody clones obtained above were evaluated by FACS, and it was shown that all clones bound to WT R.g but not to ΔIbpAB R.g.

(1-2) Evaluation of binding to IbpA C200, IbpA N200, and IbpA CR by ELISA Next, in the ELISA screening, the reactivity to various truncated proteins was examined by ELISA, and clones that showed high reactivity to IbpA C200 but no reactivity to IbpA N200 (amino acids 30-229 of WP_105084811.1) and IbpA CR (amino acids 110-378 of WP_105084811.1) were selected.

Protein solution suspended in PBS at the following ratio:
・IbpA C200 (396μg/ml)→1:80000
・IbpA N200 (384μg/ml)→1:80000
・IbpA CR (871μg/ml)→1:172000
100 μl of each was added to a 96-well plate (MAXISORP NUNC-IMMUNO PLATE) and reacted overnight at 4° C. The structure of each protein is shown in FIG.

After overnight reaction, the plate was washed three times with 200 μl of PBS containing 0.05% Tween 20, and 100 μl of PBS solution containing 5% skim milk (Wako Skim Milk Powder) was added and reacted at room temperature for 1 hour. After removing the solution, the following samples or control antibodies were added:
・Each hybridoma supernatant, 50 μl
Negative control antibody: Rat IgG (25 mg/ml) diluted 1:100, 1:1000, 1:10000 in hybridoma culture medium (RPMI), 50 μl
After addition, the mixture was incubated at room temperature for 2 hours.

The plate was washed three times with 200 μl of PBS-T, and after removing the solution, the following diluted HRP antibody solution was added:
Rat IgG HRP (Invitrogen Goat anti-Rat IgG(H+L) Secondary Antibody HRP (0.8 mg/ml)
After adding 100 μl of the above, the mixture was incubated at room temperature for 1 hour.

The plate was washed three times with 200 μl of PBS-T, and 100 μl of TMB (a 1:1 mixture of Sera care KPL TMB Peroxidase Substrate and sera care KPL Peroxidase Substrate Solution B) was prepared and added, after which the reaction was allowed to proceed in the dark at room temperature for 15 minutes. 100 μl of reaction stop solution (sera care KPL Stop Solution) was added, and readings at 450 and 620 nm were measured using a plate reader.

As a result, 27 clones were selected.

(1-3) Verification of the inhibitory effect on histamine production by LAD2 cells Finally, the inhibitory effect on histamine production was examined.

Human mast cell line LAD2 cells were seeded at 1x105 cells/well in StemPro-34 SFM (1x) (Gibco), L-glutamine (2 mM) (GIBCO), penicillin (100 U/ml)/streptomycin (100 μg/ml) (GIBCO, recombinant human SCF (Peprotech) (100 ng/ml)). ^The cells were sensitized with purified native human IgE protein (Abcam ab65866) at a final concentration of 10 μg/ml for 2 hours at 37°C _under 5% CO2.

In a 1.5 ml tube, 100 μl of medium, hybridoma supernatant or human IgG isotype control antibody (final concentration 5 μg/ml) was added to the medium, and Ibp protein (final concentration IbpA: 2.4 μg/ml, IbpB: 2.1 μg/ml, total Ibp protein 4.5 μg/ml) or IbpA C200 (final concentration 1.5 μg/ml) was added and reacted at 37 ° C during sensitization. These pre-reacted samples were added to the sensitized cells, respectively, and reacted for 2 hours at 37 ° C under 5% CO _2. Cells and culture medium were collected from each well and centrifuged at 3000 rpm, 4 ° C, for 5 minutes to separate the cells and culture supernatant.

The amount of degranulated histamine produced in the culture medium was evaluated for the culture supernatant. Specifically, the amount of histamine produced in the culture supernatant was quantified using a histamine EIA (BECKMAN COULTER: IM2015).

The inhibitory effect varied depending on the clone, and the top eight hybridomas with the highest inhibitory effect were selected, as well as two hybridomas with unique characteristics to be used as comparative antibodies in future studies, and a total of 10 hybridomas (clone No. 1 to 10) were finally cloned.

Example 2: Examination of the effect of neutralizing antibodies against the binding of human IgE and Ibp in vitro In this example, the 10 antibody clones obtained in Example 1 were examined to determine whether they had neutralizing antibody activity against the binding of human IgE and Ibp in vitro.

In order to select an antibody with a higher inhibitory effect from the 10 monoclonal antibodies prepared in Example 1, in vitro histamine production was examined using a human mast cell line (LAD2 cells). Human mast cell line LAD2 cells were seeded in a 96-well plate at a cell number of 1×105 cells/well in StemPro-34 SFM (1×) ( ^Gibco ) (L-glutamine (2 mM) (GIBCO), penicillin (100 U/ml)/streptomycin (100 μg/ml) (GIBCO, recombinant human SCF (Peprotech) (100 ng/ml)). The cells were sensitized for 2 hours at 37°C under 5% _CO2 by adding purified native human IgE protein (Abcam ab65866) at a final concentration of 10 μg/ml to a 1.5 ml tube. 100 μl of medium, hybridoma supernatant (antibody concentration 50 The Ibp proteins (final concentrations IbpA: 2.4μg/ml, IbpB: 2.1μg/ml, total Ibp protein 4.5μg/ml) were added to the sensitized cells (diluted with medium to 10ng/ml) and reacted at 37℃ during sensitization. These pre-reacted samples were added to the sensitized cells and reacted for 2 hours at 37℃ under 5% _CO2 . The cells and culture medium were collected from each well and centrifuged at 3000 rpm, 4℃, for 5 minutes to separate the cells and the culture supernatant. The amount of histamine produced in the culture medium by degranulation was evaluated for the obtained culture supernatant. Specifically, the amount of histamine produced in the culture supernatant was quantified using histamine EIA (BECKMAN COULTER: IM2015).

The results showed that the antibody from Clone A had the strongest inhibitory effect (Figure 2). Furthermore, No. 5 (hereafter referred to as Clone B) was selected because it produced the greatest amount of antibody into the culture medium among the 10 clones and the antibody subclass was IgG. The antibody subclasses of Clone A and Clone B were as follows.

Example 3: Evaluation of the effect of anti-Ibp antibodies in inhibiting the binding of human IgE to Ibp in vivo In this example, a study was conducted using SPF mice to evaluate whether antibody Clone A, selected based on the in vitro study in Example 2, also exhibits an inhibitory effect on allergic reactions in vivo.

An outline of the experimental procedure is shown in Figure 3. BALB/cA mice were divided into two groups of three mice each. The test group received anti-Ibp antibody of Clone A in PBS at 4 mg/kg (equivalent to 100 μg/mouse) intravenously via the tail vein, and one day later (24 hours later), a mixed solution of IbpA and IbpB dissolved in PBS (2.5 μg IbpA + 2.5 μg IbpB/mouse) was administered via the tail vein (anti-Ibp antibody-Ibp group). For comparison, the control group received 100 μl of PBS alone without anti-Ibp antibody of Clone A, and 24 hours later, the same amount of Ibp protein as the test group was administered (PBS-Ibp group).

For mice in each group, rectal temperature was measured using a data logger (AND AD-1687) before administration of Ibp protein (indicated as 0 minutes on the graph), and 5 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes after administration to confirm whether a decrease in rectal temperature occurred in the mice due to an allergic reaction.

The results are shown in Figure 4. In the control PBS-administered group, administration of Ibp protein caused anaphylactic shock and a drop in rectal temperature was observed. On the other hand, in the group treated with antibody (anti-Ibp antibody-Ibp-administered group), the drop in rectal temperature was alleviated and the allergic reaction was suppressed (Figure 4).

Example 4: Sequence analysis of antibodies that inhibit the binding of human IgE to Ibp In this example, sequence analysis was performed on the anti-Ibp antibody Clone A selected based on the in vitro studies in Example 2, to examine the regions important for antigen binding.

Cell preparation for antibody sequence analysis
Hybridoma cells of Clone A were incubated with the following reagents:
・RPMI Medium 1640 (x1): 500 ml
・FBS (Heat inactivated) (gibco): 50 ml
・Penicillin-Streptomycin-Glutamine (100×) (gibco): 5.5 ml
・Sodium Pyruvate (100 mM) (gibco): 5.5 ml
The hybridomas were cultured in a non-coated culture flask using a hybridoma culture medium prepared by mixing the above. The hybridoma culture medium was centrifuged (1000 rpm/5 min), the supernatant was removed, the cells were suspended in PBS, the number of cells was counted, 2 x ¹⁰⁶ cells were centrifuged again, the supernatant was removed, and the pellet was suspended in a storage solution for sending to an analytical institution.

For antibody sequence analysis, the hybridoma cells prepared as described above were sent to an analytical institution (Biopeak Co., Ltd.) for sequence analysis.

The amino acid sequence of the antibody variable region that was determined is as follows. In this sequence, the underlined highlighted portion indicates the complementarity determining region (CDR), which is the binding site between Ibp and the antibody.

・VH amino acid sequence:
EVMLVESGGG LVQPGRSMKL SCAAS GFTFS NYGMA WVRQA PTKGLEWVA T ISYDGSSTYY RDSVKG RFTI SRDNAKSTLY LQMNSLRSED TATYYCTR ES FGTTVVPHYF DY WGQGVMVT VSS (SEQ ID NO: 7)
・VL amino acid sequence:
DIQMTQSPAS LSASLEEIVT ITC QASQDIG NWLS WYQQKP GKSPQLLIY G ATSLAD GVPS RFSGSRSGTQ YSLKISRLQV EDIGIYYC LQ AYSAPWT FGG GTKLELK (SEQ ID NO: 8)

Example 5: Preparation of humanized antibodies In this example, humanized antibodies were prepared based on the antibody Clone A sequenced in Example 4.

The humanized antibodies were produced by CDR grafting of the heavy chain complementarity determining regions, CDR1 (GFTFSNYGMA, SEQ ID No.: 1), CDR2 (TISYDGSSTYYRDSVKG, SEQ ID No.: 2), and CDR3 (ESFGTTVVPHYFDY, SEQ ID No.: 3), and the light chain complementarity determining regions, CDR1 (QASQDIGNWLS, SEQ ID No.: 4), CDR2 (GATSLAD, SEQ ID No.: 5), and CDR3 (LQAYSAPWT, SEQ ID No.: 6), whose sequences were confirmed in Example 4, from the donor monoclonal antibody Clone A, using the framework region (FR) of a human antibody as an acceptor.

Subsequently, based on sequence analysis, computational modeling, and antigen/antibody structural information, we designed FR back mutations and created antibody derivatives in which mutations were introduced into non-conserved positions in the FR of the humanized antibody.

Three types of heavy chain variable region VH domains and three types of light chain variable region VL domains were created using the amino acid sequences shown below. In these sequences, the underlined highlighted parts indicate the complementarity determining regions (CDRs), which are the binding sites between Ibp and the antibody.

- Humanized VH domain (HuVHv1):
QVQLVESGGG VVQPGRSLRL SCAAS GFTFS NYGMA WVRQA PGKGLEWVA T ISYDGSSTYY RDSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCTR ES FGTTVVPHYF DY WGQGTLVT VSS (SEQ ID NO: 9);
- Humanized VH domain (HuVHv2):
QVQLVESGGG VVQPGRSLRL SCAAS GFTFS NYGMA WVRQA PTKGLEWVA T ISYDGSSTYY RDSVKG RFTI SRDNSKNTLY LQMNSLRSED TAVYYCTR ES FGTTVVPHYF DY WGQGTLVT VSS (SEQ ID NO: 10);
- Humanized VH domain (HuVHv3):
QVQLVESGGG VVQPGRSLRL SCAAS GFTFS NYGMA WVRQA PGKGLEWVA T ISYDGSSTYY RDSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCTR ES FGTTVVPHYF DY WGQGVMVT VSS (SEQ ID NO: 11);
- Humanized VL domain (HuVLv1):
DIQMTQSPSS LSASVGDRVT ITC QASQDIG NWLS WYQQKP GKSPKLLIY G ATSLAD GVPS RFSGSRSGTD YTLTISSLQP EDFATYYC LQ AYSAPWT FGG GTKVELK (SEQ ID NO: 12);
- Humanized VL domain (HuVLv2):
DIQMTQSPSS LSASVGDIVT ITC QASQDIG NWLS WYQQKP GKSPQLLIY G ATSLAD GVPS RFSGSRSGTD YTLTISSLQP EDFATYYC LQ AYSAPWT FGG GTKVELK (SEQ ID NO: 13);
- Humanized VL domain (HuVLv3):
DIQMTQSPSS LSASVGDIVT ITC QASQDIG NWLS WYQQKP GKSPQLLIY G ATSLAD GVPS RFSGSRSGTQ YTLTISSLQP EDIATYYC LQ AYSAPWT FGG GTKVELK (SEQ ID NO: 14).

In this example, these three types of heavy chain variable region VH domains and three types of light chain variable region VL domains were arbitrarily combined to produce nine types of humanized antibodies (i.e., hClone A_H1L1 (HuVHv1×HuVLv1); hClone A_H1L2 (HuVHv1×HuVLv2); hClone A_H1L3 (HuVHv1×HuVLv3); hClone A_H1L4 (HuVHv4×HuVLv4); The following clones were produced: hClone A_H2L1 (HuVHv2 x HuVLv1); hClone A_H2L2 (HuVHv2 x HuVLv2); hClone A_H2L3 (HuVHv2 x HuVLv3); hClone A_H3L1 (HuVHv3 x HuVLv1); hClone A_H3L2 (HuVHv3 x HuVLv2); hClone A_H3L3 (HuVHv3 x HuVLv3)).

The binding affinity of these antibodies to IbpB protein was measured using surface plasmon resonance (SPR) method, taking hClone A_H1L1 and hClone A_H3L1 as examples. The antibodies were captured on the surface of a Protein A chip at a capture level of approximately 300 RU, and then various concentrations (listed in Table 2) of the analyte (IbpB protein) diluted in running buffer were injected for affinity and reaction rate measurements. Using a 1:1 binding model, the association rate constant (ka value, 1/Ms) and dissociation rate constant (kd value, 1/s) were measured, and the dissociation constant KD value (M) was calculated based on these values according to the formula KD = kd/ka, and the binding affinity was calculated. The binding affinity was measured and calculated in the same manner using Clone A antibody and Clone A_hIgG1 antibody as control antibodies.

The measured binding affinities are shown in Table 2 below. The results demonstrated that the humanized antibody has strong affinity for the IbpB protein.

　Based on the findings that allergic reactions occurring in the body can be prevented by substances that bind not directly to IgE but to a region important for IgE binding in the Ibp protein of Lachnospiraceae bacteria (e.g., Ruminococcus bacteria), an enterobacteria associated with allergic diseases, the present invention provides an antibody or antibody derivative that binds to the Ibp protein of Lachnospiraceae bacteria or a modified version thereof and can prevent or treat allergic reactions occurring in the body. This antibody or antibody derivative can suppress allergic reactions by exerting an inhibitory effect on allergens. Therefore, it does not bind to human IgE and can suppress allergic reactions without inhibiting the host's immune system, thereby reducing the problem of side effects.

Claims (12)

(1) heavy chain complementarity determining regions, CDR1 (GFTFSNYGMA, SEQ ID No.: 1), CDR2 (TISYDGSSTYYRDSVKG, SEQ ID No.: 2), and CDR3 (ESFGTTVVPHYFDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (QASQDIGNWLS, SEQ ID No.: 4), CDR2 (GATSLAD, SEQ ID No.: 5), and CDR3 (LQAYSAPWT, SEQ ID No.: 6);
An antibody or antibody derivative having binding activity against an Ibp antigen, comprising the heavy and light chain complementarity determining regions of Ibp. The antibody or antibody derivative according to claim 1, wherein the antibody or antibody derivative is human. The antibody or antibody derivative according to claim 2, wherein the human antibody derivative is selected from a human antibody variant or a functional fragment thereof selected from a humanized antibody, a chimeric antibody, a multivalent antibody, and a multispecific antibody. The antibody or antibody derivative according to claim 3, wherein the functional fragment is a Fab, Fab', F(ab')2, single chain variable region fragment (scFv) or disulfide stabilized (dsFv) antibody. The amino acid sequence of the heavy chain variable region VH domain of an antibody or human antibody derivative having binding ability to an Ibp antigen is
(VH-1-1) EVMLVESGGG LVQPGRSMKL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNAKSTLY LQMNSLRSED TATYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 7) or an amino acid sequence of SEQ ID NO: 7 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-2) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 9) amino acid sequence, or an amino acid sequence of SEQ ID NO: 9 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-3) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PTKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRSED TAVYYCTRES FGTTVVPHYF DYWGQGTLVT VSS (SEQ ID NO: 10) amino acid sequence, or an amino acid sequence of SEQ ID NO: 10 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
(VH-1-4) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYGMAWVRQA PGKGLEWVAT ISYDGSSTYY RDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRES FGTTVVPHYF DYWGQGVMVT VSS (SEQ ID NO: 11) amino acid sequence, or an amino acid sequence containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the amino acid sequence of SEQ ID NO: 11 other than CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2), and CDR3 (SEQ ID NO: 3);
3. The antibody or antibody derivative of claim 1 or 2, selected from the group consisting of: The amino acid sequence of the light chain variable region VL domain of an antibody or human antibody derivative having binding ability to an Ibp antigen is
(VL-1-1) DIQMTQSPAS LSASLEEIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YSLKISRLQV EDIGIYYCLQ AYSAPWTFGG GTKLELK (SEQ ID NO: 8), or an amino acid sequence of SEQ ID NO: 8 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-2) DIQMTQSPSS LSASVGDRVT ITCQASQDIG NWLSWYQQKP GKSPKLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 12) amino acid sequence, or an amino acid sequence of SEQ ID NO: 12 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-3) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTD YTLTISSLQP EDFATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 13) amino acid sequence, or an amino acid sequence containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the amino acid sequence of SEQ ID NO: 13 other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
(VL-1-4) DIQMTQSPSS LSASVGDIVT ITCQASQDIG NWLSWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ YTLTISSLQP EDIATYYCLQ AYSAPWTFGG GTKVELK (SEQ ID NO: 14) amino acid sequence, or an amino acid sequence of SEQ ID NO: 14 containing one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6);
3. The antibody or antibody derivative of claim 1 or 2, selected from the group consisting of: An antibody or antibody derivative according to claim 1 or 2, which binds to Ibp and inhibits the binding of Ibp to IgE in vivo. The antibody or antibody derivative according to claim 7 for preventing or treating an allergic disease. The antibody or antibody derivative according to claim 8, wherein the allergic disease is atopic dermatitis. (1) heavy chain complementarity determining regions, CDR1 (GFTFSNYGMA, SEQ ID No.: 1), CDR2 (TISYDGSSTYYRDSVKG, SEQ ID No.: 2), and CDR3 (ESFGTTVVPHYFDY, SEQ ID No.: 3), and light chain complementarity determining regions, CDR1 (QASQDIGNWLS, SEQ ID No.: 4), CDR2 (GATSLAD, SEQ ID No.: 5), and CDR3 (LQAYSAPWT, SEQ ID No.: 6);
A pharmaceutical composition for preventing or treating an allergic disease, comprising an antibody or antibody derivative having binding affinity to an Ibp antigen and comprising the heavy and light chain complementarity determining regions of Ibp. The pharmaceutical composition for preventing or treating an allergic disease according to claim 10, wherein the antibody or antibody derivative is human. 12. The pharmaceutical composition for preventing or treating an allergic disease according to claim 11, wherein the allergic disease is atopic dermatitis.