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WO2024248127A1 - Médicament pour la prévention ou le traitement de maladies allergiques - Google Patents

Médicament pour la prévention ou le traitement de maladies allergiques Download PDF

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Publication number
WO2024248127A1
WO2024248127A1 PCT/JP2024/020020 JP2024020020W WO2024248127A1 WO 2024248127 A1 WO2024248127 A1 WO 2024248127A1 JP 2024020020 W JP2024020020 W JP 2024020020W WO 2024248127 A1 WO2024248127 A1 WO 2024248127A1
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seq
antibody
amino acid
acid sequence
ibp
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Japanese (ja)
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真嗣 福田
学 中藤
拓 中原
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Metagen Inc
Metagen Therapeutics Inc
Kanagawa Institute of Industrial Science and Technology
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Metagen Inc
Metagen Therapeutics Inc
Kanagawa Institute of Industrial Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria

Definitions

  • 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.
  • 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.
  • Non-Patent Document 2 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).
  • Ibp immunoglobulin-binding proteins
  • 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.
  • [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
  • 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.
  • 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.
  • 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.
  • CDR1 to CDR3 heavy and light chain complementarity determining regions
  • 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.
  • the Ibp protein of bacteria of the genus Ruminococcus binds to IgE in the body and induces histamine production from mast cells, resulting in allergies.
  • 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.
  • 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.
  • a structural region on the Ibp protein is the C-terminal region of the IbpA protein (WP_105084811.1) or the IbpB protein (WP_105084812.1).
  • 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.
  • CDR1 to CDR3 represent heavy and light chain complementarity determining regions represented by specific amino acid sequences.
  • 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.
  • 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).
  • VH-1-1 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.
  • 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 FGTTVVP
  • VH-1-2 amino acid sequence of the heavy chain variable region VH domain other than the sequences of CDR1 to CDR3
  • 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.
  • 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 ISY
  • 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).
  • VL-1-1 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.
  • 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 (
  • 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.
  • 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 ATSLADG
  • 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).
  • VH-1-1 A combination of the VH domain (VH-1-1) and the VL domain (VL-1-1): a rat antibody
  • VH-1-1 A combination of the VH domain (VH-1-1) and any of the VL domains (VL-1-2) to (VL-1-4)
  • VH-1-1 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
  • 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.
  • 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.
  • 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.
  • CDR1 to CDR3 heavy and light chain complementarity determining regions
  • 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.
  • 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.
  • the antibody or antibody derivative of the present invention when 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.
  • the Lachnospiraceae bacteria that produce the target Ibp protein are enterobacteria, it can also be administered by routes such as rectal administration.
  • DDS drug delivery system
  • 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.
  • 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.
  • 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.
  • 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 ⁇ IbpAB R.g.
  • Rat IgG HRP Invitrogen Goat anti-Rat IgG(H+L) Secondary Antibody HRP (0.8 mg/ml) After adding 100 ⁇ l of the above, the mixture was incubated at room temperature for 1 hour.
  • the plate was washed three times with 200 ⁇ l of PBS-T, and 100 ⁇ l 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 ⁇ l of reaction stop solution (sera care KPL Stop Solution) was added, and readings at 450 and 620 nm were measured using a plate reader.
  • TMB a 1:1 mixture of Sera care KPL TMB Peroxidase Substrate and sera care KPL Peroxidase Substrate Solution B
  • 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 ⁇ g/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 ⁇ g/ml for 2 hours at 37°C under 5% CO2.
  • 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
  • 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.
  • 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 ⁇ g/ml) (GIBCO, recombinant human SCF (Peprotech) (100 ng/ml)).
  • StemPro-34 SFM (1 ⁇ ) ( Gibco ) (L-glutamine (2 mM) (GIBCO), penicillin (100 U/ml)/streptomycin (100 ⁇ g/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 ⁇ g/ml to a 1.5 ml tube. 100 ⁇ l of medium, hybridoma supernatant (antibody concentration 50 The Ibp proteins (final concentrations IbpA: 2.4 ⁇ g/ml, IbpB: 2.1 ⁇ g/ml, total Ibp protein 4.5 ⁇ g/ml) were added to the sensitized cells (diluted with medium to 10ng/ml) and reacted at 37°C during sensitization. These pre-reacted samples were added to the sensitized cells and reacted for 2 hours at 37°C under 5% CO2 .
  • purified native human IgE protein Abcam ab65866
  • the cells and culture medium were collected from each well and centrifuged at 3000 rpm, 4°C, 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).
  • Example 3 Evaluation of the effect of anti-Ibp antibodies in inhibiting the binding of human IgE to Ibp in vivo
  • 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.
  • 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 ⁇ g/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 ⁇ g IbpA + 2.5 ⁇ g IbpB/mouse) was administered via the tail vein (anti-Ibp antibody-Ibp group).
  • the control group received 100 ⁇ l 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).
  • 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.
  • Example 4 Sequence analysis of antibodies that inhibit the binding of human IgE to Ibp
  • 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.
  • 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.
  • 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.
  • CDR complementarity determining region
  • ⁇ 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
  • 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.
  • CDR1 GTFSNYGMA, SEQ ID No.: 1
  • CDR2 TISYDGSSTYYRDSVKG, SEQ ID No.: 2
  • CDR3 EDFGTTVVPHYFDY, SEQ
  • CDRs complementarity determining regions
  • hClone A_H1L1 Humanized antibodies
  • hClone A_H1L2 HumanVHv1 ⁇ HuVLv2
  • hClone A_H1L3 HumanVHv1 ⁇ HuVLv3
  • hClone A_H1L4 HumanVHv4 ⁇ 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_H3
  • association rate constant ka value, 1/Ms
  • dissociation rate constant kd value, 1/s
  • KD dissociation constant
  • 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.

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Abstract

La présente invention aborde le problème de développement d'une substance pouvant prévenir ou traiter des maladies allergiques, et une composition pharmaceutique contenant ladite substance, sur la base de la découverte précédente que des réactions de maladie allergique se produisant dans un corps vivant peuvent être empêchées par une substance qui se lie à une protéine Ibp de bactéries appartenant à la famille des Lachnospiraceae.　Les inventeurs de la présente invention ont découvert qu'un anticorps ou un dérivé d'anticorps est créé en tant que substance qui, au lieu de se lier directement à IgE, se lie à une région importante pour se lier à IgE dans une protéine Ibp produite par des bactéries appartenant à la famille des Lachnospiraceae, qui sont des bactéries intestinales associées à des maladies allergiques, et une composition pharmaceutique pour prévenir ou traiter des maladies allergiques, contenant ledit anticorps ou dérivé d'anticorps, peut être obtenue.
PCT/JP2024/020020 2023-06-02 2024-05-31 Médicament pour la prévention ou le traitement de maladies allergiques Pending WO2024248127A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016171107A1 (fr) * 2015-04-20 2016-10-27 第一三共株式会社 Détection de fgfr2
WO2021020282A1 (fr) * 2019-07-26 2021-02-04 学校法人埼玉医科大学 Anticorps reconnaissant la région extracellulaire d'alk2/acvr1
WO2023100977A1 (fr) * 2021-12-02 2023-06-08 地方独立行政法人神奈川県立産業技術総合研究所 Mise en place d'une thérapie et d'un diagnostic pour des maladies allergiques par le biais de la régulation de la protéine de liaison à l'immunoglobuline

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016171107A1 (fr) * 2015-04-20 2016-10-27 第一三共株式会社 Détection de fgfr2
WO2021020282A1 (fr) * 2019-07-26 2021-02-04 学校法人埼玉医科大学 Anticorps reconnaissant la région extracellulaire d'alk2/acvr1
WO2023100977A1 (fr) * 2021-12-02 2023-06-08 地方独立行政法人神奈川県立産業技術総合研究所 Mise en place d'une thérapie et d'un diagnostic pour des maladies allergiques par le biais de la régulation de la protéine de liaison à l'immunoglobuline

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BOROWSKA, M. T. ET AL.: "The molecular characterization of antibody binding to a superantigen-like protein from a commensal microbe", PNAS, vol. 118, no. 39, 2021, pages e2023898118, XP093070063, DOI: 10.1073/pnas.2023898118 *

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