Pharmaceutical compositions containing PD-1 agonists for treating or preventing Th2-mediated diseases

Abstract

In the present invention, novel uses for PD-1 agonists have been developed. Provided is a pharmaceutical composition for treating or preventing Th2-mediated diseases, the composition containing an effective amount of a PD-1 agonist.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition containing a PD-1 agonist for treating or preventing a Th2-mediated disease. [Background technology]

[0002] Inappropriate control of the immune response can lead to disease. An insufficient immune response against pathogens such as bacteria and viruses that invade the body can lead to infectious diseases, while an autoimmune disease can develop if a harmful immune response is initiated against one's own tissues. To prevent these pathological conditions and ensure effective operation of the immune system, the immune system possesses both mechanisms to activate immune cell function, thereby promoting the immune response, and mechanisms to suppress immune function, thereby reducing the immune response. Diseases resulting from insufficient or excessive immune responses are thought to be partly attributable to disorders in these endogenous control mechanisms.

[0003] A prime example of this is cancer immunotherapy, which has rapidly become a reality in recent years. This new approach, distinct from conventional "immunotherapy," employs molecules such as CTLA-4 and PD-1, which are part of an endogenous immune suppressive mechanism known as an immune checkpoint. Among these mechanisms, their impact is particularly striking. Cancer can be viewed as a condition in which cancer cells, which should be eradicated, proliferate due to an insufficient immune response. The cause of this insufficiency has been identified as the presence of various immune suppressive mechanisms within cancer tissue, which means that cancer tissues actively utilize the body's inherent immune suppressive mechanisms to evade immune attack. Blocking CTLA-4 and PD-1, two of these representative immune suppressive mechanisms, activates previously suppressed anti-tumor immunity, demonstrating therapeutic efficacy. This demonstrates the profound influence of these endogenous immune control mechanisms and their potential as important targets for disease treatment by correcting imbalances in the immune response.

[0004] The indispensable importance of endogenous immunosuppressive mechanisms is also demonstrated by the significantly stronger inflammatory responses observed when even one of these mechanisms is absent. For example, it has been shown that PD-1-deficient mice spontaneously develop various inflammatory diseases (Okazaki et al. Nat. Immunol., 2013). Furthermore, adverse inflammatory reactions resulting from excessive immune responses have been observed in a certain percentage of cases during cancer treatment with PD-1 inhibitors (Young et al. Cancer Immunol. Res., 2018). These findings suggest that the immunosuppressive mechanisms actively utilized in cancer tissues are actually physiological feedback mechanisms designed to protect normal tissues from excessive inflammatory responses. Indeed, the effects of drugs, such as anti-PD-1 antibodies, in cancer treatment suggest that the strength of the immune response can be regulated by controlling PD-1 function. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Okazaki T, Chikuma S, Iwai Y, Fagarasan S, Honjo T. A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat. Immunol. 14:1212-8 (2013). [Non-patent document 2] Young A, Quandt Z, Bluestone JA. The Balancing Act between Cancer Immunity and Autoimmunity in Response to Immunotherapy. Cancer Immunol. Res. 6:1445-1452 (2018). Summary of the Invention [Problem to be solved by the invention]

[0006] As shown above, drugs that inhibit immunosuppressive mechanisms have been used in cancer treatments, which require enhanced immune responses. However, the treatment of inflammatory diseases requires a completely opposite approach, actively stimulating immunosuppressive mechanisms to suppress unnecessarily enhanced immune responses. PD-1 is expressed on activated T cells, and upon antigen recognition, if ligand molecules such as PD-L1 and PD-L2 are expressed on the surface of the target cell, it interacts with these molecules to block T cell activation signals. Given that PD-1 itself has the ability to induce immunosuppressive signals, artificial stimulation of this signal may potentially lead to the treatment of inflammatory diseases. While anti-PD-1 antibodies used in cancer treatment inhibit ligand binding, PD-1 agonists, which can bind to PD-1 and induce its function, are effective for active immunosuppression.

[0007] One of the objectives of the present invention is to explore new physiological activities of agonist antibodies against PD-1 and to find new uses based on those activities. [Means for solving the problem]

[0008] Numerous inflammatory diseases are caused by an inappropriately enhanced immune response, and new, more effective therapies are needed for most of these diseases. We developed an effective treatment targeting the immunoregulatory molecule PD-1 for inflammatory diseases such as type I allergies and eosinophilic diseases, which are mediated by Th2 cells. Screening of anti-human PD-1 antibodies yielded numerous antibodies with agonistic activity that induce the immunosuppressive activity of PD-1. Among these, we found that anti-PD-1 agonist antibodies with high agonistic activity were particularly effective in suppressing Th2 cell differentiation. Based on this finding, administration of anti-PD-1 agonist antibodies to a mouse model of allergic asthma, a Th2-mediated disease, resulted in significant anti-inflammatory effects in addition to suppression of Th2-mediated immune responses. These results suggest that PD-1 agonists may be useful as anti-allergy drugs and therapeutic agents for eosinophilic diseases.

[0009] The present invention has been completed based on these findings, and the gist of the present invention is as follows. (1) A pharmaceutical composition for treating or preventing a Th2-mediated disease, comprising an effective amount of a PD-1 agonist. (2) The pharmaceutical composition according to (1), wherein the PD-1 agonist is an anti-PD-1 agonist antibody or a functional fragment thereof. (3) The pharmaceutical composition according to (1) or (2), wherein the Th2-mediated disease is type I allergy. (4) The pharmaceutical composition according to (1) or (2), wherein the Th2-mediated disease is an eosinophilic disease. (5) The pharmaceutical composition according to (1) or (2), wherein the Th2-mediated disease is a disease selected from bronchial asthma, atopic dermatitis, allergic rhinitis, drug allergy, food allergy, anaphylaxis, allergic conjunctivitis, urticaria, eosinophilic sinusitis, eosinophilic gastrointestinal disease, and allergic bronchopulmonary aspergillosis. (6) A method for preventing and / or treating a Th2-mediated disease, comprising administering an effective amount of a PD-1 agonist to a subject. (7) A PD-1 agonist for use in the prevention and / or treatment of Th2-mediated diseases. (8) Use of PD-1 agonists for the prevention and / or treatment of Th2-mediated diseases. (9) A PD-1 agonist and its use for suppressing IgE production by suppressing Th2-type cytokines. (10) A PD-1 agonist and its use for suppressing eosinophil activation by suppressing Th2-type cytokines. [Effects of the Invention]

[0010] According to the present invention, naive CD4 + The functional differentiation of T cells into Th2 cells can be suppressed, and Th2-mediated diseases can be treated or prevented. This specification includes the contents disclosed in the specification and / or drawings of Japanese Patent Application No. 2020-90526, which is a priority document of this application. [Brief explanation of the drawings]

[0011] [Figure 1] A system for assessing the suppression of T cell cytokine production by PD-1 stimulation. (A) DO11.10 T cell hybridoma expressing human PD-1 (hPD-1) and IIA1.6 B lymphoma cells were used. DO11.10 T cell hybridoma was activated in response to the OVA323-339 peptide presented by MHC class II molecules (I-Ad) on IIA1.6 cells, but activation was suppressed by PD-1 stimulation. (B) IL-2 production from DO11.10 T cell hybridoma expressing human PD-1 was suppressed upon interaction with PD-L1 on IIA1.6 cells. IL-2 suppression was not observed in the absence of PD-1 or PD-L1 expression. (C) Addition of EH12.2H7, a blocking antibody against human PD-1, to the reaction system reversed the PD-L1-mediated suppression. This experimental system allows the detection of anti-PD-1 antibodies with immunomodulatory activity. [Figure 2] Evaluation of the agonist activity of anti-human PD-1 antibodies. (A) Anti-human PD-1 antibodies were screened using an evaluation system combining DO11.10 T cell hybridoma expressing human PD-1 with IIA1.6 B lymphoma cells expressing FcγRIIB but not PD-L1, using their inhibitory activity against IL-2 production as an indicator. (B) As a result, approximately 30 clones with immunosuppressive activity were obtained, ranging from highly to weakly potent. The figure shows only the clones with immunosuppressive activity among these anti-human PD-1 antibodies. [Figure 3]Comparison of the activity of anti-human PD-1 agonist antibodies. (A) Among the anti-human PD-1 antibodies found to have immunosuppressive activity, those with relatively high activity were selected and IC50 values ​​were calculated. The novel antibodies, as well as commercially available antibodies (J116, MIH4) found to have agonist activity, are all mouse IgGs. Based on the concentration-dependent inhibition curves of IL-2 production by these antibodies, the concentration at which 50% inhibition was achieved was calculated, with no IL-2 production representing 0%. (B, C) The Fv portion of the novel antibodies was chimeric with the Fc portion of human IgG1-K322A (B) or human IgG4-S228P (C). Other known anti-PD-1 antibodies reported as agonists (PD1AB6 and PD1-17: JP2018-533973 and JP2006-521783) were also used to determine IC50 values ​​based on the antibody concentration-dependent inhibition of IL-2 production. [Figure 4] Immunosuppressive effect of anti-human PD-1 agonist antibody on human T cells. Primary cultured human CD4+ T cells were activated by stimulation with anti-CD3 antibody for 3 days, then mixed with THP-1 cells expressing human FcγRIIB and stimulated with CytoStim (Miltenyi Biotec). Anti-human PD-1 agonist antibody was added to this culture system to observe the suppression of cytokine production. [Figure 5]Suppression of Th2 cell induction by PD-1 stimulation. CD4+ CD62L+ cells prepared from splenocytes of DO11.10 mice were stimulated with OVA323-339 using PD-L1(-) FcγRIIB(+) IIA1.6 cells as antigen-presenting cells. Th1 or Th2 cells were selectively induced by adding cytokines and anti-cytokine antibodies. After restimulation, IFN-γ and IL-4 production were analyzed by intracellular staining to determine the ratio of Th1 to Th2 cells induced. When Th1 and Th2 cells were induced in the presence of an anti-PD-1 agonist antibody, the proportion of Th1 cells, as indicated by IFN-γ production, was not significantly affected (A), but the proportion of IL-4-producing Th2 cells was significantly suppressed (B). Th2 functional differentiation is particularly sensitive to PD-1 stimulation, suggesting the possibility of artificially adjusting the Th1 / Th2 balance using an anti-PD-1 agonist antibody. Similar changes were observed when PD-1 was stimulated using PD-L1 (+) IIA1.6 cells, as with anti-PD-1 agonist antibodies. [Figure 6] Antibody production in mice immunized with an antigen and the effect of an anti-PD-1 agonist antibody on antibody production. Human PD-1 knock-in mice were immunized with NP-OVA (4-hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin) and simultaneously administered with an anti-PD-1 agonist antibody (HM266). HM266 was administered again 3 and 7 days later. 10 days later, hapten-specific antibody titers in the blood were measured and compared between IgG subclasses. (A) Antigen-specific antibody titers in the blood after 10 days. HM266 administration reduced both IgG1 and IgG2c antibody concentrations, with a particularly pronounced inhibitory effect on IgG1 antibodies. (B) IgG1 / IgG2c ratio of antigen-specific antibody titers in the blood after 10 days. HM266 administration reduced the IgG1 / IgG2c ratio. [Figure 7]Induction of allergic asthma in mice and the anti-inflammatory effect of J116. Human PD-1 knock-in mice were sensitized with house dust mite antigen (HDM), and one week later, allergic asthma was induced by intranasal administration of the same antigen. Seven days after daily intranasal administration of HDM, the infiltration of inflammatory cells in alveolar exudates was examined. The induction of allergic asthma increased the total number of infiltrating inflammatory cells, with eosinophils and CD4+ T cells accounting for the majority of these. However, administration of J116 to this inflammatory model significantly suppressed the infiltration of inflammatory cells, particularly eosinophils, into the alveoli. [Figure 8] J116 suppresses the increase in Th2-type cells in mice with allergic asthma. Allergic asthma was induced as shown in Figure 7, and alveolar exudates were obtained 7 days after daily intranasal administration. The cytokine production ability of infiltrating CD4+ cells was examined by intracellular staining, and a significant decrease in IL-5 and IL-13-producing cells was observed in the J116-treated group. [Figure 9] Induction of allergic asthma in mice and the anti-inflammatory effect of HM266. Allergic asthma was induced in human PD-1 knockin mice by exposure to dust mite antigen using the same method as in Figure 7. HM266 was administered to observe the suppression of inflammation. Four doses (HM266 (x4)) similar to Figure 7 suppressed the infiltration of eosinophils and CD4+ T cells into the alveoli. Furthermore, when HM266 was administered only once (HM266 (x1)) three days after the induction of inflammation in lung tissue to observe the therapeutic effect, the infiltration of inflammatory cells into the alveoli was also suppressed. [Figure 10]Anti-inflammatory effects of HM266 on allergic asthma and suppression of Th2-type immune responses. Allergic asthma was induced using the same schedule as in Figure 9, and alveolar exudates and lung tissues were collected. Cytokine production from CD4+ cells infiltrating the alveolar exudates or lung tissue was examined by intracellular staining. A decrease in IL-4, IL-5, IL-10, and IL-13-producing cells was observed in the four-times HM266 administration group (HM266 (x4)). Furthermore, HM266 administration significantly reduced blood levels of dust mite antigen-specific IgE. A similar trend was observed when HM266 was administered only once (HM266 (x1)) 3 days after the start of lung inflammation induction, suggesting the usefulness of anti-PD-1 agonist antibodies for the treatment of allergic diseases. [Figure 11] Anti-inflammatory effect of HM266 on atopic dermatitis. Allergic dermatitis was induced in human PD-1 knock-in mice by applying MC903 to the ears once every three days. (A) Concurrent administration of MC903 and an anti-PD-1 agonist antibody (HM266) significantly suppressed ear swelling. (B) Time course of IgE levels after MC903 administration. Continuous application of MC903 significantly increased IgE levels, but simultaneous administration of HM266 (prophylactic administration) significantly reduced this. Furthermore, in a therapeutic setting, HM266 administration, starting on day 12 after ear swelling had occurred, also significantly reduced IgE levels. (C, D) HM266 administration significantly suppressed the number of eosinophils infiltrating into the ear tissue (C) and scratching behavior (D) in mice. Groups 1, 2, 3, and 4 in Figure 11C and D represent vehicle only, MC903, MC903 + HM266 (preventive), and MC903 + HM266 (therapeutic), respectively. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail.

[0013] The present invention provides a pharmaceutical composition for treating or preventing a Th2-mediated disease, comprising an effective amount of a PD-1 agonist, particularly an anti-PD-1 agonist antibody. The present invention provides a method for preventing and / or treating a Th2-mediated disease, comprising administering an effective amount of a PD-1 agonist to a subject. The present invention provides a PD-1 agonist for use in the prevention and / or treatment of a Th2-mediated disease. The present invention provides use of a PD-1 agonist for the prevention and / or treatment of a Th2-mediated disease.

[0014] PD-1 (Programmed cell death 1) is a receptor expressed on the surface of activated T cells. On the other hand, its ligands, PD-L1 and PD-L2, are expressed on the surface of antigen-presenting cells. When PD-L1 or PD-L2 binds to PD-1, the immune activity of T cells is suppressed via a signal transduction pathway.

[0015] A PD-1 agonist is a substance that exhibits agonistic activity (enhancement of activation signals) against the PD-1 molecule, similar to PD-L1, and the pharmaceutical composition of the present invention contains a PD-1 agonist as an active ingredient. The PD-1 agonist is not particularly limited, and any PD-1 agonist may be used, including antibodies, antigen-binding fragments thereof, solubilized PD-L1 / L2 (e.g., Fc fusion proteins), peptides, nucleic acid molecules, other compounds, and PD-1 ligand-expressing cells in which PD-L1 / L2 expression has been artificially enhanced, with antibodies (anti-PD-1 agonist antibodies) or functional fragments thereof being preferred.

[0016] The present inventors have demonstrated that the anti-human PD-1 antibody binds to OVA, an ovalbumin-derived peptide antigen, by combining DO11.10 T cell hybridoma expressing human PD-1 with IIA1.6 B lymphoma cells expressing human FcγRIIB but not PD-L1. 323-339As a result of evaluating the agonist activity under stimulation with peptides, several clones with immunosuppressive activity were obtained, ranging from high to low (IL-2 production inhibitory activity) (see Examples below). In this assay system, the commercially available anti-human PD-1 monoclonal antibody J116 exhibited IL-2 production inhibitory activity and IC 50 Therefore, for example, in the above assay system, the IC value of J116 for the inhibitory activity of IL-2 production was about 1000 ng / ml. 50 The inhibitory activity of IL-2 production is equal to or greater than that of J116 under conditions calculated to be 50 ng / ml to 5000 ng / ml (i.e., IC50 equal to or less than that of J116). 50 Antibodies that exhibit this value can be said to be "anti-PD-1 agonist antibodies."

[0017] PD-1 agonists other than antibodies are acceptable as long as they are scientifically recognized to have IL-2 production inhibitory activity equal to or greater than that of J116.

[0018] An anti-PD-1 agonist antibody or a functional fragment thereof may bind only to region #7 of human PD-1 (the region from amino acid 38 to amino acid 48 of the amino acid sequence of SEQ ID NO: 24), or may also bind to regions such as region #6 (the region from amino acid 109 to amino acid 120 of the amino acid sequence of SEQ ID NO: 24) and region #1 (the region from amino acid 129 to amino acid 139 of the amino acid sequence of SEQ ID NO: 24). Preferably, the antibody binds only to region #7 of human PD-1. Substituting region #7 of human PD-1 (SEQ ID NO: 24) with the corresponding region of the amino acid sequence of mouse PD-1 (SEQ ID NO: 25) reduces the binding ability of the anti-human PD-1 antibody, thereby defining the antibody as binding to region #7 of human PD-1 (SEQ ID NO: 24). In this case, binding to region #7 is determined independently of binding to other regions of human PD-1. Binding to regions other than region #7 of human PD-1 is similarly defined. The registered database and registration number for the amino acid sequence of human PD-1 are NCBI accession number: NP_005009.2, and the registered database and registration number for the amino acid sequence of mouse PD-1 are NCBI accession number: NP_032824.1. Preferably, the anti-PD-1 agonist antibody or a functional fragment thereof also binds to a mouse PD-1 substitution mutant (mouse PD-1 (hu38-48)) in which the #7 domain of mouse PD-1 has been replaced with the amino acid sequence of the #7 domain of human PD-1, and / or human PD-1 (R143A point mutant) in which arginine at position 143 of human PD-1 has been mutated to alanine.

[0019] In the Examples described below, commercially available anti-human PD-1 monoclonal antibodies MIH4 and J116 demonstrated PD-1 agonistic activity. Furthermore, the inventors' newly generated anti-human PD-1 monoclonal antibodies HM266, HM647, and HM698 demonstrated favorable agonistic activity. These antibodies are useful as anti-PD-1 agonistic antibodies and can be used as active ingredients in the pharmaceutical compositions of the present invention. MIH4, J116, HM266, and HM268 bind to the #7 domain of human PD-1, while HM647 binds to the #6 and #7 domains of human PD-1.

[0020] The amino acid and nucleotide sequences of the heavy chain variable region and light chain variable region of HM266, HM647, and HM698 are shown in the sequence listing with SEQ ID NOs. as follows: JPEG0007754455000001.jpg114165

[0021] HM266, HM647, and HM698 can be produced using hybridomas, but they can also be produced as recombinant antibodies using genetic engineering techniques. That is, the heavy and light chain genes of the antibody are synthesized, inserted into a vector (e.g., a plasmid), and then introduced into host cells (e.g., CHO cells, HEK cells, etc.). The host cells are then cultured, and recombinant antibodies can be collected from the culture. When synthesizing the heavy and light chain genes of the antibody, it is preferable to optimize codons.

[0022] Anti-PD-1 agonist antibodies may be either polyclonal or monoclonal, but monoclonal antibodies are preferred. Polyclonal antibodies can be produced by repeatedly injecting an animal with an antigen (PD-1) to induce antibody production in the blood, then collecting the blood (plasma or serum) and purifying the antibody from it. Monoclonal antibodies can be produced by injecting an animal with the antigen (PD-1), extracting antibody-producing B cells from the spleen or lymph nodes, artificially fusing them with immortalized cancer cells (myeloma) to create hybridomas, selecting monoclonal antibody-producing cells from these hybridomas, and allowing these cells to produce monoclonal antibodies. Animals used for immunization include mice, rabbits, and various mammals, birds, and fish, including rats, hamsters, guinea pigs, chickens, goats, sheep, donkeys, llamas, and sharks. Monoclonal antibodies can also be produced as recombinant antibodies using genetic engineering techniques. Monoclonal antibodies may be antibodies derived from non-human animals (e.g., various mammals, birds, and fish, such as mice, rabbits, rats, hamsters, guinea pigs, goats, sheep, donkeys, llamas, camels, chickens, ostriches, and sharks), chimeric antibodies, humanized antibodies, or fully human antibodies. For example, the variable region (Fv) of an anti-PD-1 agonist antibody may be the Fv of an antibody derived from a non-human animal (e.g., mice, rabbits, rats, hamsters, guinea pigs, goats, sheep, donkeys, llamas, camels, chickens, ostriches, and sharks), or the Fab regions of the heavy and / or light chains of an antibody derived from a non-human animal may be humanized. Humanization can be achieved by grafting the CDRs of the VH and VL of an antibody derived from a non-human animal onto the VH and VL frameworks of a human antibody (Nature, 332, 323-327, 1988). Humanization may be achieved by maintaining the CDR sequences, but may also involve identifying amino acid residues directly involved in binding to the antigen, amino acid residues interacting with the CDR, and amino acid residues involved in maintaining the three-dimensional structure of the CDR, and substituting these with amino acid residues from a non-humanized antibody to improve antigen binding (MABS, 8(7), 1302-1318, 2016).In the Examples described below, chimeric antibodies were produced in which the variable regions of novel antibodies (HM266, HM647, and HM698) were linked to the constant region of human IgG1 or human IgG4. The amino acid and nucleotide sequences of the heavy chain constant region of human IgG1 (IgG1-K322A) used to produce the chimeric antibodies are shown in SEQ ID NOs: 17 and 18, respectively. The amino acid and nucleotide sequences of the heavy chain constant region of human IgG4 (IgG4-S228P) are shown in SEQ ID NOs: 19 and 20, respectively. The amino acid and nucleotide sequences of the human immunoglobulin light chain (κ chain: Igκ) constant region are shown in SEQ ID NOs: 21 and 22, respectively.

[0023] Anti-PD-1 agonist antibodies may be functionally modified. Techniques for functionally modifying antibodies include amino acid mutations, subclass substitutions, antibody-drug conjugates, glycosylated antibodies, and combinations thereof. In the amino acid mutations, mutations that improve affinity for the Fc receptor of the human or non-human animal to which the anti-PD-1 agonist antibody is to be administered are preferably introduced. The Fc receptor is preferably an Fcγ receptor, more preferably FcγRII, and even more preferably FcγRIIB. The binding ability (binding affinity) of anti-PD-1 agonist antibodies to human Fc receptors can be assessed by Fc receptor binding affinity assays using surface plasmon resonance technology with Biacore 8K (Cytiva) or by binding assays against human Fc receptor-expressing cell lines using a flow cytometer. When measured in a binding assay for human Fc receptor-expressing cell lines, the affinity of an anti-PD-1 agonist antibody for human FcγRIIB can be expressed as the GMFI ratio relative to an antibody (control antibody) containing the Fc region of human IgG1-K322A. Under measurement conditions where the GMFI without addition of antibody is approximately 40 and the GMFI with addition of the control antibody is 300-1500, the anti-PD-1 agonist antibody exhibits a GMFI that is at least 2-fold higher, preferably at least 5-fold higher, and more preferably at least 20-fold higher, than that with addition of the control antibody containing the same Fv region. When measured in an Fc receptor binding affinity assay using surface plasmon resonance technology, the affinity of an anti-PD-1 agonist antibody for human FcRIIB can be expressed as the ratio of the equilibrium dissociation constant (Kd) to that of an antibody containing the Fc region of human IgG1-K322A (control antibody), and the anti-PD-1 agonist antibody has an affinity that is 1.5-fold or greater than that of the control antibody, preferably 2-fold or greater than that of the control antibody, and more preferably 2.5-fold or greater than that of the control antibody.

[0024] The anti-PD-1 agonist antibody preferably has the Fc region of a human antibody (e.g., IgG1, IgG4, etc.) (a region corresponding to the sequence from amino acid 100 to amino acid 330 in the amino acid sequence of IgG1 shown in SEQ ID NO: 17, or the sequence from amino acid 100 to amino acid 327 in the amino acid sequence of IgG4 shown in SEQ ID NO: 19), and more preferably the Fc region of the human antibody is an Fc region variant that has been modified to improve affinity for human Fc receptors. Affinity for human Fc receptors can be improved by modifying the Fc region of an antibody. Affinity for human Fc receptors can also be improved by defucosating an antibody.

[0025] In anti-PD-1 agonist antibodies, the affinity for human Fc receptors can be improved by, for example, introducing a mutation into one or more of positions 236, 268, 239, 328, 332, 233, 237, 238, 271, 330, 267, 326, 234, 323, and 296 (according to Kabat EU numbering; the same applies below) in the amino acid sequence of the Fc region of human IgG1, preferably a combination thereof. Furthermore, these mutations may be combined with defucosylation.

[0026] When a mutation is introduced into the Fc region of human IgG1, it may be combined with other mutations, such as K322A, a mutation known to inhibit complement-dependent cytotoxicity (CDC) activity by reducing complement C1q binding, or E293A, a mutation known to inhibit ADCC activity by reducing FcγRIIIA binding. In the Examples described below, a human IgG1 heavy chain constant region having the amino acid mutation Lys322Ala, which is thought to inhibit complement-dependent cytotoxicity, was used, but it is understood that these mutations do not have a significant effect in the test system shown in the Examples below, which uses an antibody having a human heavy chain constant region.

[0027] To improve the binding ability to human Fc receptors, for example, a mutation may be introduced into one or more of positions 236, 239, 268, 328, and 332 (according to Kabat EU numbering; the same applies below) in the amino acid sequence of the Fc region of human IgG4, preferably a combination of these. These mutations may also be combined with defucosylation.

[0028] When a mutation is introduced into the Fc region of human IgG4, it may be combined with another mutation, such as S228P, which is known to have the effect of further improving antibody stability. In the Examples described below, a human IgG4 heavy chain constant region having the amino acid mutation Ser228Pro, which is thought to improve antibody stability, was used, but it is understood that these mutations do not have a significant effect in the test system shown in the Examples below, which uses an antibody having a human heavy chain constant region.

[0029] The anti-PD-1 agonist antibody may have a humanized Fab region in each of the heavy and light chains of an antibody derived from a non-human animal (e.g., a mouse) (which exhibits PD-1 agonist activity), and an Fc region that is a modified Fc region of human IgG1 or IgG4.

[0030] Functional fragments of anti-PD-1 agonist antibodies are derived from antibody molecules and refer to protein fragments capable of binding to PD-1, as well as fusion proteins containing such protein fragments. Examples include, but are not limited to, bispecific antibodies and small molecules (e.g., scFv, Fv, F(ab')2, Fab', Fab, and diabody). Examples include molecules having one or more scFv, Fv, F(ab')2, Fab', and Fab of an anti-PD-1 agonist antibody that also bind to an Fc receptor. Examples include, but are not limited to, antibody-drug conjugates of an anti-PD-1 agonist antibody and a drug, polypeptides having an scFv of an anti-PD-1 agonist antibody and an scFv of an anti-Fc receptor antibody, and fusion proteins of an scFv of an anti-PD-1 agonist antibody and an Fc region. When the functional fragment has an Fc region, such as a fusion protein of the scFv of an anti-PD-1 agonist antibody with the Fc region, the Fc region is preferably an Fc region of an Fc region variant described herein. The protein improvement and optimization techniques described above can also be applied to functional fragments.

[0031] The anti-PD-1 agonist antibody may be an immunoglobulin molecule, preferably an immunoglobulin molecule of the animal species in which the antibody is to be used. The immunoglobulin molecule may be of any class, and in humans, is preferably IgG, and may be of any subclass, with IgG1 or IgG4 being preferred.

[0032] The affinity of anti-PD-1 agonist antibodies to PD-1 is such that the binding constant (Kd) is 10 -7 M or less, and preferably 10 -8 The binding constant is preferably measured by the surface plasmon resonance (SPR) method, but can also be determined simply by flow cytometry from the concentration dependence of binding to PD-1-expressing cells.

[0033] The present inventors found that anti-PD-1 agonist antibodies can suppress the induction of Th2 cells by PD-1 stimulation (see the Examples below, Figure 5). Th2 cells produce IL-4, IL-5, and IL-13, and immune function is regulated through these cytokines.

[0034] The pharmaceutical compositions of the present invention can be used to treat or prevent Th2-mediated diseases. In the present invention, Th2-mediated diseases refer to diseases associated with immune responses induced by a central role played by cells (e.g., Th2 cells) that produce Th2-type cytokines such as IL-4, IL-5, and IL-13. Specific examples include inflammatory diseases caused by type I allergic reactions and eosinophilic diseases (diseases in which functions such as eosinophil proliferation, granule protein release, and migration, controlled by Th2-type cytokines, are involved in the pathology), which are considered to be directly related to Th2-type immunity. Examples of such diseases include bronchial asthma, atopic dermatitis, allergic rhinitis (e.g., hay fever), drug or food allergies, anaphylaxis, allergic conjunctivitis, urticaria, eosinophilic sinusitis, eosinophilic digestive diseases, and allergic bronchopulmonary aspergillosis.

[0035] The pharmaceutical compositions of the present invention can be administered to a subject (human or non-human animal) systemically or locally, orally or parenterally.

[0036] The pharmaceutical composition of the present invention may contain an effective amount of a PD-1 agonist, and can be prepared by formulating the PD-1 agonist together with a pharmaceutically acceptable carrier by mixing, dissolving, emulsifying, encapsulating, lyophilizing, or the like.

[0037] The pharmaceutical composition of the present invention may be administered either orally or parenterally. Suitable formulations for oral administration include liquids in which an effective amount of an anti-PD-1 agonist antibody is dissolved in a diluent such as water or physiological saline; capsules, granules, powders, or tablets containing an effective amount as a solid or granule; suspensions in which an effective amount is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0038] For parenteral administration, anti-PD-1 agonist antibodies can be formulated into dosage forms such as injectable solutions, suspensions, emulsions, creams, ointments, inhalants, and suppositories, along with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. For injection formulations, the antibodies of the present invention can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. Furthermore, the medicaments of the present invention can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the antibodies of the present invention can be prepared in powder form, and an aqueous solution or suspension can be prepared using sterile water or the like before use. For inhalation administration, the antibodies of the present invention can be powdered and mixed with a suitable base such as lactose or starch to form a powder mixture. Suppositories can be prepared by mixing the antibodies of the present invention with a conventional suppository base such as cocoa butter. Furthermore, the therapeutic agents of the present invention can be encapsulated in a polymer matrix or the like to form sustained-release formulations.

[0039] The dosage for a human adult is approximately 0.1 to 100 mg / kg (body weight) per dose, which may be administered in a single dose or multiple doses at intervals of one day to six months. The route of administration may be either oral or parenteral, and parenteral administration may include intravenous, intramuscular, subcutaneous, rectal, nasal, oral, and transdermal administration.

[0040] Non-human animals to which the pharmaceutical composition of the present invention can be administered include mammals such as dogs, cats, cows, pigs and horses, and birds.

[0041] The pharmaceutical composition of the present invention may be used alone or in combination with other therapeutic agents such as antihistamines, antiallergic drugs, vasoconstrictor nasal drops, steroids, small molecule immunosuppressants (cyclosporine, tacrolimus, etc.), antibody drugs (anti-IgE antibodies, anti-IL-4 antibodies, anti-IL-5 antibodies, anti-IL-13 antibodies, anti-IL-4 receptor antibodies, anti-IL-5 receptor antibodies, anti-IL-22 antibodies, anti-IL-25 antibodies, anti-IL-33 antibodies, anti-TSLP antibodies, anti-BAFF antibodies, etc.), recombinant soluble fusion proteins (CTLA-4-Ig, etc.), etc., which is expected to produce synergistic drug effects. [Example]

[0042] The present invention will be described in more detail below with reference to examples. Example 1 Methods Commercially available antibodies Anti-human PD-1 antibody (clone name: EH12.2H7, Biolegend), anti-human PD-1 antibody (clone name: J116, Invitrogen), anti-human PD-1 antibody (clone name: MIH4, Invitrogen), control mouse IgG1 (clone name: MOPC-21, Biolegend), control human IgG1 (clone name: QA16A12, Biolegend), control human IgG4 (clone name: QA16A15, Biolegend)

[0043] Obtaining and purifying new antibodies Techniques for producing anti-human PD-1 mouse monoclonal antibodies are known in the art, and the method described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986) was used. A / J and BALB / c mice were used as immunization hosts, and the immunogens used were a plasmid vector (15-50 μg) expressing the full-length human PD-1 protein (NCBI accession number: NP_005009.2) or a point mutant thereof, a fusion protein (25 μg) of the recombinant human PD-1 extracellular domain with human IgG1-Fc, or 293T cells (5 x 10 ) transiently expressing human PD-1 or a point mutant thereof. 7 Each of these was administered intramuscularly, intradermally, intraperitoneally, or intravenously at 10-50 day intervals. When an adjuvant was used, the Sigma adjuvant system (S6322-1VL; Sigma-Aldrich) was used. Three days after the final immunization, spleen cells were fused with P3U1 mouse myeloma cells to generate hybridomas. Hybridomas producing anti-human PD-1 antibodies were screened by adding the culture supernatant of each hybridoma to HEK293 cells overexpressing human PD-1. The cells were stained with R-phycoerythrin-conjugated goat anti-mouse IgG (H+L) F(ab')2 fragment (115-116-146; Jackson ImmunoResearch) as a secondary antibody and analyzed using a flow cytometer. The finally selected hybridomas were cloned by limiting dilution and cultured at high density in a cell line bioreactor (Wheaton). Anti-human PD-1 antibodies were purified from the culture supernatant using Ab-Capture ExTra (P-003-10; Proteinova).

[0044] Antibody sequence The variable region sequences of the anti-human PD-1 antibodies HM647 and HM698 were determined by sending frozen hybridomas to Visicom Japan, Inc., and using the contract analysis service of Fusion Antibodies. Furthermore, the variable region sequences of the anti-human PD-1 antibodies HM266 and HM698 were determined by the method described in A Doenecke, EL Winnacker, and M Hallek, Leukemia (1997) 11, 1787-1792. Briefly, total RNA was prepared from hybridoma cells, and cDNA encoding the antibody variable regions was PCR amplified using the 5'-RACE method with the SMARTer™ RACE cDNA Amplification Kit (Clontech). The cDNA sequence was then determined. The resulting sequence was expressed as a recombinant human IgG1 antibody, and specific binding to human PD-1 was confirmed. The variable region sequence of HM698 was identical for both methods.

[0045] Recombinant antibody preparation and purification A heavy chain expression vector was constructed using DNA encoding the heavy chain sequence and an expression vector (pcDNA3.4, Thermo Fisher Scientific). Similarly, a light chain expression vector was constructed using DNA encoding the light chain sequence and an expression vector (pcDNA3.4, Thermo Fisher Scientific). The above two expression vectors were introduced into CHO cells or HEK293 cells by lipofection, and the transformed cells were cultured. After the cells were removed by centrifugation and filtration, the culture medium was collected. Antibodies were purified by a combination of affinity chromatography using a Protein A or Capture Select kappaXL column and gel filtration chromatography. The combinations of antibodies and purification methods are as follows: Protein A → purified by gel filtration: HM266-hIgG4-S228P, HM647-hIgG1-K322A, PD1AB6-hIgG1-K322A, PD1AB6-hIgG4-S228P, PD1-17 Capture Select kappaXL → Gel filtration purified: HM266-hIgG1-K322A, HM647-hIgG4-S228P, HM698-hIgG1-K322A, HM698-hIgG4-S228P

[0046] Activity evaluation using cell lines The activity of anti-human PD-1 antibodies was assessed by measuring their effect on cytokine production induced by the interaction between human PD-1-expressing T cells and antigen-presenting cells (APCs). The DO11.10 T cell hybridoma line (from the Department of Immunogenomics, Kyoto University Graduate School of Medicine) was used as T cells. Mouse PD-1 was knocked out using Cas9 (Invitrogen), and human PD-1 was overexpressed in this line. Similarly, the IIA1.6 B cell line (from the Department of Immunogenomics, Kyoto University Graduate School of Medicine) was used as APCs. Mouse PD-L1 was knocked out in this line, and human PD-L1 or mouse FcγRIIB was overexpressed in this line. Mouse FcγRIIB-expressing IIA1.6 cells were used to assess human PD-1 agonist activity, and human PD-L1-expressing IIA1.6 cells were used to assess antagonist activity. Human PD-1-expressing DO11.10 T cell hybridoma and each IIA1.6 cell were suspended in RPMI1640 medium containing 10% fetal bovine serum, and DO11.10 T cell hybridoma was diluted to 5x10 4 1x10 IIA1.6 cells / well / 50 μl 4 The cells were seeded in a round-bottom 96-well plate at 50 μl per well. Anti-human PD-1 antibody was added at 50 μl per well to a final concentration of 5, 0.5, 0.05, or 0.005 μg / ml. OVA was then added as an antigen. 323-339 The peptide (Eurofin) was added at a final concentration of 3 μg / ml at 50 μl / well. After 18 hours, the IL-2 concentration in the culture supernatant was measured using a mouse IL-2 DuoSet ELISA (R&D Systems).

[0047] For the evaluation of human PD-1 agonist activity, the cytokine suppression obtained using human PD-L1-expressing IIA1.6 served as a positive control, and the results using DO11.10 T cell hybridoma, which does not express human PD-1, served as a negative control. For the evaluation of human PD-1 antagonist activity, an anti-human PD-1 antibody (clone name: EH12.2H7) with known antagonist activity was used.

[0048] Activity evaluation using human T cells We evaluated the activity of anti-human PD-1 antibodies against actual human T cells. Specifically, we investigated the activity of human CD4 T cells isolated from peripheral blood by negative selection. + T cells (LONZA) were plated at 1x10 in a 24-well plate pre-coated with anti-CD3 antibody (clone name: OKT3, BioLegend). 6 The cells were seeded at 1 x 10 cells / well in 1 ml and stimulated for 3 days to activate and induce PD-1 expression. The antigen-presenting cells were THP-1 human monocyte-derived cell line (ATCC) strains, which were forced to express human FcγRIIB by electroporation. THP-1 cells were grown at 1 x 10 cells / well in medium supplemented with 500 μg / ml mitomycin C. 7 The cells were suspended at 37°C for 2 hours to stop proliferation and then used as antigen-presenting cells. + T cells and mitomycin-treated THP-1 cells were suspended in medium at 5x10 4 , 2.5x10 4 Cells were seeded into a round-bottom 96-well plate at 50 μl per well. Anti-human PD-1 antibody was added at 50 μl per well to final concentrations of 5, 0.5, 0.05, or 0.005 μg / ml. Cytostim (Miltenyi Biotec) was then added as an antigen at 50 μl per well to a final concentration of 0.2 μl per well. After 18 hours, IL-2 levels in the culture supernatant were measured using an ELISA MAX Standard Set Human (BioLegend).

[0049] Naive helper T cells (CD4 + CD62L + ) recovery DO11.10 mice (OVA 323-339 Spleens were collected from peptide-specific T cell receptor transgenic mice (The Jackson Laboratory), and cell suspensions were prepared and then hemolyzed. The resulting cells were stained with FITC-labeled antibodies against CD8, CD19, CD49b, and IA / IE. After washing, anti-FITC antibodies labeled with magnetic beads were bound to the cells, and they were magnetically separated to separate CD4 + The T cell fraction was separated. + The T cell fraction was stained with PerCp-Cy5.5-labeled anti-CD4 antibody and APC-labeled anti-CD62L antibody, and CD4 was detected by FACS. + CD62L + Naive helper T cells were obtained by collecting the cells, and the resulting naive helper T cells were stimulated with Dynabeads mouse T activator (Invitrogen) for 24 hours to express human PD-1.

[0050] Forced expression of human PD-1 by retroviral infection Retroviral supernatant was prepared by transfecting Plat-E cells with MSCV-hPD-1-IRES-Thy1.1. The retroviral supernatant was added to a RetroNectin (registered trademark)-coated culture plate and centrifuged at 32°C, 2500 rpm for 2 hours. The centrifuged plate was washed with PBS and then incubated with CD4 + T cells were added and centrifuged at 2000 rpm at 32°C for 10 minutes. This retroviral infection procedure was repeated twice at 24-hour intervals to force expression of human PD-1.

[0051] Preparation of antigen-presenting cells 2x10 llA1.6-mFcγRIIb(+) PD-L1(-) and hPD-L1(+) cells 7The cells were adjusted to a concentration of 1000 cells / mL, mixed with an equal volume of mitomycin C (1 mg / mL), and treated for 2 hours at 37°C. These cells were washed three times with PBS and used as antigen-presenting cells.

[0052] Induction of Th1 / Th2 cells CD4 cells overexpressing human PD-1 + T cells (1x10 6 ) to antigen-presenting cells (1x10 6 OVA 323-339 Peptide (5 μg / mL final concentration) and anti-human PD-1 antibody (5 μg / mL final concentration) were added to the culture plate. To induce Th1 cells, IL-2 (2 ng / mL final concentration; Peprotech), IL-12 (1 ng / mL final concentration; Peprotech), IFN-γ (1 ng / mL final concentration; Peprotech), and anti-IL-4 neutralizing antibody (5 μg / mL final concentration; clone name 11B11, BioXcell) were added. To induce Th2 cells, IL-2 (2 ng / mL final concentration), IL-4 (1 ng / mL final concentration; Peprotech), anti-IFN-γ neutralizing antibody (5 μg / mL final concentration; clone name XMG1.2, BioXcell), and anti-IL-12 neutralizing antibody (5 μg / mL final concentration; clone name C17.8, BioXcell) were added. Cultures were performed in 12-well plates in 2 mL of culture medium.

[0053] After 72 hours from the start of stimulation, half of the culture supernatant was discarded, and mitomycin C-treated antigen-presenting cells (1 x 10 6 pieces), OVA 323-339 Peptide (final concentration: 5 μg / mL), anti-human PD-1 antibody (final concentration: 5 μg / mL), and Th1 and Th2 cytokine / neutralizing antibody cocktail were added to a final volume of 2 mL. FACS analysis was performed 96 hours after the start of stimulation.

[0054] FACS analysis of intracellular cytokine staining The Th1 / Th2 cells induced by the above method were counted, and 2.5 x 10 cells were plated in a flat-bottom 96-well plate pre-coated with anti-CD3 antibody (2 μg / mL).5 After incubation at 37°C for 4 hours, brefeldin A (10 μg / mL) was added and incubation continued for an additional 2 hours. Cells were then harvested from the plate, Fc receptors were blocked using anti-mouse CD16 / 32 antibodies, the cell surface was stained with PerCp-Cy5.5-anti-mouse CD4 antibodies and Fixable Viability Dye eFluor 780, washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes. The fixed cells were washed with PBS, permeabilized for 10 minutes, washed again with PBS, and stained with FITC-labeled anti-IFN-γ and PE-labeled anti-IL-4 antibodies for 45 minutes. Analysis was performed by FACS.

[0055] Generation of human PD-1 knock-in mice using genome editing A donor vector containing gRNA (5'-GCCAGGGGCTCTGGGCATGT-3') (SEQ ID NO: 23) and the human PD-1 gene was microinjected into pronuclear fertilized eggs from C57BL / 6N mice together with Cas9 protein (Invitrogen), and then transplanted into the oviducts of foster mother mice. From the resulting mice (F0), indel mice were selected and crossed with wild-type mice to generate F1 mice. The F1 mice confirmed to have been transgenic were further crossed to obtain homozygotes, which were used as human PD-1 knock-in mice for this experiment.

[0056] Evaluation of the effects of anti-PD-1 agonist antibodies on antigen-specific antibody subclasses NP-OVA (4-hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin) was co-precipitated with alum adjuvant, and 100 μg of this was intraperitoneally administered to hPD-1 knock-in mice. At the same time, 500 μg of anti-human PD-1 antibody was intraperitoneally administered (day 0). The same amount of anti-human PD-1 antibody was administered intraperitoneally again 3 and 7 days later. Ten days later, blood was collected from the orbit, and plasma fractions were collected from the peripheral blood by centrifugation. IgG1 and IgG2c antibody titers against the hapten were measured by ELISA.

[0057] Evaluation of PD-1 agonists in a house dust mite extract (HDM)-induced allergy model hPD-1 knock-in mice were intraperitoneally injected with 400 ng of HDM (D. pteronyssinus; Greer) total protein (10 ng of Derp1 equivalent) and 500 μg of anti-human PD-1 antibody (day 0). The same amount of anti-human PD-1 antibody was then intraperitoneally administered 3, 7, and 10 days later. In a therapeutic setting, anti-human PD-1 antibody was administered only once on day 10. Starting 7 days after intraperitoneal HDM administration, mice were intranasally administered 25 μg / 25 μL of HDM total protein under anesthesia. This intranasal administration was continued for 8 consecutive days. Four hours after the final intranasal administration, the mice were bled and euthanized. Bronchoalveolar lavage fluid and lung tissue were collected. The bronchoalveolar lavage fluid was centrifuged and mononuclear cells were counted. The lungs were treated with enzymes and centrifuged on a density gradient to separate mononuclear cells, which were then counted and analyzed by FACS to detect CD4 + T cells, CD8 + T cells, γδTCR + Cells, eosinophils (CD11c - SiglecF + ), neutrophils (CD11c - SiglecF - Ly-6G + ), alveolar macrophages (CD11c + SiglecF + ) and other factors were calculated. Intracellular cytokine staining was performed on mononuclear cells derived from bronchoalveolar lavage fluid and lung mononuclear cells using the same method. Blood levels of HDM-specific IgE were measured by ELISA (mouse serum anti-HDM IgE antibody assay kit; Chondrex).

[0058] Evaluation of PD-1 agonists in the MC903-induced atopic dermatitis model MC903 was dissolved in ethanol and applied to both ears of hPD-1 knock-in mice at a concentration of 5 nmol / 10 μL. At the same time, 500 μg of anti-human PD-1 antibody was administered intraperitoneally (day 0). The same amount of MC903 and anti-human PD-1 antibody were administered every three days from day 0 to day 27. In a therapeutic setting, anti-human PD-1 antibody administration began on day 12. Ear thickening was measured every three days throughout the study period. On day 29, the number of scratching behaviors was visually counted over a 10-minute period. Blood samples were collected on days 9, 15, 24, and 30, and plasma IgE concentrations were measured by ELISA (ELISAMAX Standard Set Mouse IgE; BioLegend). After day 30, mice were euthanized, and ear samples were collected. The ears were cut into pieces and treated with enzymes to collect immune cells. FACS analysis was performed to identify eosinophils (CD45 + CD11b + SiglecF + ) and other numbers were calculated.

[0059] Results and Discussion To identify functional anti-PD-1 antibodies, we utilized the established DO11.10 T cell hybridoma line (Figure 1). Because PD-1 signaling activity manifests as suppression of T cell activation, the prerequisites for this experimental system are sufficient T cell activation and PD-1-dependent immunosuppression. While these conditions can be achieved using primary T cells and antigen-presenting cells, PD-1 protein is not expressed on T cells prior to activation, and the effects of PD-1 signaling can only be examined after stimulation. The cell populations obtained in this manner are not homogeneous in many ways, resulting in poor experimental stability. Furthermore, due to inevitable inter-individual variability, the use of primary cells presents a drawback in terms of experimental system stability and reproducibility. Therefore, we chose to use an established cell line for antibody screening, which offers these advantages.

[0060] DO11.10 T cell hybridoma is a peptide derived from ovalbumin (OVA) 323-339 ) recognizing MHC class II (IA d ) CD4 of DO11.10 mice expressing a restricted T cell receptor + DO11.10 T cell hybridoma is a T cell hybridoma derived from T cells. DO11.10 T cell hybridoma expresses MHC class II (IA) expressed on the B lymphoma cell line IIA1.6. d ) and produce IL-2 in response to antigenic peptides presented on IIA1.6 (Figure 1A). This means that T cell activation in this combination is triggered by antigen recognition, offering the advantage of recapitulating a physiological activation mechanism that cannot be achieved in experimental systems that promote nonspecific T cell activation. Furthermore, gene knockout and forced expression are possible, so by forcing human PD-1 expression into the DO11.10 T cell hybridoma, which lacks mouse PD-1, it can be used as an experimental system to examine the function of anti-human PD-1 antibodies. IL-2 production was clearly reduced when the DO11.10 T cell hybridoma expressed human PD-1 and was stimulated with PD-L1-expressing IIA1.6, confirming that this change was a PD-1-specific response (Figure 1B). PD-1 and PD-L1 expressed on the cell membrane should be in their native three-dimensional conformation, which also provides an advantage over artificial experimental systems using solubilized membrane proteins. Indeed, when an anti-human PD-1 antibody (EH12.2H7), which has already been established as a blocking antibody, was added to this experimental system, the IL-2 levels that had been reduced by the action of PD-L1 were significantly restored, demonstrating that this experimental system can reliably detect blocking antibodies (Fig. 1C). Based on these findings, we concluded that this experimental system, which utilizes cell-cell interactions, is an extremely suitable combination for observing PD-1-mediated T cell suppression, and we decided to use this system as the basis for further experiments.

[0061] Detection of agonistic antibodies with immunosuppressive activity must be performed under conditions in which PD-L1 inhibition is absent. Therefore, a separate system was prepared for detecting blocking antibodies. This system used a combination of DO11.10 T cell hybridoma expressing human PD-1 and IIA1.6 cells lacking PD-L1 and expressing FcγRIIB. Using these two systems, one for detecting blocking antibodies and the other for detecting agonistic antibodies, we screened for anti-human PD-1 monoclonal antibodies. The monoclonal antibodies used were generated from mice immunized with human PD-1. As a result, several monoclonal antibodies with various levels of activity were obtained (Figure 2). Furthermore, commercially available anti-human PD-1 monoclonal antibodies, such as MIH4 and J116, were found to have agonistic activity. Comparing the agonistic activity of these antibodies, HM266, HM647, HM698, and MIH4 showed particularly favorable activity (Figure 3).

[0062] In addition to the studies using mouse cells, these anti-PD-1 agonist antibodies were also confirmed to have immunosuppressive effects on human T cells. + IL-2 production from T cells was significantly suppressed by HM266 (Fig. 4).

[0063] CD4 + The functions of helper T cells are diverse, but these diverse functions are covered by subsets of helper T cells that play specialized roles in each immune function. Typical examples of these are known to be cell groups with different roles, such as Th1, Th2, Th17, and Treg. For example, CD8 +Helper T cell subsets are specialized for immune functions with very different characteristics. Th1 cells promote cellular immunity, primarily through the direct cytotoxic activity of T cells and NK cells, while Th2 cells promote allergic reactions by class switching to IgE through enhanced humoral immunity and activating eosinophils. Each helper T cell subset produces significantly different cytokines. Th1 cells are characterized by IFN-γ, while Th2 cells produce IL-4, IL-5, and IL-13, and Th17 cells produce IL-17. Each helper T cell subset has a distinctive cytokine production pattern. Through these cytokines, helper T cell subsets regulate different immune functions.

[0064] Cytokine production by helper T cell subsets is also a function under the control of PD-1. Numerous studies have shown that cytokine production increases upon blockade of PD-1 or its ligands (PD-L1 and PD-L2). In many cases, the increase in cytokine production is consistent across cytokines. Blockade of PD-1 signaling has been shown to increase IFN-γ, TNF-α, IL-4, IL-10, and IL-13 levels in peripheral blood lymphocytes from allergy-related patients after restimulation with the allergen [1]. Furthermore, studies have shown that PD-1 blockade results in increased cytokine production by IFN-γ over IL-4, IL-5, and IL-13, resulting in Th1 cell dominance over Th2 cells [2-5]. Active stimulation of PD-1 with PD-L1 or PD-L2 suppresses cytokine production from PD-1-expressing T cells, but no significant differences have been observed between Th1 and Th2 cytokines [6-8]. Experiments using PD1-17 (Wyeth) as an hPD-1 agonist antibody have also reported consistent suppression of IFN-γ, IL-2, IL-10, and IL-13 [9]. These experimental results suggest that PD-1 / PD-1 ligands function in suppressing cytokine production from Th1 and Th2 cells already present in the sample. However, prior to the emergence of such Th1 and Th2 cells, there is a process by which these cells are formed, and further studies are needed to clarify the effects of PD-1 stimulation at this stage.

[0065] Helper T cell subsets, such as Th1 and Th2, are thought to be derived from naive helper T cells of the same origin. For example, the same naive helper T cells can become Th1 cells when activated in the presence of IL-12, but become Th2 cells when activated in the presence of IL-4. This is functional differentiation. This fact means that naive helper T cells can become either Th1- or Th2-dominant depending on the environment at the time of activation. Therefore, the functional differentiation of naive helper T cells determines whether the relative immune response is directed toward inflammation due to cytotoxic immune responses or toward allergic inflammatory responses dominated by antibodies and eosinophils. Therefore, if PD-1 stimulation alters the balance between Th1 and Th2, not only will the immune response be quantitatively suppressed, but also qualitatively altered.

[0066] Therefore, we investigated the effect of PD-1 agonist stimulation on the functional differentiation of CD4+ helper T cells. Naive helper T cells from DO11.10 mice were retrovirally expressed with hPD-1. These naive helper T cells were then specifically induced into Th1 and Th2 cells under different cytokine conditions. Anti-human PD-1 agonist antibodies (HM266, HM647, and J116) were added to these cells. The results showed that all anti-human PD-1 agonist antibodies significantly reduced the proportion of IL-4-producing Th2 cells (Figure 5). In contrast, the differentiation into IFN-γ-producing Th1 cells was resistant to these antibodies. Control antibodies did not inhibit Th2 differentiation. While stimulation with PD-L1-expressing cells also significantly reduced the proportion of Th2 cells, the effect on Th1 cell induction was relatively mild, similar to the effect of anti-human PD-1 agonist antibodies.

[0067] These results consistently demonstrate that there are differences in sensitivity of helper T cell subsets to the inhibitory effects of PD-1 agonists during functional differentiation, with Th2 differentiation being more sensitive to PD-1 signaling.

[0068] Very few studies have addressed the role of PD-1 in functional differentiation into Th1 / Th2 cells. Such studies have utilized immobilization of PD-L1 on plastic surfaces, resulting in suppression of Th1 (and Th17) cells, but not inhibition of Th2 differentiation [10, 11]. Unlike these studies, our experimental system reproduces antigen-specific activation mediated by the interaction between T cells and antigen-presenting cells, and the results demonstrate that functional differentiation into Th2 cells is particularly sensitive to PD-1 stimulation.

[0069] In vivo experiments also demonstrated the suppression of Th2-type immune responses by PD-1 agonists. Antibodies produced by B cells in response to antigens undergo maturation through mechanisms such as class switching with the help of T cells. It is known that class switching to IgG2a and IgG2c is specifically induced by Th1 cells, while class switching to IgG1 and IgE is specifically induced by Th2 cells. Human PD-1 knock-in mice were immunized with NP-OVA and NP-specific IgG production was analyzed. Administration of an anti-PD-1 agonist antibody suppressed overall production. However, focusing on the IgG subclass, the suppressive effect was predominantly on IgG1 over IgG2c, resulting in a decrease in the IgG1 / IgG2c ratio (Figure 6). These results suggest that PD-1 agonists may actually alter the Th1 / Th2 balance in immune responses in vivo and achieve highly selective suppression of Th2-type immunity.

[0070] Given our findings that PD-1 agonist stimulation strongly suppresses functional differentiation into Th2 cells, allergic diseases are one potential target for PD-1 agonist therapy. Allergic diseases, including asthma, atopic dermatitis, hay fever, and food and drug allergies, are extremely prevalent. The number of patients has been rapidly increasing worldwide in recent years, necessitating the development of effective treatments. These allergic diseases are immune responses induced by Th2 cytokine production, characterized by eosinophil-dominated inflammation and increased circulating IgE antibody levels. Increased IgE promotes mast cell activation and exacerbates inflammation. While increased IgE antibody levels are directly caused by an increase in IgE-producing B cells, class switching of antibodies to IgE is induced by the action of cytokines such as IL-4 and IL-13 released by Th2 cells. Th2 cells can also induce eosinophil activation by producing IL-5. In other words, Th2 cells increase IgE production in B cells through IL-4 and IL-13, and at the same time, promote eosinophil-dominated inflammation through IL-5, so it is thought that an increase in Th2 cells plays a central role in the pathogenesis of allergic diseases. Therefore, if the balance of functional differentiation of helper T cells becomes significantly biased toward Th2 dominance, it is thought that it will soon lead to the onset of allergic diseases. Based on this background, it is suggested that suppressing excessive Th2-type immune responses is effective in treating allergic diseases.

[0071] Based on our new findings that PD-1 agonists have particularly strong inhibitory effects on Th2 cell differentiation, we anticipate that PD-1 agonists may be effective in preventing and treating allergic diseases, which are driven by a central role played by Th2 cells. Therefore, we investigated the efficacy of PD-1 agonists in allergic asthma using an animal model of asthma induced in mice by inhaling house dust mite antigens. This experiment was conducted by sensitizing mice with house dust mite antigens and then inhaling the same antigen daily one week later. To investigate the efficacy of anti-PD-1 agonist antibodies against this disease, we began administering them at the time of sensitization. Subsequently, we continued administering the anti-PD-1 agonist antibody twice weekly. Two weeks after the start of sensitization, mice were euthanized and analyzed. J116 administration significantly reduced eosinophil infiltration into the alveoli (Figure 7). Correspondingly, the CD4+ cells infiltrating the alveoli were also significantly reduced. + Analysis of T cells revealed that the number of cells producing IL-5 and IL-13 was reduced (Fig. 8).

[0072] When HM266, which has stronger agonist activity, was used instead of J116, the anti-inflammatory effect against allergic inflammation was also enhanced. + T cell infiltration into the alveoli is suppressed, and CD4 + The T cells producing IL-4, IL-5, and IL-13 were all reduced (Figs. 9 and 10). The blood levels of mite antigen-specific IgE also significantly decreased, correlating with the decrease in Th2 cells, demonstrating that Th2-type immunity was indeed suppressed (Fig. 10). Furthermore, we verified the effectiveness of anti-PD-1 agonist antibodies in a therapeutic setting. In this case, HM266 was administered only once, three days after the start of daily inhalation of the antigen, but it significantly reduced eosinophils and CD4 T cells that produce Th2-type cytokines. + T cell infiltration was significantly suppressed (Figures 9 and 10). These results suggest that PD-1 agonists are useful as preventive and therapeutic agents against allergic inflammation.

[0073] We investigated the anti-inflammatory effect of an anti-PD-1 agonist antibody on atopic dermatitis induction, a different type I allergic disease model. When MC903 was applied to the ears of human PD-1 knockout mice to induce allergic dermatitis, concurrent administration of HM266 significantly suppressed ear swelling (Figure 11). The suppression of the allergic inflammatory response was remarkable, as evidenced by a significant decrease in blood IgE levels, the number of eosinophils infiltrating into the ear tissue, and a reduction in scratching behavior by the mice. Furthermore, to examine the therapeutic effects of HM266, we initiated administration after ear swelling was induced (day 12). In this therapeutic setting, we also observed a significant decrease in IgE levels and eosinophil counts (Figure 11). These results suggest that PD-1 agonists may be useful for the treatment of Th2-mediated diseases, including type I allergies.

[0074] Previous studies have investigated the blockade of PD-1, PD-L1, or PD-L2 in animal models of asthma, but the results have been mixed, even contradictory. Therefore, the role of PD-1 in this pathogenesis remains controversial. One study reported that PD-1 / PD-L1 blockade exacerbated allergic inflammation, but the authors concluded that this was due to an enhancement of Th17 immunity rather than an enhancement of Th2 immunity

[11] . In contrast, other studies reported that PD-1 / PD-L1 blockade did not exacerbate inflammation. In these cases, PD-L2 blockade enhanced inflammation [12-14] and resulted in a Th2-dominant immune state, as evidenced by increased IL-5 and IL-13 and decreased IFN-γ

[12] . However, the same study did not demonstrate any effect of PD-1 blockade on inflammation, leading the authors to conclude that the changes induced by PD-L2 blockade were PD-1-independent. Regarding asthma and PD-L2, there has also been research into the administration of PD-L2-Fc. This paper reported that PD-L2-Fc administered in vitro suppressed cytokine production from T cells. However, when the same substance was administered in vivo, it instead aggravated inflammation and increased IL-5, IL-13, and IgE, which is inconsistent with the in vitro results. [6] Furthermore, these results are not consistent with reports that PD-L2 blockade enhances inflammation.

[0075] To summarize existing reports, PD-1 stimulation has been known to suppress various cytokines, but there have been no reports of it selectively suppressing Th2 cells. + Regarding the role of PD-1 agonists in the functional differentiation of T cells, no studies have suggested that they are particularly sensitive to Th2 differentiation. Furthermore, the role of PD-1 in asthma models has not been fully established, as conflicting results have been presented. However, there are no reports demonstrating that PD-1 stimulation suppresses Th2-type immunity, and thus asthma or allergic dermatitis.

[0076] In response to this situation, our research findings revealed that Th1 and Th2 cells differ in their sensitivity to PD-1 stimulation and that functional differentiation into Th2 cells is strongly inhibited by PD-1 stimulation. We demonstrated that the use of PD-1 agonists, particularly anti-PD-1 agonist antibodies, can alter the Th1 / Th2 immune balance and control Th2-mediated responses. Indeed, in vivo studies demonstrated that administration of anti-PD-1 agonist antibodies inhibited the establishment of Th2-type immunity in mice and significantly suppressed allergic inflammation in tissues. These results suggest that anti-PD-1 agonist antibodies can correct the Th2-biased immune balance and are useful for the prevention and treatment of Th2-mediated diseases, particularly type I allergies and eosinophilic disorders.

[0077] References 1. Rosskopf S, Jahn-Schmid B, Schmetterer KG, Zlabinger GJ, Steinberger P. PD-1 has a unique capacity to inhibit allergen-specific human CD4+ T cell responses. Sci. Rep. 8:13543 (2018) 2. Rosenblatt J, Glotzbecker B, Mills H, Vasir B, Tzachanis D, Levine JD, Joyce RM, Wellenstein K, Keefe W, Schickler M, Rotem-Yehudar R, Kufe D, Avigan D. PD-1 blockade by CT-011, anti-PD-1 antibody, enhances ex vivo T-cell responses to autologous dendritic cell / myeloma fusion vaccine. J. Immunother. 34:409-18 (2011). 3. Dulos J、Carven GJ、van Boxtel SJ、Evers S、Driessen-Engels LJ、Hobo W、Gorecka MA、de Haan AF、Mulders P、Punt CJ、Jacobs JF、Schalken JA、Oosterwijk E、van Eenennaam H、Boots AM. PD-1 blockade augments Th1 and Th17 and suppresses Th2 responses in peripheral blood from patients with prostate and advanced melanoma cancer. J. Immunother. 35:169-78 (2012). 4. Wang S、Zhu X、Xu Y、Zhang D、Li Y、Tao Y、Piao H、Li D、Du M. Programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) regulate CD4+ T cells to induce Type 2 helper T cell (Th2) bias at the maternal-fetal interface. Hum. Reprod. 31:700-11 (2016). 5. Rajamanickam A、Munisankar S、Dolla C、Nutman TB、Babu S. Cytotoxic T-Lymphocyte-Associated Antigen 4 (CTLA-4) and Programmed Death 1 (PD-1) mediated regulation of mono- and dual functional CD4+ and CD8+ T-cell responses in a chronic helminth infection. Infect. Immun. 87:e00469-19 (2019). 6. Oflazoglu E、Swart DA、Anders-Bartholo P、Jessup HK、Norment AM、Lawrence WA、Brasel K、Tocker JE、Horan T、Welcher AA、Fitzpatrick DR. Paradoxical role of programmed death-1 ligand 2 in Th2 immune responses in vitro and in a mouse asthma model in vivo. Eur. J. Immunol. 34:3326-36 (2004). 7. Zhu B、Guleria I、Khosroshahi A、Chitnis T、Imitola J、Azuma M、Yagita H、Sayegh MH、Khoury SJ. Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis. J. Immunol. 176:3480-9 (2006). 8. Kubo S、Yamada T、Osawa Y、Ito Y、Narita N、Fujieda S. Cytosine-phosphate-guanosine-DNA induces CD274 expression in human B cells and suppresses T helper type 2 cytokine production in pollen antigen-stimulated CD4-positive cells. Clin. Exp. Immunol. 169:1-9 (2012). 9. Chemnitz JM、Parry RV、Nichols KE、June CH、Riley JL. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation、but only receptor ligation prevents T cell activation. J. Immunol. 173:945-54 (2004). 10. Herold M、Posevitz V、Chudyka D、Hucke S、Groβ C、Kurth F、Leder C、Loser K、Kurts C、Knolle P、Klotz L、Wiendl H. B7-H1 Selectively Controls TH17 Differentiation and Central Nervous System Autoimmunity via a Novel Non-PD-1-Mediated Pathway. J. Immunol. 195:3584-95 (2015). 11. McAlees JW、Lajoie S、Dienger K、Sproles AA、Richgels PK、Yang Y、Khodoun M、Azuma M、Yagita H、Fulkerson PC、Wills-Karp M、Lewkowich IP. Differential control of CD4(+) T-cell subsets by the PD-1 / PD-L1 axis in a mouse model of allergic asthma. Eur. J. Immunol. 45:1019-29 (2015). 12. Matsumoto K, Inoue H, Nakano T, Tsuda M, Yoshiura Y, Fukuyama S, Tsushima F, Hoshino T, Aizawa H, Akiba H, Pardoll D, Hara N, Yagita H, Azuma M, Nakanishi Y. B7-DC regulates asthmatic response by an IFN-gamma-dependent mechanism. J. Immunol. 172:2530-41 (2004). 13. Akbari O, Stock P, Singh AK, Lombardi V, Lee WL, Freeman GJ, Sharpe AH, Umetsu DT, Dekruyff RH. PD-L1 and PD-L2 modulate airway inflammation and iNKT-cell-dependent airway hyperreactivity in opposing directions. Mucosal Immunol. 3:81-91 (2010). 14. Lewkowich IP, Lajoie S, Stoffers SL, Suzuki Y, Richgels PK, Dienger K, Sproles AA, Yagita H, Hamid Q, Wills-Karp M. PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production. Mucosal Immunol. 6:728-39 (2013). All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0078] The present invention can be used to treat and / or prevent Th2-mediated diseases such as type I allergies and eosinophilic diseases. [Sequence List Free Text]

[0079] Clone name: HM-266 VH Amino Acid Sequence QVQLQQSGPELVKPGASVKMSCKASGYTFTSYYIQWVKQRPGQGLEWIGWIYPGDGSSKYNEKFKGKTTLTADKSSSTAYMLLSSLTSEDSGIYFCASYYGSSFDYWGQGTTLTVSS (SEQ ID NO: 1) Origin: Mus musculus VH DNA Sequence caggtccagctgcagcagtctggacctgagctggtgaagcctggggcttcagtgaagatgtcctgcaaggcttctggctacaccttcacaagctactatatacagtgggtgaagcagaggcctggacagggacttgagtggattggatggatttatcctggagatggtagtagcaagtacaatgagaagttcaagggcaagaccacactgactgcagacaaatcctccagcacagcctacatgttgctcagcagcctgacctctgaggactctgggatctatttctgtgcaagttactacggtagtagttttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 2) Origin: Mus musculus VL Amino Acid Sequence DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKFLIYYSSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGSTLPFTFGGGTKLEIK (SEQ ID NO: 3) Origin: Mus musculus VL DNA Sequence gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaaccagatggaactgttaaattcctaatatactactcatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaggaagatattgccacttacttttgccagcagggtagtacgcttccgttcacgttcggaggggggaccaagctggaaataaaa(SEQ ID NO: 4) Origin: Mus musculus Clone name : HM-647 VH Amino Acid Sequence EVLLQQSGPELVKPGASVKITCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGGTVYNQKFKDKASLTVDKSSSTGYMELRSLTSEDTAVYYCARWRSAMDYWGQGTSVTVSS(SEQ ID NO: 5) Origin: Mus musculus VH DNA Sequence gaggtcctgctgcaacagtctggacctgagctggtgaagcctggggcttcagtgaagataacctgcaaggcttctggatacacattcactgactacaacatggactgggtgaagcagagccatggaaagagccttgagtggattggagatattgatcctaacaatggtggtactgtctacaaccagaagttcaaggacaaggcctcattgactgtagacaagtcctccagcacaggctacatggagctccgcagcctgacatctgaggacactgcggtctattactgtgcaagatggcggagtgctatggactactggggtcaaggaacctcagtcaccgtctcctca(SEQ ID NO: 6) Origin: Mus musculus VL Amino Acid Sequence DIQMTQPTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLISYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQVDIATYFCQQYSTLPWTFGGGSKLEIK (SEQ ID NO: 7) Source: Mus musculus VL DNA Sequence gatatccagatgacacagcctacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcaacagaaaccagatggaactgttaaactcctgatctcctacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaggtagatattgccacttacttttgccaacagtatagtacgcttccgtggacgttcggtggaggctccaagctggaaatcaaa (SEQ ID NO: 8) Source: Mus musculus Clone name : HM-698 VH Amino Acid Sequence EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDIDPNNGGTVYNQKFKGKASLTVDKSSSTAYMELRSLTSEDTAVYYCARWRSAMDYWGQGTSVTVSS (SEQ ID NO: 9) Source: Mus musculus VH DNA Sequence Derived from Mus musculus: gaggtcctgctgcaacagtctggacctgaactagtgaagcctggggcttcagtgaagataccctgcaaggcttctggatacacattcactgactacaacatggactgggtgaagcagagccatggaaagagccttgagtggattggagatattgatcctaacaatggtggtactgtctacaaccagaagttcaagggcaaggcctcattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggacactgcggtctattactgtgcaagatggcggagtgctatggactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO: 10) VL Amino Acid Sequence Derived from Mus musculus: DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQYNTLPWTFGGGTKLEIK (SEQ ID NO: 11) VL DNA Sequence Derived from Mus musculus: gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagtataatacgcttccgtggacgttcggtggaggcaccaaactggaaatcaaa (SEQ ID NO: 12) Mouse IgG1 CH Amino Acid Sequence AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO: 13) Origin: Mus musculus DNA Sequence gccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctggagtctgacctctacactctgagcagctcagtgactgtcccctccagccctcggcccagcgagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatgaacacgaatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaa(SEQ ID NO: 14) Origin: Mus musculus Mouse Igk CL Amino Acid Sequence Derived from Mus musculus: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 15) DNA Sequence Derived from Mus musculus: cgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt (SEQ ID NO: 16) Human IgG1-K322A CH Amino Acid Sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 17) derived from: artificial IgG1-K322A CH sequence IgG4-S228P CH amino acid sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGR* (SEQ ID NO: 19) derived from: artificial IgG4-S228P CH sequence Human Igk CL amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 21) from: Homo sapiens CL base sequence cgaactgtagccgcaccaagcgtgttcatctttccgccatccgatgaacagctgaagtcaggcacagcgtcagttgtctgtctcctcaacaacttctaccccagagaggccaaagtgcagtggaaagtggacaatgccctgcagtcaggcaattctcaggaatctgtga cagagcaggactccaaagacagtacctatagcctgtctagcacactgacgctctctaaggccgactatgagaagcacaaggtctatgcctgtgaagtgacacatcaagggctgagcagtccagtcactaagagcttcaatcgtggggaatgctga (SEQ ID NO: 22) Origin: Homo sapiens gRNA (5'-GCCAGGGGCTCTGGGCATGT-3') (SEQ ID NO: 23) derived from: artificial Amino acid sequence of human PD-1 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 24) Mouse PD-1 amino acid sequence MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMVIGIMSALVGIPVLLLLAWALAVFCSTSMSEARGAGSKDDTLKEEPSAAPVPSVAYEELDFQGREKTPELPTACVHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHEDGHCSWPL (SEQ ID NO: 25)

Claims

1. A pharmaceutical composition for treating or preventing a Th2-mediated disease, comprising an effective amount of an anti-PD-1 agonist antibody or a functional fragment thereof.

2. The pharmaceutical composition according to claim 1, wherein the Th2-mediated disease is type I allergy.

3. The pharmaceutical composition according to claim 1, wherein the Th2-mediated disease is an eosinophilic disease.

4. 2. The pharmaceutical composition according to claim 1, wherein the Th2-mediated disease is a disease selected from bronchial asthma, atopic dermatitis, allergic rhinitis, drug allergy, food allergy, anaphylaxis, allergic conjunctivitis, urticaria, eosinophilic sinusitis, eosinophilic gastrointestinal disease, and allergic bronchopulmonary aspergillosis.

5. A composition for suppressing IgE production by suppressing Th2-type cytokines, the composition comprising an anti-PD-1 agonist antibody or a functional fragment thereof.

6. A composition for suppressing eosinophil activation by suppressing Th2-type cytokines, the composition comprising an anti-PD-1 agonist antibody or a functional fragment thereof.

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