Allergen-binding antibodies suitable for treating tree pollen allergies

Abstract

Tree pollen allergy is a seasonal allergy caused by various tree pollens suspended in the air. This allergy affects a significant proportion of the population, particularly in Europe and North America, and is characterized by symptoms such as sneezing, nasal congestion, runny nose, itchy or watery eyes, and throat inflammation. These symptoms typically occur in spring when trees such as birch, alder, hazel, and oak release pollen into the air. Tree pollen allergy is a type 1 hypersensitivity reaction mediated by immunoglobulin E (IgE) antibodies. This invention relates to antibodies and their antigen-binding fragments that bind to one or more tree pollen allergens, Bet v 1, Aln g 1, Cor a 1, and Que a 1, and inhibit the IgE-mediated allergic reaction.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to antibodies that bind to one or more tree pollen allergens, Bet v 1, Aln g 1, Cor a 1, and Que a 1, and their antigen-binding fragments, therapeutic compositions containing these antibodies, and methods for using these antibodies. [Background technology]

[0002] Background of the Invention Tree pollen allergy is a seasonal allergy caused by various airborne tree pollens. This allergy affects a significant portion of the population, particularly in Europe and North America, and is characterized by symptoms such as sneezing, nasal congestion, runny nose, itchy and watery eyes, and throat inflammation. These symptoms typically manifest in the spring when birch, alder, hazel, and oak trees release pollen into the air. Tree pollen allergy is a type 1 hypersensitivity reaction mediated by immunoglobulin E (IgE) antibodies. IgE is produced by B cells in response to the presence of allergens, such as Bet v 1, the major allergen found in birch pollen (D'Amato et al., 2007).

[0003] Bet v 1 belongs to a family of proteins known as pathogenicity-associated proteins 10 (PR-10), and is present in various tree pollens, where it has been shown to cause cross-reactivity between different allergens. For example, Que a 1, Aln g 1, and Cor a 1 are homologous allergens found in oak, alder, and hazelnut pollen, respectively, and share significant structural and immunological similarities with Bet v 1. Therefore, individuals allergic to any of the above allergens may also experience allergic reactions to other cross-reactive allergens, potentially complicating the diagnosis and management of allergies (Asam et al., 2015).

[0004] The symptoms of tree pollen allergy are caused by cross-linking of tree pollen allergen-specific IgE bound to high-affinity epsilon receptors (FcεRI) on the surface of mast cells and basophils, which triggers the release of inflammatory mediators such as histamine, leukotrienes, and cytokines. These mediators cause characteristic allergic symptoms and may induce bronchial hypersensitivity or asthma in some patients (Gould and Sutton, 2008).

[0005] Current treatment options for tree pollen allergy include allergen avoidance, pharmacotherapy, and allergen-specific immunotherapy (AIT). Allergen avoidance involves minimizing exposure to tree pollen through measures such as staying indoors during periods of high pollen count, wearing masks, and keeping windows closed. Pharmacotherapy options include antihistamines, nasal corticosteroids, leukotriene receptor antagonists, and decongestants, which alleviate allergy symptoms but do not have long-term disease-modifying effects. AIT is a disease-modifying treatment that induces immune tolerance by gradually increasing the dose of the allergen, thereby reducing the severity of the allergy. AIT can be administered subcutaneously or sublingually and has been shown to reduce symptoms of tree pollen allergy and improve patients' quality of life (Canonica et al., 2014).

[0006] Allergen-specific antibodies have long been proposed as an allergy treatment because they may block the entry of allergen molecules into mucosal tissue, or they may bind to allergens before the allergens bind to IgE bound to high-affinity receptors on mast cells and basophils, thereby preventing the release of histamine and other inflammatory mediators from these cells.

[0007] Allergen-specific antibodies for the treatment of IgE-mediated allergies have already been reported (US 5,670,626 and US 6,849,259), including antibodies that block the binding of allergens to mast cells (US 2010 / 0034812).

[0008] Patent application WO 2018 / 222854 describes a treatment for birch allergy by using an antibody composition consisting of two or more antibodies that bind to different epitopes on the same Bet v 1 allergen, thereby more efficiently blocking allergen-mediated cell activation. Of the Bet v 1-binding antibodies disclosed in WO 2018 / 222854, only one can also bind to other tree pollen allergens, Aln g 1 and Cor a 1. Therefore, this antibody exhibits cross-reactivity with the allergens Aln g 1 and Cor a 1.

[0009] This strategy of combining antibodies specific to the same allergen is also described in patent applications WO 2013 / 166236 and WO 2018 / 234383, which relate to the treatment of cat allergy by binding to the allergen Fel d 1 and the treatment of peanut allergy by binding to the allergen Ara h 2, respectively.

[0010] However, the tree pollen dispersal period is not a single event, but rather an overlapping event in which the different tree pollen allergens described herein peak at different times of the year. Therefore, treatments targeting a single allergen cannot effectively treat the symptoms of patients who are allergic to two or more allergens of the PR-10 family.

[0011] The present invention aims to overcome the above-mentioned problems. [Overview of the Initiative]

[0012] The present invention relates to an antibody or antigen-binding fragment thereof that can bind to a tree pollen allergen selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1.

[0013] The inventors have provided a class of antibodies that have the potential to treat pollen allergies caused by four important tree pollen allergens. As shown herein, these antibodies can individually bind with good affinity to all four allergens: Bet v 1, Aln g 1, Cor a 1, and Que a 1, and are thought to inhibit the binding of these allergens to IgE antibodies in humans when administered in therapeutically effective doses. As shown in Example 2, a number of individual antibodies shown in Table 1, such as seven antibodies 2E02, 2C10, 2B04, 2E0, A07, B10, and 2_10, were all able to bind to all four tree pollen allergens (Bet v 1, Aln g 1, Cor a 1, and Que a 1) and showed higher affinity than conventional antibodies such as REGN5713, REGN5714, and REGN5715 described in WO 2018 / 222854. Furthermore, as shown in Example 5, combining two or three antibodies improved their ability to block the binding of individual tree pollen allergens to IgE compared to individual antibodies, and inhibited the activation of basophils, which are a major factor in allergies as described above.

[0014] Therefore, in a first embodiment, the present invention relates to antibodies 2E02, 2C10, 2B04, 2E0, A07, B10, and 2_10, or their antigen-binding fragments. Accordingly, the first embodiment relates to antibodies or their antigen-binding fragments comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein, The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO: 1, the HCDR2 amino acid sequence contained in SEQ ID NO: 1, the HCDR3 amino acid sequence contained in SEQ ID NO: 1, the LCDR1 amino acid sequence contained in SEQ ID NO: 2, the LCDR2 amino acid sequence contained in SEQ ID NO: 2, and the LCDR3 amino acid sequence contained in SEQ ID NO: 2; or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 21, the HCDR2 amino acid sequence contained in SEQ ID NO: 21, the HCDR3 amino acid sequence contained in SEQ ID NO: 21, the LCDR1 amino acid sequence contained in SEQ ID NO: 22, the LCDR2 amino acid sequence contained in SEQ ID NO: 22, and the LCDR3 amino acid sequence contained in SEQ ID NO: 22; or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 41, the HCDR2 amino acid sequence contained in SEQ ID NO: 41, the HCDR3 amino acid sequence contained in SEQ ID NO: 41, the LCDR1 amino acid sequence contained in SEQ ID NO: 42, the LCDR2 amino acid sequence contained in SEQ ID NO: 42, and the LCDR3 amino acid sequence contained in SEQ ID NO: 42; or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 61, the HCDR2 amino acid sequence contained in SEQ ID NO: 61, the HCDR3 amino acid sequence contained in SEQ ID NO: 61, the LCDR1 amino acid sequence contained in SEQ ID NO: 62, the LCDR2 amino acid sequence contained in SEQ ID NO: 62, and the LCDR3 amino acid sequence contained in SEQ ID NO: 62; or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 81, the HCDR2 amino acid sequence contained in SEQ ID NO: 81, the HCDR3 amino acid sequence contained in SEQ ID NO: 81, the LCDR1 amino acid sequence contained in SEQ ID NO: 82, the LCDR2 amino acid sequence contained in SEQ ID NO: 82, and the LCDR3 amino acid sequence contained in SEQ ID NO: 82; or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 101, the HCDR2 amino acid sequence contained in SEQ ID NO: 101, the HCDR3 amino acid sequence contained in SEQ ID NO: 101, the LCDR1 amino acid sequence contained in SEQ ID NO: 102, the LCDR1 amino acid sequence contained in SEQ ID NO: 102, and the LCDR3 amino acid sequence contained in SEQ ID NO: 102; or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 121, the HCDR2 amino acid sequence contained in SEQ ID NO: 121, the HCDR3 amino acid sequence contained in SEQ ID NO: 121, the LCDR1 amino acid sequence contained in SEQ ID NO: 122, the LCDR2 amino acid sequence contained in SEQ ID NO: 122, and the LCDR3 amino acid sequence contained in SEQ ID NO: 122.

[0015] Each of the above CDRs (HCDR1, HCDR2, and HCDR3, LCDR1, LCDR2, and LCDR3) may be determined according to the IMGT method, the Kabat method, or the Chothia method.

[0016] Optionally, each CDR region determined by the IMGT, Kabat, or Chothia method may have one, two, or three amino acid substitutions. For example, each HCDR1 may contain one, two, or three amino acid substitutions, each HCDR2 may contain one, two, or three amino acid substitutions, each HCDR3 may contain one, two, or three amino acid substitutions, each LCDR1 may contain one, two, or three amino acid substitutions, each LCDR2 may contain one, two, or three amino acid substitutions, and / or each LCDR3 may contain one, two, or three amino acid substitutions.

[0017] In other words, the first aspect relates to an antibody or its antigen-binding fragment, where, The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 3, the HCDR2 amino acid sequence of SEQ ID NO: 4, the HCDR3 amino acid sequence of SEQ ID NO: 5, the LCDR1 amino acid sequence of SEQ ID NO: 6, the LCDR2 amino acid sequence of SEQ ID NO: 7, and the LCDR3 amino acid sequence of SEQ ID NO: 8. The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 23, the HCDR2 amino acid sequence of SEQ ID NO: 24, the HCDR3 amino acid sequence of SEQ ID NO: 25, the LCDR1 amino acid sequence of SEQ ID NO: 26, the LCDR2 amino acid sequence of SEQ ID NO: 27, and the LCDR3 amino acid sequence of SEQ ID NO: 28; or · The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 43, the HCDR2 amino acid sequence of SEQ ID NO: 44, the HCDR3 amino acid sequence of SEQ ID NO: 45, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 47, and the LCDR3 amino acid sequence of SEQ ID NO: 48; or · The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 63, the HCDR2 amino acid sequence of SEQ ID NO: 64, the HCDR3 amino acid sequence of SEQ ID NO: 65, the LCDR1 amino acid sequence of SEQ ID NO: 66, the LCDR2 amino acid sequence of SEQ ID NO: 67, and the LCDR3 amino acid sequence of SEQ ID NO: 68; or · The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 83, the HCDR2 amino acid sequence of SEQ ID NO: 84, the HCDR3 amino acid sequence of SEQ ID NO: 85, the LCDR1 amino acid sequence of SEQ ID NO: 86, the LCDR2 amino acid sequence of SEQ ID NO: 87, and the LCDR3 amino acid sequence of SEQ ID NO: 88; or · The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 103, the HCDR2 amino acid sequence of SEQ ID NO: 104, the HCDR3 amino acid sequence of SEQ ID NO: 105, the LCDR1 amino acid sequence of SEQ ID NO: 106, the LCDR2 amino acid sequence of SEQ ID NO: 107, and the LCDR3 amino acid sequence of SEQ ID NO: 108. Or · The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 123, the HCDR2 amino acid sequence of SEQ ID NO: 124, the HCDR3 amino acid sequence of SEQ ID NO: 125, the LCDR1 amino acid sequence of SEQ ID NO: 126, the LCDR2 amino acid sequence of SEQ ID NO: 127, and the LCDR3 amino acid sequence of SEQ ID NO: 128, each HCDR1 may contain 1, 2, or 3 amino acid substitutions, each HCDR2 may contain 1, 2, or 3 amino acid substitutions, each HCDR3 may contain 1, 2, or 3 amino acid substitutions, each LCDR1 may contain 1, 2, or 3 amino acid substitutions, each LCDR2 may contain 1, 2, or 3 amino acid substitutions, and / or each LCDR3 may contain 1, 2, or 3 amino acid substitutions.

[0018] In other words, the first aspect relates to an antibody or an antigen-binding fragment thereof, where · the antibody or an antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 1 and a LCVR having the amino acid sequence of SEQ ID NO: 2. Or · the antibody or an antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 21 and a LCVR having the amino acid sequence of SEQ ID NO: 22. Or · the antibody or an antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 41 and a LCVR having the amino acid sequence of SEQ ID NO: 42. Or · the antibody or an antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 61 and a LCVR having the amino acid sequence of SEQ ID NO: 62. Or · the antibody or an antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 81 and a LCVR having the amino acid sequence of SEQ ID NO: 82; or · the antibody or an antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 101 and a LCVR having the amino acid sequence of SEQ ID NO: 102; or · the antibody or an antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 121 and a LCVR having the amino acid sequence of SEQ ID NO: 122, each HCVR may contain 1, 2, or 3 amino acid substitutions, and / or each LCVR may contain 1, 2, or 3 amino acid substitutions.

[0019] In a second aspect, the invention relates to a combination of two different antibodies or antigen-binding fragments thereof, e.g., a composition comprising at least two antibodies or antigen-binding fragments thereof. Preferably, the composition comprises three antibodies, e.g., at least three antibodies, and optionally, the composition comprises four antibodies or antigen-binding fragments thereof, where the antibodies and antigen-binding fragments thereof are selected from the antibodies or antigen-binding fragments thereof described herein, particularly selected from the group of seven antibodies or antigen-binding fragments thereof: 2E02, 2C10, 2B04, 2E0, A07, B10, and 2_10.

[0020] In a preferred embodiment, the composition comprises two antibodies or antigen-binding fragments according to the present invention.

[0021] In another preferred embodiment, the composition comprises three antibodies or antigen-binding fragments according to the present invention.

[0022] In a third embodiment, the present invention relates to a pharmaceutical composition comprising one or more antibodies selected from antibodies or antigen-binding fragments as defined herein, or a therapeutically effective amount thereof, together with one or more pharmaceutically acceptable excipients.

[0023] In a preferred embodiment, the present invention relates to a pharmaceutical composition comprising three antibodies or antigen-binding fragments selected from antibodies or antigen-binding fragments as defined herein, together with one or more pharmaceutically acceptable excipients. [Brief explanation of the drawing]

[0024] Legend for the figure [Figure 1] Figure 1 shows the epitope binning matrix of selected antibodies against Bet v 1. Black squares indicate that the mAb does not bind pairwise to Bet v 1. White squares indicate that the mAb binds pairwise to Bet v 1. * = Epitope bins representing mAbs 1_4 and 1_11. ** = Epitope bins representing mAbs 1_5, 2_6, 2_7, 2_11, 2_13, and 2_18. [Figure 2] Figure 2 shows an overview of the antibodies obtained as described in the examples. Each ring represents one of the antibodies in Table 1. Overlapping rings indicate that the antibody binds to overlapping epitopes on Bet v 1, while non-overlapping rings indicate that the antibody binds to different, non-overlapping epitopes. [Figure 3]Figure 3 shows the sensorgram when three mAbs are sequentially coupled to Bet v 1. 0-60 sec: baseline. 60-480 sec: mAb A07 loaded onto Protein A sensor chip. 480-510 sec: baseline. 510-810 sec: blocking mAb. 810-840 sec: baseline. 840-1140 sec: Bet v 1. 1140-1200 sec: baseline. 1200-1500 sec: mAb A07. 1500-1560 sec: baseline. 1560-1860 sec: mAb 2B04. 1860-1910 sec: baseline. 1910-2210 sec: mAb 2E02. 2210-2270 sec: baseline. [Figure 4-1] Figure 4 shows the inhibition of basophil activation by nBet v 1 stimulation. A mixture containing 4 nM mAbs was pre-incubated with nBet v 1 at concentrations shown on the X axis and then added to PBMCs. Basophil activation was measured by flow cytometry as described in Example 5. Activated basophils are shown on the Y axis as the percentage of CD63-positive cells. Data from six donors (Figure 4A-F) are shown. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1] Figure 5 shows the inhibition of basophil activation by nAln g 1 stimulation. A mixture containing 4 nM mAbs was pre-incubated with nAln g 1 at the concentrations shown on the X axis and then added to PBMCs. Basophil activation was measured by flow cytometry as described in Example 5. Activated basophils are shown on the Y axis as the percentage of CD63-positive cells. Data from six donors (Figure 5A-F) are shown. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6-1]Figure 6 shows the inhibition of basophil activation by nCor a 1 stimulation. Mixtures containing 4 nM mAbs each were pre-incubated with different concentrations of nCor a 1, shown on the X axis, before being added to PBMCs. Basophil activation was measured by flow cytometry as described in Example 5. Activated basophils are shown on the Y axis as the percentage of CD63-positive cells. Data from six donors (Figure 6A-F) are shown. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 7-1] Figure 7 shows the inhibition of basophil activation by nQue a 1 stimulation. Mixtures containing 4 nM mAbs each were pre-incubated with different concentrations of nQue a 1, shown on the X axis, before being added to PBMCs. Basophil activation was measured by flow cytometry as described in Example 5. Activated basophils are shown on the Y axis as the percentage of CD63-positive cells. Data from six donors (Figures 7A-F) are shown. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 8-1] Figure 8 shows the inhibition of basophil activation by stimulation with nBet v 1, nAln g 1, nCor a 1, or nQue a 1. Allergens were pre-incubated with mAbs before PBMC stimulation. Basophil activation was measured by flow cytometry as described in Example 5. The inhibition rate against activated basophils in the absence of mAbs is shown on the Y axis. [Figure 8-2] Same as above. [Figure 9-1] Figure 9 shows the inhibition of basophil activation by stimulation with nBet v 1, nAln g 1, nCor a 1, or nQue a 1. Allergens were pre-incubated with mAbs before PBMC stimulation. Basophil activation was measured by flow cytometry as described in Example 5. The inhibition rate against activated basophils in the absence of mAbs is shown on the Y axis. [Figure 9-2] Same as above. [Modes for carrying out the invention]

[0025] Detailed disclosure of the present invention definition allergens In this invention, "allergen" refers to a compound that is recognized as a foreign substance by the immune system and triggers an immune response against it. In other words, it refers to a molecule that does not exist in the human body but may trigger an abnormal immune response and induce allergic symptoms through the activation of mast cells and basophils via IgE. Those skilled in the art will understand that in this invention, "autoantigens," i.e., molecules produced by the human body, are not considered allergens. Examples of allergens have been reported by the World Health Organization (WHO) and the Allergen Nomenclature Subcommittee of the International Union of Immunology (IUIS), and can be viewed at the web URL: http: / / allergen.org / .

[0026] antibody As used herein, the term “antibody” means any antigen-binding molecule or molecular complex that includes at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., Bet v 1). As used herein, the term “antibody” refers to an immunoglobulin molecule (i.e., a “complete antibody molecule”) consisting of four polypeptide chains, each consisting of two heavy chains (H chains) and two light chains (L chains) interconnected by disulfide bonds, as well as their polymers (e.g., IgM) or antigen-binding fragments. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (consisting of domains CH1, CH2, and CH3). Each light chain consists of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions are further subdivided into a highly variable region called the complementarity-determining region (CDR) and a more conserved region called the framework region (FR). Each VH and VL region consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. In certain embodiments of the present invention, the FRs of the antibody (or its antigen-binding fragment) may be identical to the human germline sequence, or they may be naturally or artificially modified. The amino acid consensus sequence may be defined based on parallel analysis of two or more CDRs.

[0027] Antibody variant An "antibody variant" is a protein derived from an antibody that possesses the same binding specificity as the antibody, but is not a naturally occurring product in mammals. Therefore, this term refers not only to various fragments of antibodies but also to artificial antibody analog forms. Furthermore, this term also refers to antibody forms that exist in nature but are not common in mammals, such as heavy chain antibodies and IgY found in birds and reptiles. These antibody forms are created by manipulating the CDR of mammalian antibodies or combinatorially produced antibodies into an antibody form different from its original origin.

[0028] antigen binding fragment "Antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.

[0029] Examples of antibody-binding fragments include Fab fragments (monovalent fragments consisting of VL, VH, CL, and CHI domains), F(ab’)2 fragments (bivalent fragments consisting of two Fab fragments linked by a disulfide bond in the hinge region), single-chain Fv (scFv), disulfide-bonded Fv (dsFv), complementarity-determining regions (CDR), VL (light-chain variable region), VH (heavy-chain variable region), sdAbs (single-domain antibodies), and any combination thereof, or other functional portions of immunoglobulin peptides capable of binding to a target antigen, but are not limited thereto. Antibodies and their antigen-binding sites include domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, sdAbs (e.g., monovalent sdAbs, bivalent sdAbs, etc.), small modular immunopharmaceuticals (SMIP), and shark variable IgNAR domains.

[0030] Binding affinity The term "binding affinity" refers to the affinity of a test compound (such as an antibody or its antigen-binding fragment disclosed herein) for binding to a specific allergen. The ability of a test compound to bind to a specific allergen may be tested using the Biolayer Interference (BLI) method described herein. Briefly, this assay measures the binding and dissociation of a test molecule to a specific allergen. Based on these measurements, k on , k off , the ratio k off / k on (=K D ) may be determined. k on is a constant representing the rate at which a test molecule binds to an allergen, and k off represents the rate at which a test molecule dissociates from an allergen. The ratio k off / k on is the equilibrium dissociation constant K D . K DThe lower the value, the higher the affinity of the test molecule to the allergen. Generally, K values ​​in the sub-nanometer range, especially in the sub-nM affinity range of 1 to 10 nM, are considered to be high. D The value may be desirable. Other IgE affinity binding assays can also be used and are known to those skilled in the art.

[0031] Blocking / neutralizing antibodies As used herein, “blocking antibody” or “neutralizing antibody” (or “antibody that neutralizes the activity of an allergen”) refers to an antibody or its antigen-binding portion that binds to one of the allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1 and inhibits at least one biological activity of that allergen. For example, the antibody of the present invention may help prevent a primary allergic reaction caused by Bet v 1. Alternatively, the antibody of the present invention may exhibit the ability to prevent a secondary allergic reaction caused by Bet v 1, or the ability to reduce, decrease, or inhibit at least one symptom of an allergic reaction caused by Bet v 1, including sneezing, coughing, asthma, or anaphylactic reaction caused by Bet v 1. The inhibition of the biological activity of Bet v 1 can be evaluated by measuring one or more indicators of Bet v 1 biological activity by one or more of several standard in vitro or in vivo assays (such as the passive cutaneous anaphylaxis assay described herein) or by other in vivo assays known in the art.

[0032] control antibody "Control antibody" refers to the antibody used in the laboratory as a comparison target for the antibody of the present invention. Specifically, the three antibodies REGN5713, REGN5714, and REGN5715, described in WO 2018 / 222854, are used as control antibodies. The VH and VL amino acid sequences of each comparison antibody are shown below. [ka] [ka]

[0033] Epitope The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site (called a paratope) in the variable region of an antibody molecule. A single antigen may have multiple epitopes. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" may also refer to a site on an antigen to which B cells and / or T cells react. It may also refer to a region of an antigen to which an antibody binds. Epitopes may be either linear or conformal. Linear epitopes are formed by adjacent amino acid residues in a polypeptide chain. Conformal epitopes are formed by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. In certain embodiments, an epitope may include a determinant that is a chemically active surface substrate of a molecule, such as an amino acid, sugar side chain, phosphorylated group, or sulfonyl group, and in certain embodiments may have specific three-dimensional structural properties and / or specific charge properties. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes formed from consecutive amino acids are usually retained by exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents.

[0034] full length antibody As used herein, the term "full-length antibody" refers to an antibody (e.g., parental or mutant antibody) that contains all the constant and variable heavy and light chain domains corresponding to those typically found in the wild-type antibody of the isotype in question.

[0035] Human-derived In the present invention, the term "human-derived" means that at least the CDR of the antibody is derived from a human antibody or a humanized transgenic animal, but a "human-derived" antibody may contain further elements that are different from the human antibody or humanized transgenic animal antibody from which the CDR is derived. Examples include part or the entire framework of the heavy chain and / or light chain variable region, and / or part or all of the heavy chain and / or light chain constant region. For example, the variable region, part thereof, or CDR may be derived from a human IgE antibody and transplanted onto the backbone of an IgG or IgA antibody, such as a human IgG or IgA antibody. The peptide sequence of a human-derived antibody may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identical to the sequence of an antibody extracted from a human.

[0036] Preferably, the peptide sequence of the human-derived antibody may be at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the sequence of the antibody extracted from a human.

[0037] IMGT In this invention, the term "IMGT" refers to a method for determining the CDR region. Here, the amino acid residues of the immunoglobulin monovariate domain can be numbered using conserved amino acids that always have the same position. For example, cysteine ​​23, tryptophan 41, leucine 89, and cysteine ​​104. FR1 of VHH consists of amino acid residues 1-26, CDR1 consists of amino acid residues 27-38, FR2 consists of amino acid residues 39-55, CDR2 consists of amino acid residues 56-65, FR3 consists of amino acid residues 66-104, CDR3 consists of amino acid residues 105-117, and FR4 consists of amino acid residue 118 and the remaining sequence. The maximum length of the CDR is as described above. If the CDR is shorter than this, a gap is created (Lefranc et al., 2002).

[0038] multispecific antibodies In this invention, the term "multispecific antibody or multispecific antigen-binding fragment" refers to an antibody or antigen-binding fragment that can bind to at least two different epitopes on different antigens or on the same antigen. Conventional monospecific antibodies typically have two identical epitope-binding sites (paratopes), one in each Fab region, and are therefore specific to only one epitope (two paratopes and one specificity). A multispecific antibody or multispecific antigen-binding fragment means that it has at least two different types of paratopes and can bind to at least two different types of epitopes.

[0039] In particular, a multispecific antibody may contain two or more paratopes, one or more of which are identical, so that all paratopes in the construct belong to at least two different types of paratopes, and therefore the antibody may have at least two specificities. For example, a multispecific antibody may contain four paratopes, two of which are identical and thus have the same specificity. Therefore, a multispecific antibody is bispecific and tetravalent. Thus, a monospecific antibody refers to one or more paratopes having the same specificity. Bispecificity means an antibody that has one, two, three, or other paratopes, but has a total of two specificities. For example, a bispecific antibody may have one paratope for each specificity. In another embodiment, a multispecific antibody may have two or more paratopes for each specificity.

[0040] Therefore, in one embodiment, the multispecific antibody may be bispecific, triplicate, or quadruplicate.

[0041] Paratope The term "paratope" refers to the antigen-binding (or epitope-binding) site of an antibody.

[0042] Recombinant host cells Where used herein, the term “recombinant host cell” (or “host cell”) is intended to refer to a cell into which an expression vector has been introduced. It should be understood that these terms refer not only to specific target cells but also to the offspring of such cells. Such offspring may not be identical to the parent cells in practice, as certain modifications may occur in subsequent generations due to mutation or environmental influences, but they are still included in the scope of the term “host cell” where used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK-293 cells, PER.C6 cells, NSO cells, and lymphocytes; prokaryotic cells such as Escherichia coli; and other eukaryotic hosts such as plant cells and fungi.

[0043] Therapeutic effective dose The term "therapeutic dose" refers to the amount of medication administered that produces the desired effect. The exact amount depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0044] Tree pollen allergen. In this specification, the term “tree pollen allergens” means allergens derived from at least birch, alder, hazel, and / or oak pollen. Of these tree pollen allergens, the most clinically relevant are the major allergens Bet v 1, Aln g 1, Cor a 1, and Que a 1. The prefix “n” preceding the allergen name (e.g., nBet v 1) indicates that the allergen is in a naturally occurring form (including isoforms and various natural post-translational derivatizations (e.g., glycosylation)). Natural allergens may also be indicated with the prefix (n) to distinguish them from recombinant allergens (e.g., nBet v 1 and rBet v 1) which are preceded by the prefix (r). Furthermore, the term “natural allergens” is used to refer to allergens purified from naturally derived raw materials.

[0045] As used herein, the term “tree pollen allergens” typically refers to the group of allergens including Bet v 1, Aln g 1, Cor a 1, and Que a 1.

[0046] vector As used herein, the term “vector” refers to a nucleic acid molecule capable of inducing the transcription of a nucleic acid segment ligated to a vector. One type of vector is a “plasmid,” which takes the form of a circular double-stranded DNA loop. Another type of vector is a viral vector, in which the nucleic acid segment may be ligated to a viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication or episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), upon introduction into a host cell, are integrated into the host cell's genome and replicate together with the host genome. Furthermore, certain vectors can induce the expression of functionally ligated genes. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology often take the form of plasmids. Hereinafter, “plasmid” and “vector” may be used interchangeably, as plasmids are the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, adeno-associated viruses).

[0047] Short sequences not listed in the WIPO Standard 26-compliant sequence listing, which constitute part of the description. The following sequences are not listed in the sequence list because they are too short. [Table A]

[0048] Specific Embodiments of the Invention antibody According to a first aspect of the present invention, an antibody or antigen-binding fragment thereof is provided that can bind to one or more tree pollen allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1. Thus, a single antibody may be specific to one, two, three, or four allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1. Each antibody described herein, either alone or in combination of two, three, four, or more antibodies, may promote the elimination of one, two, three, or any tree pollen allergen selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1, or block the binding of one, two, three, or any tree pollen allergen selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1. The present invention reduces the amount of IgE already formed in the subject, thereby decreasing the activation of mast cells and basophils. In an interesting embodiment, an antibody, or its antigen-binding fragment, or a combination of antibodies or a combination of its antigen-binding fragments may reduce or prevent one or more symptoms in a subject who is allergic to or sensitized to one or more tree pollen allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1.

[0049] In a preferred embodiment, at least the CDR region is derived from a human antibody, such as a human IgE antibody, IgG1 antibody, or IgG4 antibody. Therefore, the CDR region means that it has 100% amino acid sequence alignment with the originally detected human antibody. In a more preferred embodiment, the antibody disclosed herein preferably has a full-length human IgG skeleton, such as IgG1 or IgG4, but can comprise any Fc portion of a human antibody, such as IgE-Fc, IgG-Fc, or IgA-Fc. The hinge region may contain the S228P mutation to avoid Fab arm replacement. Thus, the antibody disclosed herein may be a complete human antibody, but may optionally contain a small number of amino acid substitutions, deletions, or additions, such as one, two, or three, to obtain an antibody with desirable properties, such as extended half-life. In an alternative embodiment, the antibody may comprise only the antigen-binding portion, e.g., Fab.

[0050] In one embodiment, the antibody or its antigen-binding fragment can bind to at least two allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1, for example, it can bind to each of the allergens Bet v 1 and Aln g 1. Preferably, the antibody or its antigen-binding fragment can bind to at least three allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1, for example, it can bind to each of the allergens Bet v 1, Aln g 1, and Cor a 1, and more preferably, the antibody or its antigen-binding fragment can bind to each of the allergens Bet v 1, Aln g 1, Cor a 1, and Que a 1.

[0051] In another embodiment, the antibody or its antigen-binding fragment is an allergen Bet v 1 with affinity of 1 nM or less; and / or Aln g 1 with affinity of 1 nM or less; and / or • Cor a 1 with affinity of 300 nM or less; and / or • Que a 1 with affinity of 300 nM or less It can be combined with.

[0052] Affinity may be determined by a biolayer interference (BLI) assay.

[0053] In a preferred embodiment, the Fc portion is IgG4-Fc.

[0054] The affinity of each antibody or its antigen-binding fragment is used to represent the binding strength to the target (here, binding to the tree pollen allergen described herein), and in a preferred embodiment, it may be less than 1000 nM, e.g., 300 nM, e.g., 100 nM, e.g., 10 nM, preferably 1 nM, more preferably 0.1 nM, and even more preferably 0.01 nM.

[0055] The present invention relates to an antibody or its antigen-binding fragment capable of binding to an allergen selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1.

[0056] In one embodiment, the antibody or its antigen-binding fragment is capable of binding to at least two allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1, for example, it can bind to allergens Bet v 1 and Aln g 1, respectively. Preferably, the antibody or its antigen-binding fragment is capable of binding to at least three allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1, for example, it can bind to allergens Bet v 1, Aln g 1, and Cor a 1, and more preferably, the antibody or its antigen-binding fragment can bind to allergens Bet v 1, Aln g 1, Cor a 1, and Que a 1. In another embodiment, the antibody or its antigen-binding fragment is capable of binding to allergens Bet v 1 with affinity of 1 nM or less; and / or Aln g 1 with affinity of 1 nM or less; and / or • Cor a 1 with affinity of 300 nM or less; and / or • Que a 1 with affinity of 300 nM or less It can be combined.

[0057] Affinity may be measured by a BLI assay.

[0058] In a further embodiment, the antibody or its antigen-binding fragment can bind to the allergen Bet v 1 with an affinity of 1000 nM or less. For example, 300 nM or less, 100 nM or less, or 10 nM or less.

[0059] In another embodiment, the antibody or its antigen-binding fragment can bind to the allergen Aln g 1 with an affinity of 1000 nM or less, for example, 300 nM or less, for example, 100 nM or less, for example, 10 nM or less.

[0060] In another embodiment, the antibody or its antigen-binding fragment can bind to the allergen Cor a 1 with an affinity of 1000 nM or less, for example, 300 nM or less, for example, 100 nM or less, for example, 10 nM or less.

[0061] In another embodiment, the antibody or its antigen-binding fragment can bind to the allergen Que a 1 with an affinity of 1000 nM or less, for example, 300 nM or less, for example, 100 nM or less, for example, 10 nM or less.

[0062] In yet another embodiment, the antibody or its antigen-binding fragment can bind to each of the allergens Bet v 1, Aln g 1, Cor a 1, and Que a 1 with an affinity of 1000 nM or less, for example 300 nM or less, for example 100 nM or less, for example 10 nM or less.

[0063] In one embodiment, the antibody or its antigen-binding fragment can bind to the allergen Bet v 1 with an affinity of 1 nM or less.

[0064] In one embodiment, the antibody or its antigen-binding fragment can bind to the allergen Aln g 1 with an affinity of 1 nM or less.

[0065] In another embodiment, the antibody or its antigen-binding fragment can bind to the allergen Cor a 1 with an affinity of 300 nM or less.

[0066] In a further embodiment, the antibody or its antigen-binding fragment can bind to the allergen Que a 1 with an affinity of 300 nM or less.

[0067] As mentioned above, affinity may be measured by BLI.

[0068] Where applicable, antibodies or their antigen-binding fragments may be obtained from humans. Humans from whom antibodies or their antigen-binding fragments are extracted may be selected from those suffering from birch pollen allergy, hazel pollen allergy, alder pollen allergy and / or oak pollen allergy, those sensitized to Bet v 1, Que a 1, Aln g 1 and / or Cor a 1, preferably those sensitized who do not have clinically relevant allergic symptoms; or those who have undergone allergen-specific immunotherapy for birch pollen allergy, hazel pollen allergy, alder pollen allergy and / or oak pollen allergy.

[0069] Therefore, in a preferred embodiment, the antibody of the present invention is a human antibody.

[0070] Therefore, in one embodiment, the antibody is of human origin, such as a human antibody, and its antigen-binding fragment is derived from a human amino acid sequence.

[0071] In another embodiment, the antibody is a human antibody, and its antigen-binding fragment contains a human amino acid sequence.

[0072] In some embodiments, the antibody is derived from a human sensitized to Bet v 1 and / or Cor a 1 and / or Aln g 1 and / or Que a 1. In one embodiment, the antibody is derived from a human sensitized to Bet v 1. In another embodiment, the antibody is derived from a human sensitized to Cor a 1. In a further embodiment, the antibody is derived from a human sensitized to Aln g 1. In yet another embodiment, the antibody is derived from a human sensitized to Que a 1.

[0073] The allergen-binding site of each antibody of the present invention consists of six individual CDR regions (three in the heavy chain variable region (HCVR) and three in the light chain variable region (LCVR)), which together are called the "fab" domain. Furthermore, the fab domain may be linked to a constant FC region.

[0074] Table 1 shows non-restrictive exemplary antibodies defined by the fab region. Table 1 shows the full-length amino acid sequences of the individual HCVRs and LCVRs of each antibody, which, when combined, constitute the fab region. [Table 1]

[0075] Therefore, one embodiment of the present invention relates to an antibody or its antigen-binding fragment selected from the group consisting of 2E02, 2C10, 2B04, 2E07, A07, B10, and 2_10, preferably selected from the group consisting of 2E02, 2C10, 2B04, and 2E07.

[0076] In an embodiment of the first aspect of the present invention, the antibody or its antigen-binding fragment is SEQ ID NO: 1 and 2 (HCVR and LCVR of antibody 2E02, respectively); 21 and 22 (HCVR and LCVR of antibody 2C10, respectively); 41 and 42 (HCVR and LCVR of antibody 2B04, respectively). 61 and 62 (HCVR and LCVR of antibody 2E07, respectively); 81 and 82 (HCVR and LCVR of antibody A07, respectively); 101 and 102 (HCVR and LCVR of antibody B10, respectively); or The combination of HCVR and LCVR has amino acid sequences 121 and 122 (HCVR and LCVR of antibody 2_10, respectively), where each amino acid sequence may have 1, 2, or 3 amino acid substitutions.

[0077] In further embodiments, each combination of HCVR and LCVR listed in Table 1 is linked to an FC domain, preferably an IgG-Fc domain, and more preferably IgG4-Fc.

[0078] Since such antibodies may undergo affinity maturation, humanization, or other amino acid changes in their amino acid sequence, further embodiments include the sequence number. 1 and 2; 21 and 22; 41 and 42; 61 and 62; 81 and 82; 101 and 102; or 121 and 122 This relates to antibodies having an amino acid sequence that is at least 80%, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, identical to an amino acid sequence selected from the group consisting of the above.

[0079] Therefore, further embodiments of the first aspect of the present invention relate to an antibody or an antigen-binding fragment thereof, wherein the antibody or fragment comprises HCVR and LCVR, The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 2 (HCVR and LCVR of antibody 2E02, respectively), and each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 21 and LCVR having the amino acid sequence of SEQ ID NO: 22 (HCVR and LCVR of antibody 2C10, respectively), and each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42 (HCVR and LCVR of antibody 2B04, respectively), and each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 61 and LCVR having the amino acid sequence of SEQ ID NO: 62 (HCVR and LCVR of antibody 2E07, respectively), and each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82 (HCVR and LCVR of antibody A07, respectively), and each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 101 and LCVR having the amino acid sequence of SEQ ID NO: 102 (HCVR and LCVR of antibody B10, respectively), and each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 121 and LCVR having the amino acid sequence of SEQ ID NO: 122 (HCVR and LCVR of antibody 2_10, respectively), and each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or In a preferred embodiment, the antibody or its antigen-binding fragment comprises HCVR and LCVR defined for an antibody selected from the group consisting of 2E02, 2C10, 2B04, 2E07, and A07.

[0080] Antibodies defined by the CDR region Those skilled in the art know that each variable domain in an antibody or its antigen-binding region, including the HCVR and LCVR, consists of three complementarity-determining regions (CDRs) flanked by four framework regions (FR1-FR4).

[0081] In this specification, the antibody binding region means that it includes one or all of the six complementarity-determining regions, three of which are located on the HCVR (HCDR1, HCDR2, and HCDR3) and three of which are located on the LCVR (HCDR1, HCDR2, and HCDR3).

[0082] The primary purpose of the CDR region is to define regions that exhibit specificity for epitopes. However, not all residues within the CDR are involved in epitope binding; they merely determine structural elements (Wilton E. et al 2018, Reference 6). Furthermore, the framework region is not the only one responsible for structure. Residues in the FR adjacent to the CDR may also influence binding.

[0083] Various methods can be applied to determine the locations of the various framework regions and CDR regions of an antibody.

[0084] One method is the "Kabat numbering system" or "Kabat," first proposed by American scientist Elvis Kabat. In this method, amino acid residues of immunoglobulin single variable domains are numbered according to the general numbering of VH domains shown by Kabat et al. (Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91).

[0085] As is well known in the art, the total number of amino acid residues in each CDR may vary and may not coincide with the total number of amino acid residues indicated by the Kabat number. That is, one or more positions indicated by the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number. This generally means that the numbers indicated by the Kabat number may or may not coincide with the actual numbers of amino acid residues in the actual sequence. The total number of amino acid residues in an HCVR domain is usually in the range of 110 to 120, and often between 112 and 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described in this specification.

[0086] Another method for determining the CDR region of HCVR and LCVR is to use the Chothia numbering scheme. In this method, amino acid residues of the immunoglobulin monovariate domain can be numbered using conserved amino acids that always occupy the same position (Dondelinger M et al. frontiers in immunology 2018).

[0087] Another method for determining the CDR region of HCVR and LCVR is to use IMGT. In this method, the amino acid residues of the immunoglobulin monovariate domain can be numbered using conserved amino acids that are always in the same position, such as cysteine ​​23, tryptophan 41, leucine 89, and cysteine ​​104. The FR1 of HCVR and LCVR consists of amino acid residues at positions 1-26, the CDR1 of HCVR and LCVR consists of amino acid residues at positions 27-38, and the FR2 of LCVR consists of amino acid residues at positions 3-4. HCVR and LCVR include amino acid positions 39-55, the CDR2 of HCVR and LCVR includes amino acid positions 56-65, the FR3 of HCVR and LCVR includes amino acid positions 66-104, the CDR3 of HCVR and LCVR includes amino acid positions 105-117, and the FR4 of HCVR and LCVR includes amino acid position 118 and the remaining sequence. The maximum length of the CDR is as described above. If the CDR is short, a gap is created (Lefranc et al 2002 - Developmental and Comparative Immunology).

[0088] Another method for determining the CDR region of an antibody is to use the Aho numbering system (Mitchelle & Colwell, Proteins, 2017 and Honegger & Pluckthun et al 2001).

[0089] Interesting alternative CDR definitions, each specifically incorporated herein by reference, are: Honegger's "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool." J Mol Biol. 2001;309:657-670; Ofran et al.'s "Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes." J Immunol. 2008;181:6230-6235; Almagro's "Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires." J Mol Recognit. 2004;17:132-143; and Padlanet et al.'s "Identification of specificity-determining residues in antibodies." Faseb J. This includes, but is not limited to, 1995;9:133-139.

[0090] As mentioned above, the combination of CDR regions in the antibody determines its specificity. There are a total of six CDRs, three each in the HCVR region and the LCVR region, which can be determined by various methods such as the IMGT method, Kabat method, and Chothia method, as previously described.

[0091] Tables 2a-c show the amino acid sequences of the six CDR regions of each exemplary antibody disclosed in Table 1, determined by the IMGT, Kabat, and Chothia methods. [Table 2a] [Table 2b] [Table 2c]

[0092] Therefore, a first aspect of the present invention also relates to an antibody defined by a CDR region determined, for example, by IMGT. One aspect relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or fragment comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where; The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 3, the HCDR2 amino acid sequence of SEQ ID NO: 4, the HCDR3 amino acid sequence of SEQ ID NO: 5, the LCDR1 amino acid sequence of SEQ ID NO: 6, the LCDR2 amino acid sequence of SEQ ID NO: 7, and the LCDR3 amino acid sequence of SEQ ID NO: 8, and each amino acid sequence may have one, two, or three amino acid substitutions (CDR of antibody 2E02); or The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 23, the HCDR2 amino acid sequence of SEQ ID NO: 24, the HCDR3 amino acid sequence of SEQ ID NO: 25, the LCDR1 amino acid sequence of SEQ ID NO: 26, the LCDR2 amino acid sequence of SEQ ID NO: 27, and the LCDR3 amino acid sequence of SEQ ID NO: 28, and each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody 2C10); or The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 43, the HCDR2 amino acid sequence of SEQ ID NO: 44, the HCDR3 amino acid sequence of SEQ ID NO: 45, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 47, and the LCDR3 amino acid sequence of SEQ ID NO: 48, and each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody 2B04); or The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 63, the HCDR2 amino acid sequence of SEQ ID NO: 64, the HCDR3 amino acid sequence of SEQ ID NO: 65, the LCDR1 amino acid sequence of SEQ ID NO: 66, the LCDR2 amino acid sequence of SEQ ID NO: 67, and the LCDR3 amino acid sequence of SEQ ID NO: 68, and each amino acid sequence may have one, two, or three amino acid substitutions (CDR of antibody 2E07); or The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 83, the HCDR2 amino acid sequence of SEQ ID NO: 84, the HCDR3 amino acid sequence of SEQ ID NO: 85, the LCDR1 amino acid sequence of SEQ ID NO: 86, the LCDR2 amino acid sequence of SEQ ID NO: 87, and the LCDR3 amino acid sequence of SEQ ID NO: 88, and each amino acid sequence may have one, two, or three amino acid substitutions (CDR of antibody A07); or The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 103, the HCDR2 amino acid sequence of SEQ ID NO: 104, the HCDR3 amino acid sequence of SEQ ID NO: 105, the LCDR1 amino acid sequence of SEQ ID NO: 106, the LCDR2 amino acid sequence of SEQ ID NO: 107, and the LCDR3 amino acid sequence of SEQ ID NO: 108, and each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody B10); or The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence of SEQ ID NO: 123, the HCDR2 amino acid sequence of SEQ ID NO: 124, the HCDR3 amino acid sequence of SEQ ID NO: 125, the LCDR1 amino acid sequence of SEQ ID NO: 126, the LCDR2 amino acid sequence of SEQ ID NO: 127, and the LCDR3 amino acid sequence of SEQ ID NO: 128, and each amino acid sequence may have one, two, or three amino acid substitutions (CDR of antibody 2_10).

[0093] In one embodiment, the antibody or its antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) defined for an antibody selected from the group consisting of 2E02, 2C10, 2B04, 2E07, and A07.

[0094] CDR determined by HCVR and LCVR Alternatively, individual CDR regions may be derived from larger sequences determined by different numbering schemes, such as Kabat, Chothia, or IMTG, but not limited to these. Thus, in one embodiment, the antibody or antigen-binding fragment of the present invention comprises a combination of complementarity-determining region 1 (HCDR1), complementarity-determining region 2 (HCDR2), and complementarity-determining region 3 (HCDR3) in the HCVR and LCDR1, LCDR2, and LCDR3 in the LCVR, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise or consist of amino acid sequences determined by Kabat, Chothia, or IMTH, selected from any one of SEQ ID NOs: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121, and 122. Since such sdAb may undergo affinity maturation, humanization, or other amino acid changes of the amino acid sequence, further embodiments relate to monomer sdAb having an amino acid sequence that is at least 80%, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121, and 122.

[0095] In another embodiment, the antibody or antigen-binding fragment of the present invention comprises combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having an amino acid sequence determined by Kabat, Chothia, IMTG, or Aho, selected from any one of SEQ ID NOs: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121, and 122. Preferably, desired effects, such as changes in affinity and / or decreased immunogenicity, may be obtained by substituting one, two, or three amino acids in each CDR region determined by Kabat, Chothia, IMTG, or Aho.

[0096] Therefore, in other words, a first aspect of the present invention relates to an antibody or its antigen-binding fragment comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of an antibody selected from the group consisting of 2E02, 2C10, 2B04, 2E0, A07, B10, and 2_10.

[0097] Therefore, the first aspect also relates to an antibody or its antigen-binding fragment comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where; The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 1, the HCDR2 amino acid sequence contained in SEQ ID NO: 1, the HCDR3 amino acid sequence contained in SEQ ID NO: 1, the LCDR1 amino acid sequence contained in SEQ ID NO: 2, the LCDR2 amino acid sequence contained in SEQ ID NO: 2, and the LCDR3 amino acid sequence contained in SEQ ID NO: 2 (Antibody 2E02); or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 21, the HCDR2 amino acid sequence contained in SEQ ID NO: 21, the HCDR3 amino acid sequence contained in SEQ ID NO: 21, the LCDR1 amino acid sequence contained in SEQ ID NO: 22, the LCDR2 amino acid sequence contained in SEQ ID NO: 22, and the LCDR3 amino acid sequence contained in SEQ ID NO: 22 (Antibody 2C10); or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 41, the HCDR2 amino acid sequence contained in SEQ ID NO: 41, the HCDR3 amino acid sequence contained in SEQ ID NO: 41, the LCDR1 amino acid sequence contained in SEQ ID NO: 42, the LCDR2 amino acid sequence contained in SEQ ID NO: 42, and the LCDR3 amino acid sequence contained in SEQ ID NO: 42 (Antibody 2B04); or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 61, the HCDR2 amino acid sequence contained in SEQ ID NO: 61, the HCDR3 amino acid sequence contained in SEQ ID NO: 61, the LCDR1 amino acid sequence contained in SEQ ID NO: 62, the LCDR2 amino acid sequence contained in SEQ ID NO: 62, and the LCDR3 amino acid sequence contained in SEQ ID NO: 62 (Antibody 2E07); or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 81, the HCDR2 amino acid sequence contained in SEQ ID NO: 81, the HCDR3 amino acid sequence contained in SEQ ID NO: 81, the LCDR1 amino acid sequence contained in SEQ ID NO: 82, the LCDR2 amino acid sequence contained in SEQ ID NO: 82, and the LCDR3 amino acid sequence contained in SEQ ID NO: 82 (Antibody A07); or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 101, the HCDR2 amino acid sequence contained in SEQ ID NO: 101, the HCDR3 amino acid sequence contained in SEQ ID NO: 101, the LCDR1 amino acid sequence contained in SEQ ID NO: 102, the LCDR1 amino acid sequence contained in SEQ ID NO: 102, and the LCDR3 amino acid sequence contained in SEQ ID NO: 102 (Antibody B10); or The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 121, the HCDR2 amino acid sequence contained in SEQ ID NO: 121, the HCDR3 amino acid sequence contained in SEQ ID NO: 121, the LCDR1 amino acid sequence contained in SEQ ID NO: 122, the LCDR2 amino acid sequence contained in SEQ ID NO: 122, and the LCDR3 amino acid sequence contained in SEQ ID NO: 122 (Antibody 2_10).

[0098] Each of the above CDRs (HCDR1, HCDR2, and HCDR3, LCDR1, LCDR2, and LCDR3) may be determined according to the IMGT method, the Kabat method, or the Chothia method.

[0099] Optionally, each CDR region determined by the IMGT, Kabat, or Chothia method may have one, two, or three amino acid substitutions. For example, each HCDR1 may contain one, two, or three amino acid substitutions, each HCDR2 may contain one, two, or three amino acid substitutions, each HCDR3 may contain one, two, or three amino acid substitutions, each LCDR1 may contain one, two, or three amino acid substitutions, each LCDR2 may contain one, two, or three amino acid substitutions, and / or each LCDR3 may contain one, two, or three amino acid substitutions.

[0100] Preferably, the antibody or its antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where these CDRs are derived from antibodies selected from the group consisting of 2E02, 2C10, 2B04, 2E0, A07, B10, and 2_10.

[0101] Modification of the amino acid sequence of an antibody and its antigen-binding fragment. The amino acid sequences of the CDR, or framework regions outside the CDR (including the fab and Fc regions), may be subjected to amino acid substitutions, insertions, or deletions for the purpose of modifying biological activity, expression levels, stability, or other functional properties. In preferred embodiments, amino acid changes are incorporated only outside the CDR region.

[0102] In some embodiments, the antibody of the present invention comprises an affinity-matured human-type or humanized amino acid sequence. In further embodiments, the amino acid sequence of the CDR region does not need to change even if the antibody undergoes affinity maturation or humanization. In such embodiments, the antibody comprises an affinity-matured human-type or humanized amino acid sequence, but the CDR region involved in binding to the target is not affinity-matured.

[0103] In some embodiments, one or more CDRs of the HCVR or LCVR (i.e., CDR1, CDR2, and / or CDR3) may independently undergo amino acid substitutions, for example, by one, two, three, or more amino acid residue substitutions. The amino acid substitutions in the CDRs may be conservative amino acid substitutions. A “conservative” amino acid substitution is generally an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure and / or charge that has little or substantial effect on the function, activity, or other biological properties of the antibody binding domain described herein. Such conservative amino acid substitutions are well known in the art. For example, a conservative substitution is preferably one in which one amino acid residue from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0104] Particularly preferred conservative amino acid substitutions are as follows: Ala to Gly; Ala to Ser; Arg to Lys; Asn to Gln; Asn to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala; Gly to Pro; His to Asn; His to Gln; Ile to Leu; Ile to Val; Leu to To Ile; from Leu to Val; from Lys to Arg; from Lys to Gln; from Lys to Glu; from Met to Leu; from Met to Tyr; from Met to Ile; from Phe to Met; from Phe to Leu; from Phe to Tyr; from Ser to Thr; from Thr to Ser; from Trp to Tyr; from Tyr to Trp; from Phe to Val; from Phe to Ile and / or Phe or Leu.

[0105] Conservative amino acid substitutions may also be performed in amino acid sequences other than CDR.

[0106] In other embodiments, amino acid substitutions in the amino acid sequence of a CDR may be made with the aim of providing an antibody or antigen-binding fragment with altered, for example, increased affinity to a target. The amino acid sequence of a CDR is typically more determinant of affinity to a target than the amino acid sequence outside the CDR. Therefore, the antibodies or antigen-binding fragments described herein may also be subjected to affinity maturation by introducing one or more modifications to the amino acid sequences of one or more CDRs, thereby improving, for example, the affinity of the resulting antibody or antigen-binding fragment to a target compared to the first (parent) antibody or antigen-binding fragment. Methods for affinity maturation of antibodies or antigen-binding fragments are known in the art and are obvious to those skilled in the art.

[0107] In some cases, one or more amino acid substitutions, insertions, or deletions may have been made in framework regions outside the CDR.

[0108] Amino acid substitutions in the amino acid sequence outside the CDR typically result in less alteration of the biological activity of the antibody or its antigen-binding fragment compared to substitutions within the CDR. However, depending on the host organism used to express the antibody or its antigen-binding fragment described herein, any changes in the amino acid sequence of the antibody or its antigen-binding fragment (e.g., deletions, insertions, and / or substitutions) may be designed to improve the level of expression. For example, within the capabilities of those skilled in the art, these changes may be designed to remove one or more sites for post-translational modification (e.g., one or more glycosylation sites). Alternatively, substitutions or insertions may be designed to introduce one or more sites for adding functional groups, for example, to enable the insertion of an affinity tag (His tag) or site-specific PEGylation. The possibility of N-terminal post-translational modification may be eliminated by changing the N-terminal glutamic acid (E) to aspartic acid (D). Thus, the amino acid difference may be changing glutamic acid (Glu) at position 1 (determined according to the Kabat number) to aspartic acid (Asp).

[0109] As mentioned above, the parent CDR region may undergo sequence mutations such as substitutions, deletions, or additions of one, two, or three amino acids. The mutants may have the same, improved, or increased allergen-binding activity.

[0110] mutation Fully human monoclonal antibodies disclosed herein that specifically bind to Bet v 1, Que a 1, Aln g 1, and / or Cor a 1 may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention comprises antibodies derived from any of the amino acid sequences disclosed herein and their antigen-binding fragments, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily construct a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof using the heavy and light chain variable region sequences disclosed herein.

[0111] FC region of antibody As stated above, the antibody fragments (e.g., Fc regions) of the antibodies disclosed herein may be any antibody type (e.g., IgG, IgE, IgM, IgD, and IgA), isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass, including engineered subclasses having a modified Fc portion which may optionally provide reduced or enhanced effector cell activity, or altered in vivo distribution, serum half-life, or excretion rate. The antibody fragments may originate from any animal species, but human-derived Fc is preferred. Exemplary effector functions include C1-q binding; GDC; binding to Fc receptors; ADCC; ADCP; and downregulation of cell surface receptors (e.g., B cell receptors). These effector functions generally require the Fc region to interact with receptors, such as FcγRI, FcγRIIA, FcγRIIBI, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and / or low-affinity FcRn receptors. Furthermore, the Fc region may be "dead" Fc, which is Fc that has been mutated to maintain activity, such as prolonging the serum half-life, but does not activate high-affinity Fc receptors. Also, Fc may have reduced complement binding.

[0112] If it is desirable that Fc does not interact with its receptor (e.g., FcγRIIB), the antibody fragment may be a CH1 immunoglobulin domain (e.g., IgG1-CH1 domain or IgG4-CH1 domain). Unlike conventionally used IgG-FC domains, these domains do not bind to the inhibitory FcγRIIb receptor, do not compete with serum immunoglobulin G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism (Rozan et al., 2013).

[0113] In certain embodiments, the antibody fragment is derived from IgG4, for example, IgG4-Fcm, or for example, human IgG4-Fc.

[0114] Therefore, the Fc portion of the antibody of the present invention may be selected from an IgG4 antibody fragment.

[0115] IgG4 is unstable in vivo due to the "half-antibody exchange" phenomenon, resulting in bispecificity (or, in most cases, a functional monomer). Therefore, for therapeutic purposes, introducing a single amino acid mutation (the so-called S228P mutation) into the hinge region is considered to prevent this dissociation.

[0116] Another method to prevent the in vivo instability of IgG4 may be to apply an IgG4-Fc scaffold in a knob-into-hole (KIH) configuration. This may prevent the dissociation.

[0117] Therefore, in one embodiment, the Fc portion is IgG4 Fc.

[0118] In another embodiment, IgG4 Fc contains the S228P mutation.

[0119] Furthermore, the Fc region may be a natural sequence Fc region containing the same amino acid sequence as a naturally occurring Fc region. Natural sequence human Fc regions include natural sequence human IgG1 Fc regions (non-A and A allotypes), natural sequence human IgG2 Fc regions, natural sequence human IgG3 Fc regions, and natural sequence human IgG4 Fc regions, as well as their natural variants. Alternatively, the Fc region may be a mutant Fc region containing an amino acid sequence different from the amino acid sequence of the natural sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the mutant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, compared to the natural sequence Fc region or the Fc region of the parent polypeptide. The mutated Fc region in this specification may have at least about 80% amino acid sequence homology or sequence identity with the natural sequence Fc region and / or the Fc region of the parent antibody fragment, most preferably at least about 90% homology, and more preferably at least about 95% homology.

[0120] In one embodiment, the mutant Fc sequence may include three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions at EU index positions 330 and 331 in the complement C1-q binding site reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). In human IgG1, substitutions at positions 233–236 of the IgG2 residue and positions 327, 330, and 331 of the IgG4 residue significantly reduce ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; Shields RL. et al., 2001, J Biol Chem. 276(9):6591-604). Other Fc variants are possible, including, but are not limited to, those with deletions of regions capable of forming disulfide bonds, those with specific amino acid residues removed from the N-terminus of native Fc, or those with added methionine residues. Thus, one or more Fc portions of a molecule may contain one or more mutations in the hinge region to remove disulfide bonds. In yet another embodiment, the hinge region of Fc can be completely removed. In yet another embodiment, the molecule may contain Fc variants.

[0121] Furthermore, Fc variants can be constructed by substituting, deleting, or adding amino acid residues that affect complement binding or Fc receptor binding. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[0122] The antibody fragment, for example, the Fc portion, may be modified by substitution, deletion, or insertion of amino acid residues for structural reasons (e.g., expression yield). Non-limiting examples of mutations that can introduce expression yield are described below.

[0123] Antibody fragments may be modified to have a form with native glycans, a form with increased glycans compared to the native form, or a form with decreased glycans compared to the native form, or they may be in an aglycosylated or deglycosylated form. Increase, decrease, removal, or other modification of glycans may be achieved by chemical, enzymatic, or genetically engineered methods, such as expression in producing cell lines. Such cell lines may include microorganisms (e.g., Pichia pastris) and mammalian cell lines (e.g., CHO cells) that naturally express glycosylation enzymes. Furthermore, microorganisms or cells may be engineered to express glycosylation enzymes or to be unable to express them. As an example of cells engineered to alter sialylation activity, the alpha-2,6-sialyltransferase 1 gene has been engineered in Chinese hamster ovary cells and Sf9 cells. Constructs expressed by these engineered cells are sialized by exogenous gene products. Further methods for obtaining Fc molecules with altered sugar residue amounts compared to multiple native molecules include, for example, separating the multiple molecules into glycosylated and non-glycosylated fractions using lectin affinity chromatography. The presence of specific glycosylation sites has been shown to alter the function of immunoglobulins. For example, removing sugar chains from Fc molecules abruptly reduces the binding affinity of the first complement component C1 to the C1-q portion, reducing or eliminating antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thus preventing the induction of unwanted immune responses in vivo. Additional important modifications include sialylation and fucosylation. The presence of sialic acid in IgG correlates with anti-inflammatory activity, and removing fucose from IgG enhances ADCC activity. Constructs enhance the effector function of the Fc sequence, for example, improving its binding ability to FccyRI and increasing ADCC activity. For example, fucose attached to the V-linked glycan of Asn-297 in Fc sterically inhibits the interaction between Fc and FccyRIIIA. Furthermore, removing fucose through glycotechnology strengthens binding to FccyRIIIA, resulting in ADCC activity more than 50 times higher compared to wild-type IgG1 control.Protein engineering through amino acid mutations in the Fc region of IgG1 has generated several variants that enhance the binding affinity between Fc and FccyRIIIA. In particular, the triple alanine mutant S298A / E333A / K334A exhibits a twofold increase in binding to FcγRIIIA and ADCC function.

[0124] The S239D / I332E (2X) and S239D / I332E / A330L(3X) mutants exhibit significantly increased binding affinity to FcγRIIIA and enhanced ADCC activity in vitro and in vivo. Other Fc mutants identified by yeast display also showed improved binding to FcγRIIIA. See, for example, Liu et al. (2014) JBC 289(6):3571-90, which is specifically incorporated herein by reference.

[0125] As described above, antibodies can be manipulated to provide multispecificity to a wider range of different epitopes. Therefore, in some embodiments of the present invention, the antibody is multispecific. A multispecific antibody may comprise two or more antigen-binding fragments selected from among the antigen-binding fragments disclosed herein.

[0126] Antibody composition / combination of the second embodiment According to a second aspect of the present invention, the antibodies or antigen-binding fragments disclosed herein can be combined in a composition comprising two or more antibodies.

[0127] In one embodiment of the present invention, the composition comprises an antibody, and each antibody or antigen-binding fragment can be simultaneously bound to the same Bet v 1 molecule.

[0128] When combining different antibodies, it is important to select antibodies that can simultaneously bind to any of the target allergens, such as Bet v 1 and / or the other three tree pollen allergens. For example, distal epitope binding can be verified by epitope binning studies using BLI. Furthermore, antibodies may be selected based on their ability to bind to all target allergens, such as all four tree pollen allergens described herein.

[0129] In one embodiment, the composition comprises an antibody or antigen-binding fragment that can simultaneously bind to the same Bet v 1 molecule. In one embodiment, the composition comprises an antibody or antigen-binding fragment that can simultaneously bind to the same Que a 1 molecule. In one embodiment, the composition comprises an antibody or antigen-binding fragment that can simultaneously bind to the same Aln g 1 molecule. In one embodiment, the composition comprises an antibody or antigen-binding fragment that can simultaneously bind to the same Cor a 1 molecule.

[0130] In a preferred embodiment of the present invention, the composition comprises an antibody or antigen-binding fragment that can simultaneously bind to the same Bet v 1 and / or Cor a 1, and / or Aln g 1 and / or Que a 1.

[0131] As shown in Figure 2, the antibodies in Table 1 can be grouped according to the epitopes they bind to on allergens. When multiple antibodies bind to distant epitopes on the same target allergen (non-overlapping circles in Figure 2), these antibodies may bind to the target allergen simultaneously and reduce the allergic reaction by blocking the allergen from binding to IgE.

[0132] For example, antibodies 2B04, A07, and 2E02 (shown as bold dotted circles in Figure 2) do not bind to overlapping epitopes, as can be seen from the separation of the circles in Figure 2.

[0133] This also applies to antibodies 2B04, A07, and 2C10, which are shown as bold dotted circles in Figure 2, and these are completely isolated. Further details will be explained in Example 3.

[0134] The number of antibodies capable of blocking one or more target allergens from binding to IgE can be tested using a BLI assay. Typically, a combination of three antibodies is sufficient to effectively block IgE. However, in some cases, the desired effect may be achieved with just two different antibodies.

[0135] Accordingly, a second aspect of the present invention relates to a composition comprising two, for example, at least two antibodies or their antigen-binding fragments. Typically, the composition comprises three, for example, at least three antibodies or their antigen-binding fragments, the antibodies being selected from the antibodies or their antigen-binding fragments as defined herein.

[0136] In one embodiment, the composition comprises four different antibodies or their antigen-binding fragments as defined herein, for example, five different antibodies or their antigen-binding fragments.

[0137] In a preferred embodiment, the composition comprises two or three antibodies. As previously stated, the antibody combination must be able to prevent human-derived IgE with tree pollen allergy from binding to tree pollen allergens. Preferably, the combination must provide sufficient blocking effect against at least Bet v 1. More preferably, the combination must provide blocking effect against one or more, preferably all, tree pollen allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1. Typically, the blocking effect can be determined by performing a basophil activation test using basophils from patients with tree pollen allergy, showing a reduction in IgE binding to any or all of the four tree pollen allergens.

[0138] In some embodiments, the composition comprises two antibodies or antigen-binding fragments having an antigen-binding region such as a CDR contained in an antibody selected from the group consisting of antibodies 2B04, A07, and 2E02, or from the group consisting of antibodies 2B04, A07, and 2C10. In preferred embodiments, the composition comprises two antibodies or antigen-binding fragments having an antigen-binding region such as a CDR contained in an antibody selected from the group consisting of antibodies 2B04, A07, and 2E02, or from the group consisting of antibodies 2B04, A07, and 2C10.

[0139] Furthermore, as shown in Example 5, the combination of two antibodies can highly inhibit the binding of allergens to IgE and, consequently, the activation of basophils. It has also been shown that antibody A07 exhibits high blocking activity in basophil activation tests. Therefore, the compositions described herein may include at least an antibody or its antigen-binding fragment having an antigen-binding region such as a CDR contained in antibody A07.

[0140] Therefore, in one embodiment, the composition comprises a combination of a first antibody and a second antibody, where, The first antibody contains HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 2, and the second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42; or The first antibody contains HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 2, and the second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82; or The first antibody contains HCVR having the amino acid sequence of SEQ ID NO: 21 and LCVR having the amino acid sequence of SEQ ID NO: 22, and the second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42; or The first antibody contains HCVR having the amino acid sequence of SEQ ID NO: 21 and LCVR having the amino acid sequence of SEQ ID NO: 22, and the second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82; or The first antibody contains HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42, and the second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82.

[0141] Antibodies can be defined based on the CDR region, as described above. In the following embodiments, the HCDR and LCDR regions are determined by IMGT.

[0142] Therefore, in other embodiments, the composition comprises a combination of a first antibody and a second antibody, each containing three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where, The first antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 1, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 2; the second antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 42; or The first antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 1, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 2; the second antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 81, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 82; or The first antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22; the second antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 42; or The first antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22; the second antibody contains each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 81, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 82; or The first antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 42. The second antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 81, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 82.

[0143] In another embodiment, the composition includes a combination of three antibodies, such as a composition comprising a combination of a first antibody, a second antibody, and a third antibody, where, The first antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82; The second antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42; and The third antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 2.

[0144] In a further embodiment, the composition comprises three antibodies, The first antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82; The second antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42; and The third antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 121 and LCVR having the amino acid sequence of SEQ ID NO: 122.

[0145] In yet another embodiment, the composition comprises three antibodies, The first antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82. The second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42. The third antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 21 and LCVR having the amino acid sequence of SEQ ID NO: 22.

[0146] In yet another embodiment, the composition comprises three antibodies, The primary antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 101 and LCVR having the amino acid sequence of SEQ ID NO: 102. The second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42. The third antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 2.

[0147] In yet another embodiment, the composition comprises three antibodies, The primary antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 101 and LCVR having the amino acid sequence of SEQ ID NO: 102. The second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42. The third antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 121 and LCVR having the amino acid sequence of SEQ ID NO: 122.

[0148] In yet another embodiment, the composition comprises three antibodies, The primary antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 101 and LCVR having the amino acid sequence of SEQ ID NO: 102. The second antibody contains HCVR having the amino acid sequence of SEQ ID NO: 41 and LCVR having the amino acid sequence of SEQ ID NO: 42. The third antibody comprises HCVR having the amino acid sequence of SEQ ID NO: 21 and LCVR having the amino acid sequence of SEQ ID NO: 22.

[0149] Antibodies can be defined according to the CDR region, as described above. Therefore, in one embodiment, the composition comprises three antibodies. The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 83, 84, and 85, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 86, 87, and 88, respectively. The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively. The third antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 3, 4, and 5, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 6, 7, and 8, respectively.

[0150] In a further embodiment, the composition comprises three antibodies, The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 83, 84, and 85, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 86, 87, and 88, respectively. The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively. The third antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 123, 124, and 125, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 126, 127, and 128, respectively.

[0151] In yet another embodiment, the composition comprises three antibodies, The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 83, 84, and 85, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 86, 87, and 88, respectively. The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively, and The third antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 23, 24, and 25, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 26, 27, and 28, respectively.

[0152] In yet another embodiment, the composition comprises three antibodies, The primary antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 103, 104, and 105, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 106, 107, and 108, respectively. The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively. The third antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 3, 4, and 5, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 6, 7, and 8, respectively.

[0153] In yet another embodiment, the composition comprises three antibodies, The primary antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 103, 104, and 105, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 106, 107, and 108, respectively. The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively. The third antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 123, 124, and 125, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 126, 127, and 128, respectively.

[0154] In yet another embodiment, the composition comprises three antibodies, The first antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 103, 104, and 105, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 106, 107, and 108, respectively; The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NOs. 43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively; and The third antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 23, 24, and 25, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 26, 27, and 28, respectively.

[0155] Alternatively, the compositions described herein include a first antibody, a second antibody, and a third antibody, each comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein, The first antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 2; the second antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 42; the third antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 81, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 82; or The first antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 2; the second antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 42; the third antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 101, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 102; or The first antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 22; the second antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 42; the third antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 81, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 82; or The first antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22; the second antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence of SEQ ID NO: 42; the third antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 101, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 102; or The first antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 42; the second antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 101, and LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 102; and the third antibody contains each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 121, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 122.

[0156] In a further embodiment, the composition includes: • An antibody or antigen-binding fragment comprising HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 2; and One or more isolated human monoclonal antibodies or antigen-binding fragments capable of binding to one or more of the allergens Bet v 1, Aln g 1, Cor a 1, and Que a 1.

[0157] In another embodiment, the composition includes: • An antibody or antigen-binding fragment comprising HCVR having the amino acid sequence of SEQ ID NO: 1 and LCVR having the amino acid sequence of SEQ ID NO: 2; and An antibody or its antigen-binding fragment comprising HCVR having the amino acid sequence of SEQ ID NO: 21 and LCVR having the amino acid sequence of SEQ ID NO: 22.

[0158] Pharmaceutical composition A third aspect of the present invention relates to a pharmaceutical composition comprising one or more antibodies or antigen-binding fragments as defined herein in a therapeutically effective amount and one or more pharmaceutically acceptable excipients. Thus, an antibody according to the first aspect of the present invention or a combination of antibodies according to the second aspect of the present invention can be formulated into a pharmaceutical composition comprising an antibody comprising a multispecific antibody according to the first aspect of the present invention or a combination of antibodies according to the second aspect of the present invention and one or more pharmaceutically acceptable excipients.

[0159] Typically, pharmaceutical compositions are formulated to be administered by injection, such as subcutaneous or intramuscular injection.

[0160] Applications in treatment The antibodies of the present invention can bind to any of the various allergens described herein. Furthermore, as described in the examples, different antibodies can bind to non-overlapping epitopes and thus can bind to individual allergens simultaneously. Therefore, by binding to different allergens, individual antibodies can block IgE binding to specific allergens. By combining the binding of different antibodies, a wider range of allergens can be covered, and IgE blocking can be enhanced.

[0161] Therefore, in one embodiment, the antibody or its antigen-binding fragment according to the present invention can reduce or inhibit the binding of IgE antibodies to Bet v 1 and / or Cor a 1 and / or Aln g 1 and / or Que a 1.

[0162] Since IgE directly links allergens to allergic reactions through the activation of mast cells and basophils, inhibiting IgE binding to allergens effectively prevents cell activation and can be used in the treatment of allergies.

[0163] Accordingly, a fourth aspect of the present invention relates to a method for preventing or mitigating mast cell degranulation associated with Bet v 1 and / or Cor a 1, and / or Aln g 1 and / or Que a 1 sensitization, and / or blocking basophil activation, comprising administering the antibody or antigen-binding fragment thereof described in the first aspect, a combination of antibodies described in the second aspect, or a pharmaceutical composition described in the third aspect to a subject requiring such administration.

[0164] Furthermore, a fourth aspect of the present invention relates to a method for treating tree pollen allergy, comprising administering an antibody or antigen-binding fragment thereof described in the first aspect, a combination of antibodies described in the second aspect, or a pharmaceutical composition described in the third aspect to a subject in need thereof.

[0165] Furthermore, a fourth aspect of the present invention relates to using the antibody or antigen-binding fragment thereof described in the first aspect, the combination of antibodies described in the second aspect, or the pharmaceutical composition described in the third aspect, in a method for treating tree pollen allergy in a subject requiring such treatment.

[0166] Furthermore, a fourth aspect of the present invention relates to using the antibody or antigen-binding fragment thereof described in the first aspect, the combination of antibodies described in the second aspect, or the pharmaceutical composition described in the third aspect to manufacture a pharmaceutical for use in a method of treating tree pollen allergy in a subject requiring such treatment.

[0167] It should be understood that tree pollen allergies may be selected from a group consisting of allergies to birch pollen, alder pollen, hazel pollen, and oak pollen. It should also be understood that tree pollen allergies may be associated with allergies or sensitization to one or more allergens of Bet v 1, Cor a 1, Aln g 1, and Que a 1.

[0168] The target population may be susceptible to Bet v 1. That is, the patient may have detectable IgE antibodies against Bet v 1, for example, IgE antibodies with a serum IgE level greater than 0.7 kU.

[0169] Another embodiment relates to a method for treating a subject that is sensitive to Aln g 1, the method comprising administering one or more antibodies according to the present invention, antibody compositions described herein, or pharmaceutical compositions.

[0170] One embodiment relates to a method for treating a subject that exhibits susceptibility to Cor a 1, the method comprising administering one or more antibodies according to the present invention, antibody compositions described herein, or pharmaceutical compositions.

[0171] One embodiment relates to a method for treating a subject exhibiting susceptibility to Que a 1, the method comprising administering one or more antibodies, antibody compositions, or pharmaceutical compositions described herein according to the present invention.

[0172] In a preferred embodiment, the subject is a human.

[0173] Expression system The antibodies or antigen-binding fragments described herein can be obtained by several different methods. One option is to use host cells having an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding fragment described herein.

[0174] In this regard, those skilled in the art will readily understand that a polynucleotide encoding at least the variable domains of the light chain and / or heavy chain may encode the variable domains of both immunoglobulin chains or only one of them. Similarly, the polynucleotides may be under the control of the same promoter or may be regulated separately.

[0175] Furthermore, the present invention relates to conventionally used vectors in genetic engineering, particularly plasmids, cosmids, viruses, and bacteriophages, comprising a polynucleotide encoding the antigen of the antibody of the present invention, or preferably a variable domain of the immunoglobulin chain; optionally used in combination with the polynucleotide of the present invention encoding the variable domain of the other immunoglobulin chain of the antibody of the present invention. Preferably, the vector is an expression vector and / or a gene transfer vector or a targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses may be used to deliver the polynucleotide or vector of the present invention to a target cell population. Recombinant viral vectors can be constructed using methods well known to those skilled in the art.

[0176] The present invention further relates to host cells transformed with the polynucleotides or vectors of the present invention. The host cells may be prokaryotic or eukaryotic cells. The polynucleotides or vectors of the present invention present in the host cells may be integrated into the genome of the host cells or maintained extrachromosomally. The host cells may be any prokaryotic or eukaryotic cells, such as bacteria, insects, fungi, plants, animals, or human cells. Preferred fungal cells are, for example, cells of the genus Saccharomyces, particularly cells of the species S. cerevisiae. The term “prokaryotic cell” means all bacteria that can be transformed or transfected with DNA or RNA molecules to express the antibodies or corresponding immunoglobulin chains of the present invention. Prokaryotic hosts may include, for example, Gram-negative and Gram-positive bacteria such as Escherichia coli, Salmonella typhi, Serratia marcescens, and Bacillus subtilis. The term “eukaryotic cell” means yeast, higher plants, insect cells, preferably mammalian cells, most preferably HEK293 cells, NSO cells, and CHO cells.

[0177] A further embodiment relates to a method for preparing an anti-allergen antibody or an allergen-binding fragment thereof, comprising culturing cells containing a polynucleotide encoding the antibody or the binding fragment described herein, and isolating the antibody or the allergen-binding fragment from the cells or the cell culture medium.

[0178] In a further embodiment, the present invention relates to a method for producing an antibody or a bound fragment thereof, wherein the method comprises (a) culturing cells as described herein, and (b) comprising isolating the antibody or its bound fragment from the culture.

[0179] The transformed host can be grown in a fermenter and cultured according to techniques known in the art to achieve optimal cell proliferation. After expression, the whole antibody of the present invention, its dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc.; see Scopes, “Protein Purification”, Springer Verlag, NY (1982). The antibody of the present invention or its corresponding immunoglobulin chain can then be isolated from the growth medium, cell lysate, or cell membrane fraction. Isolation and purification of the antibody or immunoglobulin chain of the present invention, for example, recombinantly expressed antibody or immunoglobulin chain can be carried out by any conventional means, such as preparative chromatographic separation and immunological separation using monoclonal or polyclonal antibodies targeting the constant region of the antibody of the present invention, for example. It will be apparent to those skilled in the art that the antibody of the present invention can be further bound with other parts for, for example, drug targeting and imaging applications. Such binding may be performed chemically on the binding site after the expression of the antibody or antigen, or the binding product may be manipulated into the antibody or antigen of the present invention at the DNA level. These DNAs are then expressed in a suitable host system, the expressed proteins are collected, and regenerated as needed.

[0180] Therefore, one aspect of the present invention relates to a nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof according to the present invention.

[0181] One embodiment relates to an expression vector comprising a nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof as described herein.

[0182] Another embodiment relates to a host cell containing the expression vector described herein.

[0183] A further embodiment relates to a method for preparing an anti-allergen antibody or an allergen-conjugated fragment thereof, comprising culturing cells containing a polynucleotide encoding the antibody or its conjugated fragment as described herein, and isolating the antibody or its allergen-conjugated fragment from the cells or the cell culture medium. [Examples]

[0184] Production of human antibodies the purpose The objective of this study was to produce a fully human Bet v 1-specific antibody to prevent patient IgE bound to effector cells from binding to Bet v 1 and causing an immediate response.

[0185] material and method Biotinylation of nBet v 1, nQue a 1, and OVA: To enable Bet v 1 and Que a 1-specific memory B cell isolation by FACS, Bet v 1 and Que a 1 were biotinylated and complexed with streptavidin PE (SA PE). nBet v 1 and nQue a 1 were purified from pollen extracts with protein concentrations greater than 1 mg / mL. EZ-Link® Sulfo-NHS-LC-LC-Biotin (Thermo Scientific) was diluted with water and added to nBet v 1, nQue a 1, and OVA in biotin:allergen ratios of 2:1, 1:2, and 1:2, respectively, and incubated at room temperature for 30 minutes with stirring without light irradiation.

[0186] Preparation of fluorescent nBet v 1, nQue a 1, and OVA (decoy) multimers: To prepare biotinylated nBet v 1 and Que a 1 SA PE multimers, SA-PE (BioLegend) was added in ratios of 1:2 and 1:4, respectively. To prepare biotinylated OVA SA PE multimers, SA-PE (BioLegend) was added in a ratio of 1:4 (biotinylated OVA 1: SA APC 4).

[0187] Isolation and freezing of PBMCs from blood donors: Peripheral blood samples were collected from patients with birch pollen allergy. PBMCs were isolated from plasma and red blood cells using leucosep®. Cells were diluted to approximately 5 × 10⁷ cells / mL with RPMI 1640 + GlutaMAX® (Gibco), counted using NucleoCounter NC-200 (ChemoMetec) and Via1 Cassettes (ChemoMetec), and viability was confirmed.

[0188] Single-cell sorting of Bet v 1 and Que a 1-specific memory B cells: approximately 5-10 × 10⁶ mononuclear cells separated as described above. 7The following was used: OVA decoy multimers were added to cells at a final concentration of 5 nM, and the respective antigens, nBet v 1 or nQue a 1 fluorescent multimers, were added at a final concentration of 1 nM. Cells were enriched according to the protocol for lyophilized anti-PE microbeads (Miltenyi Biotec), and control samples were then stained with Brilliant Stain Buffer (BD Horizon) using IgD FITC (Biolegend, IA6-2), IgM FITC (Biolegend, MHM-88), CD19 BV650 (Biolegend, SJ25C1), CD3 BV480 (Biolegend, UCHT1), CD14 BV480 (Biolegend, M5E2), live / dead® fixable aqua dead cell kit (Thermo Scientific), CD38 BV421 (Biolegend, HIT2), and CD27 APC-H7 (Biolegend, M-T271).

[0189] 150 single antigen-specific B cells (CD3-, CD14-, CD19+, IgD+IgM-, allergen PE+, and decoy AF647-) were single-cell sorted in 96-well plates.

[0190] Single-cell RNA sequencing: cDNA and libraries were prepared from single cells obtained using the method described above, according to the SMART-Seq® Single-Cell Kit User Manual (version 101619). After amplification and purification, the cDNA was quantified using Qubit 4 (Invitrogen) and quality-checked using TapeStation 4150 (Agilent). Library preparation for Illumina sequencing was performed using NovaSeq 6000 Reagent Kits (Illumina; 20028400). The cDNA was fragmented, index-labeled, amplified, and pooled before sequencing. Sequences were then performed using Illumina NovaSeq 6000. The sequencing results of tetramer-sorted memory B cells were analyzed using RNA sequence aligner STAR (version 2.7.10a). The B cell receptor sequence was reconstructed using an improved version of the BraCeR pipeline (docker pull nielsphk / bracer:1.3) created by Lindeman et al. (2018) (Lindeman et al., 2018).

[0191] From single-cell sorted B cells, 137 heavy-chain and light-chain paired B-cell receptor sequences were generated.

[0192] Plasmid design: Human VH and VL sequences derived from selected B cells were cloned into mammalian expression vectors.

[0193] Heavy chain expression vector: Following the signal peptide, a synthetic gene consisting of VH+ human IgG4 (with the so-called S228P mutation) was cloned into the expression vector pcDNA3.1(+) as a HindIII / BamHI fragment.

[0194] Heavy chain expression vector: Following the signal peptide, a synthetic gene consisting of VH+ human IgG4 (with the so-called S228P mutation) was cloned into the expression vector pcDNA3.1(+) as a HindIII / BamHI fragment.

[0195] The sequence was codon-optimized for mammalian expression. Transfection-grade plasmid preparations were obtained from Genscript (New Jersey, USA).

[0196] mAb expression: Antibodies were expressed using a plasmid encoding heavy chain:light chain in a 1:1 ratio, according to the "Expi293® Expression System User Guide" (ThermoFisher, publication number: MAN0019402, revision number: B.0). Six days after transfection, the supernatant was centrifuged at 2700 g at 4°C for 1 hour and filtered through a 0.45 μm filter.

[0197] result 150 B cells were single-cell sorted and scRNAseq was performed. As a result, 76 heavy-chain and light-chain pair sequences were obtained, from which 70 antibodies were expressed, and 36 Bet v 1-specific antibodies were obtained. [Examples]

[0198] Affinity measurement of antibodies that bind to tree pollen allergens the purpose The objective of this embodiment is to measure the affinity of the obtained antibodies for the major tree pollen allergens: Bet v 1, Aln g 1, Cor a 1, and Que a 1.

[0199] material and method The equilibrium dissociation constants (KDs) of various related allergens that bind to monoclonal antibodies were measured using biolayer interferometry (BLI) with an Octet RED96e instrument. All binding tests were performed at 25°C and a shaking speed of 1000 rpm in kinetic buffer containing 8 mM NaH2PO4, 150 mM NaCl, 3 mM KCl, 2 mM KH2PO4, 0.1% BSA, and 0.05% v / v surfactant Tween-20 (pH 7.4). For antibodies expressed as IgG4, the monoclonal antibody was loaded onto an Octet® ProA biosensor before binding to the allergen. For antibodies expressed as IgE, biotinylated anti-IgE VHH was loaded onto an Octet® SAX biosensor to capture the monoclonal antibody before binding to the allergen. Binding studies were performed on the following allergens: birch (nBet v 1 and rBet v 1.0112), alder (Aln g 1), hazel (Cor a 1), and white oak (Que a 1). Immobilized monoclonal antibodies were immersed in 2-fold diluted allergens for 5 minutes to bind, and during this time, the dissociation of the allergen bound to the monoclonal antibody was monitored in kinetic buffer for 30 minutes. Double reference subtraction was performed. Maximum allergen capture level, overall association rate (k) a ), dissociation rate (kd), and dissociation equilibrium constant (K D The ) were determined by fitting real-time binding sensorgrams to a 1:1 fitting model using Data Analysis HT 11.1 software (Sartorius). For comparison, Regeneron antibodies REGN5713, REGN5714, and REGN5715 were added.

[0200] result The binding kinetic parameters at 25°C for various monoclonal antibodies of the present invention with natural and recombinant Bet v 1, natural Aln g 1, natural Cor a 1, and natural Que a 1 are shown in Tables 3 to 8.

[0201] The affinity (Kd) of the antibodies of the present invention for the antigen is lower than that of the control antibodies. All of the antibodies 2E02, 2C10, 2B04, 2E07, A07, B10, and 2_10 have a higher affinity for the allergens Bet v 1, Aln g 1, Cor a 1, and Que a 1 than REGN5713, REGN5714, and REGN5715.

Table 3-1

Table 3-2

[0202] As shown in Table 3, 7 out of the 36 antibodies tested were able to bind to all 4 allergens tested. Furthermore, out of the 3 control antibodies (REGN5713, REGN5714, and REGN5715), only 1 (REGN5715) was able to bind to all 4 allergens. This indicates that the binding affinity to Bet v 1 does not necessarily result in cross-reactivity with the other 3 allergens.

Table 4-1

Table 4-2

[0203] As shown in Table 4, all 36 monoclonal antibodies of the present invention showed measurable binding to recombinant Bet v 1, and the K D values were in the range of 249 pM to 468 nM. Furthermore, the antibodies 2B04, 2C10, 2E02, and A07 all showed stronger affinity compared to REGN5713, REGN5714, and REGN5715.

Table 5-1

Table 5-2

[0204] As shown in the table, all 34 of the 36 monoclonal antibodies of the present invention showed measurable binding to the natural type Bet v 1, and K D The values ​​ranged from 219 pM to 506 nM. Furthermore, antibodies 2E02, 2B04, A07, and B10 all showed higher affinity for the native Bet v 1 compared to the control antibodies REGN5713, REGN5714, and REGN5715. [Table 6-1] [Table 6-2]

[0205] As shown in the table, 15 of the monoclonal antibodies of the present invention showed measurable binding to natural Aln g 1, and K D The values ​​ranged from 626 pM to 4.34 uM. The remaining 21 antibodies did not show measurable binding to Aln g 1 under the test conditions. Furthermore, 2_9, 2A03, 2A09, 2B04, 2C10, 2E02, 2E07, A07, and B10 all showed higher affinity compared to the control antibodies REGN5713, REGN5714, and REGN5715. [Table 7-1] [Table 7-2]

[0206] As shown in the table, 14 of the monoclonal antibodies of the present invention showed measurable binding to natural Cor a 1, and K DThe values ​​ranged from 2.69 nM to 2.18 uM. The other 22 antibodies did not show measurable binding to Cor a 1 under the test conditions. 2_20, 2B04, 2C10, 2E02, 2E07, A07, and B10 all showed higher affinity for Cor a 1 compared to REGN5713, REGN5714, and REGN5715. [Table 8-1] [Table 8-2]

[0207] As shown in the table, seven of the monoclonal antibodies of the present invention showed measurable binding to natural Que a 1, and K D The values ​​ranged from 660 pM to 158 nM. The remaining 29 antibodies did not show measurable binding to Que a 1 under the test conditions. Furthermore, antibodies 2_10, 2E02, 2E07, 2C10, 2B04, and A07 all showed higher affinity for Que a 1 compared to REGN5713, REGN5714, and REGN5715.

[0208] conclusion This study demonstrates that antibodies 2_10, 2B04, 2C10, 2E02, 2E07, and A07 bind to all four tested tree pollen allergens (Bet v 1, Aln g 1, Cor a 1, and Que a 1) and exhibit high cross-reactivity compared to control antibodies REGN5713, REGN5714, and REGN5715. [Examples]

[0209] Epitope binning in Bet v 1 the purpose The objective of this study was to perform epitope binning on a subset of mAbs in Bet v 1.

[0210] material and method Epitope binning: Using the Octet RED96e platform, pairwise epitope binning was performed using BLI. It was carried out at 1000 rpm stirring and 30 °C. In all experiments, kinetic buffer (8 mM NaH2PO4, 3 mM KCl, 2 mM KH2PO4, 0.1% BSA, and 0.02% Tween-20) was used. Specifically, each mAb (position 1) was immobilized on the sensor chip and immersed in 100 nM nBet v 1 for 120 seconds. Next, the chip was individually immersed in all mAbs (position 2) at 9 μg / mL for 300 seconds to evaluate whether other mAbs could bind to the epitopes not occupied by the immobilized mAb. The same mAb was placed at positions 1 and 2 as a negative control, and by comparing the mAbs, overlapping clones and non-overlapping clones were determined. The figure shows the epitope binning matrix.

[0211] Results As shown in Figure 3, different antibodies can be grouped based on epitope specificity. Each ring represents one of the antibodies in Table 4. Overlapping rings indicate that the antibodies bind to overlapping epitopes on Bet v 1, and non-overlapping rings indicate that the antibodies bind to different non-overlapping epitopes.

[0212] Based on the binding to each epitope, antibodies can be classified into groups that can bind to Bet v 1 simultaneously.

[0213] Examples of such groups are · A07, 2B04 and 2E02 or · A07, 2B04 and 2_10 or · A07, 2B04 and 2C10 or · B10, 2B04 and 2E02 or · B10, 2B04 and 2_10 or · B10, 2B04 and 2C10 are.

[0214] Conclusion In conclusion, we were able to group individual antibodies based on their epitope specificity for Bet v 1. Furthermore, this grouping allowed us to define the group of antibodies that simultaneously bind to Bet v 1. [Examples]

[0215] Determination of the simultaneous binding of three anti-Bet v 1 monoclonal antibodies to Bet v 1. the purpose The objective of this experiment was to demonstrate, using the predictions from Example 3, that each of the three selected Bet v 1 monoclonal antibodies binds to a unique Bet v 1 epitope, and that no steric hindrance is observed in the simultaneous binding of these three antibodies, regardless of the binding order of the monoclonal antibodies. The binding order dependence of the three Bet v 1 monoclonal antibodies was also evaluated.

[0216] material and method The simultaneous binding of three anti-Bet v 1 monoclonal antibodies to Bet v 1 was measured using Octet RED96e (Sartorius) based on real-time label-free biolayer interferometry (BLI). The experiment was performed at 25°C in kinetic buffer (8 mM NaH2PO4, 3 mM KCl, 2 mM KH2PO4, 0.1% BSA, 0.02% Tween-20).

[0217] In three separate experiments, it was previously shown that the three IgG4 mAbs analyzed in this example (A07, 2B04, and 2E02) each bound to Bet v 1 with high affinity, showed high cross-reactivity with PR-10 allergens from closely related tree species (Example 2), and could bind to Bet v 1 simultaneously in a pairwise manner (Example 3). They were initially captured at the 2.5–3.3 nm level via a Protein A sensor chip (Table 5). Subsequently, the remaining Protein A binding sites were blocked with high concentrations (more than 10-fold) of unrelated non-Bet v 1 IgG4 mAbs. The sensor chips were then immersed in wells containing 4.25 μg / ml of Bet v 1 for 5 minutes and subsequently captured at the 0.3–0.5 nm level with a Protein A-binding anti-Bet v 1 antibody (Table 5). Next, the sensor chips were sequentially immersed in wells containing either the same anti-Bet v 1 mAb (negative control) directly captured on the Protein A chip, or two additional mAb clones (mAb-1 to 3, Table 5) (5 minutes each step). See Figure 4 for an example of a sensorgram.

[0218] The baseline step, which involves immersing the sensor chip in kinetic buffer, was included between all of the above steps.

[0219] result The binding level (nm) was measured using Data Analysis HT 11.1.3.50 (Molecular Devices), and the results are shown in Table 9. Signals below 0.05 nm indicate no binding, while signals above 0.1 nm indicate binding, i.e., no competition between mAbs for binding to Bet v 1. All three Bet v 1 monoclonal antibodies included in this example were able to bind to Bet v 1 simultaneously, and the binding reaction was not affected by the order in which the antibodies were added. [Table 9]

[0220] conclusion This demonstrates that a group of three antibodies with different epitope specificities can simultaneously bind to Bet v 1. Regardless of the order in which the three mAbs bind to Bet v 1, no competition was observed that prevented the simultaneous binding of the three antibodies. This suggests that mAb clones A07, 2B04, and 2E02 bind to different epitopes without steric hindrance and without overlap, as predicted in Example 4. [Examples]

[0221] Inhibition of allergen-specific IgE binding to tree pollen allergens. the purpose The purpose of this study was to determine whether the mAb of the present invention can block the binding of IgE to nBet v 1, nQue a 1, nAln g 1, and nCor a 1, and inhibit the activation of basophils in tree pollen allergy patients stimulated with any of nBet v 1, nQue a 1, nAln g 1, or nCor a 1.

[0222] material and method The mAb of the present invention was used to test for IgE epitope blocking and basophil activation inhibition. IgE epitope blocking and basophil activation inhibition are important in vitro assays for evaluating the allergic reaction suppression effect of antibody cocktails, as previously suggested (Orengo et al., 2018; Atanasio et al., 2022).

[0223] PBMCs were isolated from fresh blood collected from donors with tree pollen allergies. Cells were pre-incubated with IL-3 and then mixed with single tree pollen allergens pre-incubated for 1 hour with various mAbs (mixtures or individually). Basophil activation was performed at 37°C for 1 hour. The cells were then washed, stained with antibodies against specific cell surface proteins (CD123, CD203c, and CD63), and analyzed by flow cytometry. Basophil activation was measured by increased expression of CD63 in basophils identified within PBMCs based on CD123 and CD203c.

[0224] result Various mAb mixtures were tested (Table 10), and the effects of individual mAbs were compared. [Table 10]

[0225] This experiment was conducted using basophils isolated from six donors with tree pollen allergies. Basophils were inhibited with mAb mixes containing 4 nM of each mAb (Figures 5-8). Allergen stimulation was performed using nBet v 1 (Figures 5A-F), nAln g 1 (Figures 6A-F), nCor a 1 (Figures 7A-F), and nQue a 1 (Figures 8A-F).

[0226] As shown in Figures 5A-F, in all six donors, activation was strongly inhibited by the antibody mix compared to the buffer control.

[0227] When basophils were stimulated with nAln g 1, nCor a 1, and nQue a 1, only the mixture of ALK-1 and ALK-2 inhibited activation, whereas control 1 had no effect on activation or only a very limited effect in all six donors (Figures 6-8A~F).

[0228] Next, increasing the concentration of each mAb (from 5 × 10⁻⁴ nM to 200 nM) inhibited basophil activation (Figure 9-10 and Table 11-18). The concentrations of the allergens used to stimulate basophils (nBet v 1, nAln g 1, nCor a 1, and nQue a 1) were individually selected for each donor A-F (due to variations in donor sensitivity). Figures 9-10A-D show two representative donors (Donors D and F in Table 11-18) who were stimulated with all four allergens (one person each).

[0229] As shown in Figures 9A and 10A, ALK-1, ALK-2, and control 1 were able to inhibit nBet v 1-induced activation more strongly than the individual antibodies.

[0230] When basophils were stimulated with either nAln g 1, nCor a 1, or nQue a 1, ALK-1 and ALK-2 showed the strongest inhibition among all donors tested, compared to control 1 and the individual antibodies (Figures 9B-D, 10B-D, Tables 11-14). [Table 11] [Table 12] [Table 13] [Table 14]

[0231] The inhibitory activity of ALK-3 and ALK-4 on basophil activation after stimulation with nAln g 1, nCor a 1, and nQue was investigated (Table 15-18). In all six donors, ALK-3 and ALK-4 showed stronger inhibition of basophil activation compared to the individual antibodies. [Table 15] [Table 16] [Table 17] [Table 18]

[0232] conclusion This study demonstrated that ALK-1 and ALK-2 block the binding of IgE to allergens (nBet v 1, nAln g 1, nCor a 1, and nCor a 1), thereby inhibiting basophil activation. The inhibition of nBet v 1-induced basophil activation by ALK-1 and ALK-2 was comparable to that of the control group "Control 1" (Figures 5A-F, 9A, 10A). On the other hand, ALK-1 and ALK-2 more strongly inhibited basophil activation mediated by nAln g 1, nCor a 1, and nQue a 1 compared to "Control 1" (Figures 6B-F, 9B-D, 10B-D).

[0233] Furthermore, it was shown that mixing the two antibodies more strongly inhibited basophil activation by all four allergens than using the individual antibodies.

[0234] reference TIFF2026513548000030.tif206164 TIFF2026513548000031.tif183164

Claims

1. An antibody or its antigen-binding fragment comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein, - The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 1, the HCDR2 amino acid sequence contained in SEQ ID NO: 1, the HCDR3 amino acid sequence contained in SEQ ID NO: 1, the LCDR1 amino acid sequence contained in SEQ ID NO: 2, the LCDR2 amino acid sequence contained in SEQ ID NO: 2, and the LCDR3 amino acid sequence contained in SEQ ID NO: 2, or - The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 21, the HCDR2 amino acid sequence contained in SEQ ID NO: 21, the HCDR3 amino acid sequence contained in SEQ ID NO: 21, the LCDR1 amino acid sequence contained in SEQ ID NO: 22, the LCDR2 amino acid sequence contained in SEQ ID NO: 22, and the LCDR3 amino acid sequence contained in SEQ ID NO: 22, or - The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 41, the HCDR2 amino acid sequence contained in SEQ ID NO: 41, the HCDR3 amino acid sequence contained in SEQ ID NO: 41, the LCDR1 amino acid sequence contained in SEQ ID NO: 42, the LCDR2 amino acid sequence contained in SEQ ID NO: 42, and the LCDR3 amino acid sequence contained in SEQ ID NO: 42, or - The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 61, the HCDR2 amino acid sequence contained in SEQ ID NO: 61, the HCDR3 amino acid sequence contained in SEQ ID NO: 61, the LCDR1 amino acid sequence contained in SEQ ID NO: 62, the LCDR2 amino acid sequence contained in SEQ ID NO: 62, and the LCDR3 amino acid sequence contained in SEQ ID NO: 62, or - The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 81, the HCDR2 amino acid sequence contained in SEQ ID NO: 81, the HCDR3 amino acid sequence contained in SEQ ID NO: 81, the LCDR1 amino acid sequence contained in SEQ ID NO: 82, the LCDR2 amino acid sequence contained in SEQ ID NO: 82, and the LCDR3 amino acid sequence contained in SEQ ID NO: 82, or - The antibody or its antigen-binding fragment contains the HCDR1 amino acid sequence contained in SEQ ID NO: 101, the HCDR2 amino acid sequence contained in SEQ ID NO: 101, the HCDR3 amino acid sequence contained in SEQ ID NO: 101, the LCDR1 amino acid sequence contained in SEQ ID NO: 102, the LCDR1 amino acid sequence contained in SEQ ID NO: 102, and the LCDR3 amino acid sequence contained in SEQ ID NO: 102, or The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO: 121, the HCDR2 amino acid sequence contained in SEQ ID NO: 121, the HCDR3 amino acid sequence contained in SEQ ID NO: 121, the LCDR1 amino acid sequence contained in SEQ ID NO: 122, the LCDR2 amino acid sequence contained in SEQ ID NO: 122, and the LCDR3 amino acid sequence contained in SEQ ID NO:

122. Here, each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 may be determined by IMGT, Kabat, or Chothia, if desired. Here, each of the CDR regions determined by IMGT, Kabat, or Chothia may have one, two, or three amino acid substitutions, so that each HCDR1 may contain one, two, or three amino acid substitutions, each HCDR2 may contain one, two, or three amino acid substitutions, each HCDR3 may contain one, two, or three amino acid substitutions, each LCDR1 may contain one, two, or three amino acid substitutions, each LCDR2 may contain one, two, or three amino acid substitutions, and / or each LCDR3 may contain one, two, or three amino acid substitutions.

2. The antibody or antigen-binding fragment according to claim 1, wherein the CDR region is determined by the IMGT method.

3. Antibodies or their antigen-binding fragments, here, - The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 3, the HCDR2 amino acid sequence of SEQ ID NO: 4, the HCDR3 amino acid sequence of SEQ ID NO: 5, the LCDR1 amino acid sequence of SEQ ID NO: 6, the LCDR2 amino acid sequence of SEQ ID NO: 7, and the LCDR3 amino acid sequence of SEQ ID NO: 8; or - The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 23, the HCDR2 amino acid sequence of SEQ ID NO: 24, the HCDR3 amino acid sequence of SEQ ID NO: 25, the LCDR1 amino acid sequence of SEQ ID NO: 26, the LCDR2 amino acid sequence of SEQ ID NO: 27, and the LCDR3 amino acid sequence of SEQ ID NO: 28; or - The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 43, the HCDR2 amino acid sequence of SEQ ID NO: 44, the HCDR3 amino acid sequence of SEQ ID NO: 45, the LCDR1 amino acid sequence of SEQ ID NO: 46, the LCDR2 amino acid sequence of SEQ ID NO: 47, and the LCDR3 amino acid sequence of SEQ ID NO: 48; or - The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 63, the HCDR2 amino acid sequence of SEQ ID NO: 64, the HCDR3 amino acid sequence of SEQ ID NO: 65, the LCDR1 amino acid sequence of SEQ ID NO: 66, the LCDR2 amino acid sequence of SEQ ID NO: 67, and the LCDR3 amino acid sequence of SEQ ID NO: 68; or - The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 83, the HCDR2 amino acid sequence of SEQ ID NO: 84, the HCDR3 amino acid sequence of SEQ ID NO: 85, the LCDR1 amino acid sequence of SEQ ID NO: 86, the LCDR2 amino acid sequence of SEQ ID NO: 87, and the LCDR3 amino acid sequence of SEQ ID NO: 88; or - The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 103, the HCDR2 amino acid sequence of SEQ ID NO: 104, the HCDR3 amino acid sequence of SEQ ID NO: 105, the LCDR1 amino acid sequence of SEQ ID NO: 106, the LCDR2 amino acid sequence of SEQ ID NO: 107, and the LCDR3 amino acid sequence of SEQ ID NO: 108; or The antibody or its antigen-binding fragment includes the HCDR1 amino acid sequence of SEQ ID NO: 123, the HCDR2 amino acid sequence of SEQ ID NO: 124, the HCDR3 amino acid sequence of SEQ ID NO: 125, the LCDR1 amino acid sequence of SEQ ID NO: 126, the LCDR2 amino acid sequence of SEQ ID NO: 127, and the LCDR3 amino acid sequence of SEQ ID NO:

128. Here, each HCDR1 may contain one, two, or three amino acid substitutions, each HCDR2 may contain one, two, or three amino acid substitutions, each HCDR3 may contain one, two, or three amino acid substitutions, each LCDR1 may contain one, two, or three amino acid substitutions, each LCDR2 may contain one, two, or three amino acid substitutions, and / or each LCDR3 may contain one, two, or three amino acid substitutions.

4. Antibodies or their antigen-binding fragments, here, - The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO: 1 and an LCVR having the amino acid sequence of SEQ ID NO: 2; or - The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO: 21 and an LCVR having the amino acid sequence of SEQ ID NO: 22; or - The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO: 41 and an LCVR having the amino acid sequence of SEQ ID NO: 42; or - The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO: 61 and an LCVR having the amino acid sequence of SEQ ID NO: 62; or - The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence of SEQ ID NO: 81 and LCVR having the amino acid sequence of SEQ ID NO: 82; or - The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO: 101 and an LCVR having the amino acid sequence of SEQ ID NO: 102; or - The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence of SEQ ID NO: 121 and an LCVR having the amino acid sequence of SEQ ID NO: 122; or Here, each HCVR may contain one, two, or three amino acid substitutions, and / or each LCVR may contain one, two, or three amino acid substitutions.

5. An antibody or antigen-binding fragment according to any one of claims 1 to 4, which can reduce or inhibit the binding of an IgE antibody to one, two, three, or four allergens selected from the group consisting of Bet v 1, Cor a 1, Aln g 1, and Que a 1.

6. The antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody comprises a human-derived Fc region.

7. The antibody or antigen-binding fragment according to claim 6, wherein the Fc region is IgG1-Fc or IgG4-Fc.

8. The antibody or antigen-binding fragment according to claim 6, wherein the Fc region is IgG4-Fc.

9. The antibody or antigen-binding fragment according to claim 8, wherein the IgG4-Fc comprises the S228P mutation.

10. The antibody or its antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody is a human antibody.

11. A multispecific antibody comprising two or more antigen-binding fragments, wherein the two or more antigen-binding fragments are selected from among the antigen-binding fragments described in any one of claims 1 to 2 and claims 5 to 10 dependent on claims 1 to 2; or selected from among the antibodies or their antigen-binding fragments described in claims 3 and claims 5 to 10 dependent on claim 3; or selected from among the antibodies or their antigen-binding fragments described in claims 4 and claims 5 to 10 dependent on claim 4.

12. A composition comprising two antibodies or antigen-binding fragments thereof, wherein the two antibodies or antigen-binding fragments are selected from among the antibodies or antigen-binding fragments described in any one of claims 1 to 2 and claims 5 to 10 dependent on claims 1 to 2; or selected from among the antibodies or antigen-binding fragments described in claims 3 and claims 5 to 10 dependent on claim 3; or selected from among the antibodies or antigen-binding fragments described in claims 4 and claims 5 to 10 dependent on claim 4.

13. The composition according to claim 12, comprising at least an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein each of HCDR1, HCDR2, and HCDR3 has the amino acid sequence contained in SEQ ID NO: 1, and each of LCDR1, LCDR2, and LCDR3 has the amino acid sequence contained in SEQ ID NO:

2.

14. The antibody composition according to any one of claims 12 and 13, comprising at least an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein each of HCDR1, HCDR2, and HCDR3 has the amino acid sequence contained in SEQ ID NO: 81, and each of LCDR1, LCDR2, and LCDR3 has the amino acid sequence contained in SEQ ID NO:

82.

15. The composition according to claim 12, comprising a first antibody and a second antibody, wherein the first antibody and the second antibody each comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where, - The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 1, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 2; the second antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 42; or - The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 1, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 2; the second antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 81, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 82; or - The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22; the second antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 42; or - The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22; the second antibody comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 81, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 82; or The first antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and also contains LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO:

42. The second antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 81, and also contains LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO:

82. composition.

16. A composition according to any one of claims 12 to 14, comprising three antibodies or antigen-binding fragments thereof, wherein the three antibodies or antigen-binding fragments are selected from the antibodies or antigen-binding fragments according to any one of claims 1 to 2 and any one of claims 5 to 10 dependent on claims 1 to 2; or selected from the antibodies or antigen-binding fragments according to any one of claims 3 and any one of claims 5 to 10 dependent on claim 3; or selected from the antibodies or antigen-binding fragments according to claims 4 and any one of claims 5 to 10 dependent on claim 4.

17. The composition according to claim 16, comprising a first antibody, a second antibody, and a third antibody, wherein the first antibody, the second antibody, and the third antibody each comprise three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), where, - The first antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 2; the second antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 42; the third antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 81, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 82; or - The first antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 2; the second antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 42; the third antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 101, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 102; or - The first antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 22; the second antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 42; the third antibody comprises HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 81, and LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 82; or - The first antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22; the second antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence of SEQ ID NO: 42; the third antibody comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 101, and each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 102; or The first antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 41, and also contains LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 42; the second antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 101, and also contains LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO: 102; the third antibody contains HCDR1, HCDR2, and HCDR3, each having the amino acid sequence contained in SEQ ID NO: 121, and also contains LCDR1, LCDR2, and LCDR3, each having the amino acid sequence contained in SEQ ID NO:

122. composition.

18. A pharmaceutical composition comprising, together with one or more pharmaceutically acceptable excipients, a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 10, a multispecific antibody according to claim 11, or a composition according to any one of claims 12 to 17.

19. A method for treating tree pollen allergy, comprising administering the pharmaceutical composition according to claim 19, wherein the tree pollen allergy is related to an allergy to one or more allergens Bet v 1, Cor a 1, Aln g 1, and Que a 1.

20. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a multispecific antibody according to claim 11, or a composition according to any one of claims 12 to 17, wherein the tree pollen allergy is related to an allergy to one or more of Bet v 1, Cor a 1, Aln g 1, and Que a 1.

21. A nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof according to any one of claims 1 to 10.

22. An expression vector comprising a nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof as described in claim 21.

23. A host cell comprising the expression vector described in claim 22.

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