Allergen-binding antibodies useful for treatment of tree pollen allergy

Abstract

Tree pollen allergy is seasonal allergy caused by various tree pollen spread in air. The allergy affects a substantial proportion of populations, is particularly significant in European and North America areas, and is characterized by symptoms such as sneezing, nasal obstruction, rhinorrhea, eye itching and lacrimation, and throat irritation. The symptoms typically occur in spring, at which birch, alder, hazel and oak release pollen into the air. Tree pollen allergy is a type 1 hypersensitivity reaction mediated by an immunoglobulin E (IgE) antibody. The present invention relates to antibodies and antigen binding fragments thereof that bind to one or more of tree pollen allergens Bet v 1, Aln g 1, Cor a 1 and Quue a 1, resulting in inhibition of IgE-mediated anaphylaxis.

Description

Technical Field

[0001] The present invention relates to antibodies and antigen-binding fragments thereof that bind to one or more of tree pollen allergens (Bet v 1, Aln g 1, Cor a 1 and Que a 1), therapeutic compositions comprising said antibodies, and methods of using said antibodies. Background Technology

[0002] Tree pollen allergy is a seasonal allergy caused by various airborne tree pollens. 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 and watery eyes, and throat irritation. These symptoms typically occur in the spring months 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 v1 (a major allergen found in birch pollen) (D'Amato et al., 2007).

[0003] Bet v1 belongs to a protein family called Pathogenesis-Associated Protein 10 (PR-10), which contains proteins found in the pollen of various trees and has been shown to trigger cross-reactivity with different allergens. For example, Que a1, Aln g1, and Cor a1 are homologous allergens found in oak, alder, and hazelnut pollen, respectively, and they share significant structural and immunological similarities with Bet v1. Therefore, individuals allergic to one of these allergens may also experience allergic reactions to other cross-reactive allergens, which can complicate the diagnosis and treatment of allergies (Asam et al., 2015).

[0004] Tree pollen allergy symptoms are caused by cross-linking of tree pollen allergen-specific IgE with high-affinity ε receptors (FcεRI) on the surface of mast cells and basophils. This cross-linking triggers the release of inflammatory mediators such as histamine, leukotrienes, and cytokines. These mediators cause characteristic allergy symptoms and may induce bronchial hyperresponsiveness and asthma in some individuals (Gould and Sutton, 2008).

[0005] Current treatment options for tree pollen allergy include allergen avoidance, pharmacological therapy, and allergen-specific immunotherapy (AIT). Allergen avoidance involves minimizing exposure to tree pollen through measures such as staying indoors during high pollen counts, wearing masks, and keeping doors and windows closed. Pharmacological options include antihistamines, nasal corticosteroids, leukotriene receptor antagonists, and decongestants, which can alleviate allergy symptoms but do not provide long-term relief. AIT is a disease-modifying treatment that involves administering gradually increasing doses of the allergen to induce immune tolerance and reduce the severity of the allergy. AIT can be administered subcutaneously or sublingually and has been shown to be effective in reducing symptoms of tree pollen allergy and improving patients' quality of life (Canonica et al., 2014).

[0006] Allergen-specific antibodies have previously been proposed as a treatment for allergies because they may be able to block allergen molecules from entering mucosal tissues, or bind allergens before they have a chance to bind to IgE, which binds to high-affinity receptors on mast cells or 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 been previously described (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 the treatment of birch allergy by using an antibody composition 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). Only one of the Bet v 1 binding antibodies disclosed in WO 2018 / 222854 can also bind to other tree pollen allergens, Aln g 1 and Cor a 1. Therefore, the 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 has been further described in patent applications WO 2013 / 166236 and WO 2018 / 234383, which respectively relate to treating feline allergy by binding to allergen Fel d 1 and treating peanut allergy by binding to allergen Ara h 2.

[0010] However, tree pollen seasons are not isolated events, but rather overlapping events, with the different tree pollen allergens described in this article peaking at different times of the year. Therefore, treatment targeting a single allergen will not be effective in treating symptoms in patients allergic to two or more of the PR-10 family of allergens.

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

[0012] This invention relates to antibodies or antigen-binding fragments thereof capable of binding to tree pollen allergens selected from Bet v 1, Aln g 1, Cor a 1 and Que a 1.

[0013] The inventors have provided a selection of antibodies with the potential to treat pollen allergies caused by four important tree pollen allergens. As shown herein, the antibodies are capable of binding with good affinity to each of the four allergens, Betv1, Alng1, Cora1, and Que1, and when administered at therapeutically effective doses, their combination can compete with these allergens for binding to human IgE antibodies. As shown in Example 2, many of the single antibodies shown in Table 1, such as the seven antibodies 2E02, 2C10, 2B04, 2E0, A07, B10, and 2_10, are capable of binding to all four tree pollen allergens (Betv1, Alng1, Cora1, and Que1) with higher affinity than prior art antibodies such as REGN5713, REGN5714, and REGN5715 described in WO 2018 / 222854. Further, as described in Example 5, the combination of two or three antibodies showed an enhanced ability to block the binding between a single tree pollen allergen and IgE, compared to a single antibody, resulting in the inhibition of basophil activation, which, as previously mentioned, is a key driver of allergy.

[0014] Therefore, in a first aspect, the present invention relates to antibodies 2E02, 2C10, 2B04, 2E0, A07, B10, and 2_10, or antigen-binding fragments thereof. Therefore, the first aspect relates to antibodies or antigen-binding fragments thereof comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein;

[0015] 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

[0016] The antibody or its antigen-binding fragment comprises 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

[0017] The antibody or its antigen-binding fragment comprises 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

[0018] The antibody or its antigen-binding fragment comprises 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

[0019] The antibody or its antigen-binding fragment comprises 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

[0020] The antibody or its antigen-binding fragment comprises 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 LCDR2 amino acid sequence contained in SEQ ID NO:102, and the LCDR3 amino acid sequence contained in SEQ ID NO:102; or

[0021] 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.

[0022] Each CDR (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3) as described above can be determined according to the IMGT, Kabat or Chothia method.

[0023] Optionally, each CDR region determined by the IMGT, Kabat, or Chothia method may have 1, 2, or 3 amino acid substitutions, such as 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.

[0024] In other words, the first aspect involves an antibody or its antigen-binding fragment, wherein;

[0025] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO:3, the HCDR2 amino acid sequence contained in SEQ ID NO:4, the HCDR3 amino acid sequence contained in SEQ ID NO:5, the LCDR1 amino acid sequence contained in SEQ ID NO:6, the LCDR2 amino acid sequence contained in SEQ ID NO:7, and the LCDR3 amino acid sequence contained in SEQ ID NO:8; or

[0026] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO:23, the HCDR2 amino acid sequence contained in SEQ ID NO:24, the HCDR3 amino acid sequence contained in SEQ ID NO:25, the LCDR1 amino acid sequence contained in SEQ ID NO:26, the LCDR2 amino acid sequence contained in SEQ ID NO:27, and the LCDR3 amino acid sequence contained in SEQ ID NO:28; or

[0027] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO:43, the HCDR2 amino acid sequence contained in SEQ ID NO:44, the HCDR3 amino acid sequence contained in SEQ ID NO:45, the LCDR1 amino acid sequence contained in SEQ ID NO:46, the LCDR2 amino acid sequence contained in SEQ ID NO:47, and the LCDR3 amino acid sequence contained in SEQ ID NO:48; or

[0028] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO:63, the HCDR2 amino acid sequence contained in SEQ ID NO:64, the HCDR3 amino acid sequence contained in SEQ ID NO:65, the LCDR1 amino acid sequence contained in SEQ ID NO:66, the LCDR2 amino acid sequence contained in SEQ ID NO:67, and the LCDR3 amino acid sequence contained in SEQ ID NO:68; or

[0029] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO:83, the HCDR2 amino acid sequence contained in SEQ ID NO:84, the HCDR3 amino acid sequence contained in SEQ ID NO:85, the LCDR1 amino acid sequence contained in SEQ ID NO:86, the LCDR2 amino acid sequence contained in SEQ ID NO:87, and the LCDR3 amino acid sequence contained in SEQ ID NO:88; or

[0030] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO:103, the HCDR2 amino acid sequence contained in SEQ ID NO:104, the HCDR3 amino acid sequence contained in SEQ ID NO:105, the LCDR1 amino acid sequence contained in SEQ ID NO:106, the LCDR2 amino acid sequence contained in SEQ ID NO:107, and the LCDR3 amino acid sequence contained in SEQ ID NO:108; or

[0031] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence contained in SEQ ID NO:123, the HCDR2 amino acid sequence contained in SEQ ID NO:124, the HCDR3 amino acid sequence contained in SEQ ID NO:125, the LCDR1 amino acid sequence contained in SEQ ID NO:126, the LCDR2 amino acid sequence contained in SEQ ID NO:127, and the LCDR3 amino acid sequence contained in SEQ ID NO:128.

[0032] 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.

[0033] In addition, to put it another way, the first aspect involves antibodies or their antigen-binding fragments, wherein;

[0034] 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; or

[0035] 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; or

[0036] 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; or

[0037] 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; or

[0038] 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

[0039] 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; or

[0040] The antibody or its antigen-binding fragment comprises HCVR having the amino acid sequence SEQ ID NO:121 and LCVR having the amino acid sequence SEQ ID NO:122.

[0041] Each HCVR may contain 1, 2 or 3 amino acid substitutions, and / or each LCVR may contain 1, 2 or 3 amino acid substitutions.

[0042] In a second aspect, the present invention relates to a combination of two different antibodies or antigen-binding fragments thereof, such as a composition comprising at least two antibodies or antigen-binding fragments thereof, preferably comprising three antibodies, such as at least three antibodies, optionally comprising four antibodies or antigen-binding fragments thereof, wherein the antibodies and their antigen-binding fragments are selected from the antibodies or antigen-binding fragments thereof described herein, such particularly from the group consisting of seven antibodies 2E02, 2C10, 2B04, 2E0, A07, B10 and 2_10 or their antigen-binding fragments thereof.

[0043] In a preferred embodiment, the composition comprises two antibodies or antigen-binding fragments thereof according to the invention.

[0044] In another preferred embodiment, the composition comprises three antibodies or antigen-binding fragments thereof according to the invention.

[0045] In a third aspect, the present invention relates to pharmaceutical compositions comprising a therapeutically effective amount of one or more antibodies or antigen-binding fragments thereof, and one or more pharmaceutically acceptable excipients, wherein the one or more antibodies or antigen-binding fragments thereof are selected from antibodies or antigen-binding fragments thereof as defined herein.

[0046] In one preferred embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of three antibodies or antigen-binding fragments thereof, and one or more pharmaceutically acceptable excipients, wherein the antibodies or antigen-binding fragments thereof are selected from antibodies or antigen-binding fragments thereof as defined herein. Attached Figure Description

[0047] Figure 1 This displays the epitope binning matrix for the selected antibodies against Bet v 1. Black squares indicate that the mAb does not bind in pairs with Bet v 1. White squares indicate that the mAb binds in pairs with Bet v 1. * = represents the epitope bins for mAbs 1_4 and 1_11. ** = represents the epitope bins for mAbs 1_5, 2_6, 2_7, 2_11, 2_13, and 2_18.

[0048] Figure 2 An overview of the antibodies obtained as described in the examples is shown. Each ring represents one of the antibodies in Table 1. Overlapping rings indicate that the antibody binds to an overlapping epitope on Bet v1, while non-overlapping rings indicate that the antibody binds to a different non-overlapping epitope.

[0049] Figure 3 The sensor plots show the sequential binding of three mAbs to Bet v 1. 0-60 s: Baseline. 60-480 s: Loading mAb A07 onto the protein A sensor tip. 480-510 s: Baseline. 510-810 s: Blocking mAb. 810-840 s: Baseline. 840-1140 s: Bet v 1. 1140-1200 s: Baseline. 1200-1500 s: mAb A07. 1500-1560 s: Baseline. 1560-1860 s: mAb 2B04. 1860-1910 s: Baseline. 1910-2210 s: mAb 2E02. 2210-2270 s: Baseline.

[0050] Figure 4 shows the inhibition of basophil activation induced by nBet v1 stimulation. Before addition to PBMCs, a mixture of mAbs containing 4 nM of each mAb was pre-incubated with different concentrations of nBet v1, as shown on the X-axis. Basophil activation was measured by flow cytometry as described in Example 5 – activated basophils are shown as the percentage of CD63-positive cells on the Y-axis. Data from six donors are shown ( Figures 4A-4F ).

[0051] Figure 5 shows the inhibition of basophil activation induced by nAln g1 stimulation. Before addition to PBMCs, a mixture of mAbs containing 4 nM of each mAb was pre-incubated with different concentrations of nAln g1, as shown on the X-axis. Basophil activation was measured by flow cytometry as described in Example 5 – activated basophils are shown as the percentage of CD63-positive cells on the Y-axis. Data from six donors are shown ( Figures 5A-5F ).

[0052] Figure 6 shows the inhibition of basophil activation induced by nCor a1 stimulation. Before addition to PBMCs, a mixture of mAbs containing 4 nM of each mAb was pre-incubated with different concentrations of nCor a1 as shown on the X-axis. Basophil activation was measured by flow cytometry as described in Example 5 – activated basophils are shown as the percentage of CD63-positive cells on the Y-axis. Data from six donors are shown ( Figures 6A-6F ).

[0053] Figure 7 shows the inhibition of basophil activation induced by nQue a 1 stimulation. Before addition to PBMCs, a mixture of mAbs containing 4 nM of each mAb was pre-incubated with different concentrations of nQue a 1 as shown on the X-axis. Basophil activation was measured by flow cytometry as described in Example 5 – activated basophils are shown as the percentage of CD63-positive cells on the Y-axis. Data from six donors are shown ( Figures 7A-7F ).

[0054] Figure 8 shows the inhibition of basophil activation induced by stimulation with nBet v1, nAln g1, nCor a1, or nQue a1. The allergen was pre-incubated with the mAb before stimulation of the PBMCs. Basophil activation was measured by flow cytometry as described in Example 5. The Y-axis shows the percentage inhibition of activated basophils relative to the absence of the mAb.

[0055] Figure 9 shows the inhibition of basophil activation induced by stimulation with nBet v1, nAln g1, nCor a1, and nQue a1. The allergens were pre-incubated with mAbs before stimulation of PBMCs. Basophil activation was measured by flow cytometry as described in Example 5. The Y-axis shows the percentage inhibition of activated basophils relative to the absence of mAbs.

[0056] Detailed disclosure of the present invention

[0057] definition

[0058] allergens

[0059] In this specification, the term "allergen" refers to a compound that is recognized as a foreign substance by the immune system, thereby triggering an immune response to the allergen. In other words, it refers to a molecule that is not present in the human body and can induce an abnormal immune response through IgE-mediated activation of mast cells and basophils, thus triggering allergic symptoms. Those skilled in the art will understand that, in this specification, "autoantigens," i.e., molecules produced by the human body, are not considered allergens. Examples of allergens reported by the WHO / IUIS Allergen Nomenclature Subcommittee are available at http: / / allergen.org / .

[0060] Antibody

[0061] As used herein, the term "antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., Bet v1). As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains (two heavy chains (H) and two light chains (L) interconnected by disulfide bonds (i.e., a "complete antibody molecule"), as well as its multimers (e.g., IgM) or its antigen-binding fragments. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (composed 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 can be further subdivided. This is a hypervariable region called the complementarity-determining region (CDR), interspersed with more conserved regions called framework regions (FR). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the invention, the FR of the antibody (or its antigen-binding fragment) may be identical to the human germline sequence, or may be natural or artificially modified. A common amino acid sequence can be defined based on the side-by-side analysis of two or more CDRs.

[0062] Antibody variants

[0063] "Antibody variants" are proteins derived from antibodies that possess the same binding specificity as antibodies but are not naturally expressed products in mammals. Therefore, the term refers to various fragments of antibodies as well as artificial antibody analogues. Furthermore, the term indicates antibody forms that exist in nature but are not commonly found in mammals (such as heavy chain antibodies and IgY found in birds and reptiles), where the CDR (corresponding antibody derivative) from mammalian antibodies or combinations thereof has been engineered into the antibody form, and the CDR was not originally derived from that antibody form.

[0064] antigen-binding fragments

[0065] "Antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.

[0066] Examples of antibody-binding fragments include, but are not limited to, Fab fragments (monovalent fragments consisting of VL, VH, CL, and CHI domains), F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region), single-chain Fv (scFv), disulfide-linked Fv (dsFv), complementarity-determining regions (CDRs), VL (light chain variable regions), VH (heavy chain variable regions), sdAbs (single-domain antibodies), and any combination of these fragments or any other functional portion of an immunoglobulin peptide capable of binding to a target antigen. Antibodies and their antigen-binding portions include domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, sdAbs (e.g., monovalent sdAbs, bivalent sdAabs, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0067] Combining affinity

[0068] The term "binding affinity" refers to the affinity of a test compound (such as an antibody or its antigen-binding fragment disclosed herein) for a specific allergen. The ability of a test compound to bind to a specific allergen can be tested using the biolayer interferometry (BLI) described herein. In short, this assay measures the association and dissociation of the test molecule with a specific allergen. Based on these measurements, k can be determined. on , k off Ratio k off / k on (=KD). k on It is a constant used to characterize the rate at which a test molecule binds to an allergen, while k off Characterizes the rate at which test molecules dissociate from allergens. off / k on The ratio produces the equilibrium dissociation constant K. D K D The lower the value, the higher the affinity of the tested molecule for the allergen. Typically, K values ​​in the sub-nanomolar concentration range... D The value may be ideal, such as particularly in the sub-nanomolar concentration affinity range, such as below 1-10 nM. Other IgE affinity binding assays may be used, which are known to those skilled in the art.

[0069] Blocking / neutralizing antibodies

[0070] As used herein, "blocking antibody" or "neutralizing antibody" (or "antibody that neutralizes allergen activity") is intended to refer to an antibody or its antigen-binding portion that, upon binding to one of the allergens selected from the group consisting of Bet v1, Aln g1, Cor a1, and Que a1, results in the inhibition of at least one biological activity of that allergen. For example, the antibody of the present invention may help prevent primary allergic reactions caused by Bet v1. Alternatively, the antibody of the present invention may exhibit the ability to prevent secondary allergic reactions caused by Bet v1, or at least reduce, decrease, or inhibit one symptom of an allergic reaction caused by Bet v1, including sneezing, coughing, asthma symptoms, or an allergic reaction caused by Bet v1. Such inhibition of the biological activity of Bet v1 can be assessed by measuring one or more indicators of the biological activity of Bet v1 using several standard in vitro or in vivo assays (such as the passive cutaneous anaphylaxis assay described herein) or one or more other in vivo assays known in the art.

[0071] control antibody

[0072] The term "control antibody" refers to the antibody used as a comparison with the antibodies of the present invention in the experimental section. Specifically, three antibodies were used as control antibodies: REGN5713, REGN5714, and REGN5715, all of which are described in WO 2018 / 222854. The VH and VL amino acid sequences of each comparison antibody are listed below.

[0073] REGN5713 (H4H16992P)

[0074] VH-Cγ4 (SEQ ID NO 161):

[0075] QVQLQESGPGLVKPSETLSLTCSVSGGSITNYFWTWIRQSPGKGLEWIGYIYYSGGTNYNPSLKSRVTISIDTSKNQFSLNMNSVTAADTAVYYCAGSYYYGVDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0076] VL-Cκ (SEQ ID NO 164):

[0077] EIVLTQSPATLSLSPGERATLSCRASQSIKSFLAWYRQKPGQAPRLLIYDASNRPTGIPARFSGSGSGTDFTLTINSLESEDFAVYFCQQRNNWPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0078] REGN5714 (H4H1738P2)

[0079] VH-Cγ4 (SEQ ID NO 162):

[0080] EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSFISDSSSNIYYADSVKGRFTISRDNAKKSLYLQMTSLRAEDTAVYYCAREAIGSTSFDNWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0081] VL-Cκ (SEQ ID NO 165):

[0082] EIVMTQSPATLSVSPGERATLSCRASQSVSSSLAWYQQKPGQAPRRLIYSASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAIYYCHQYNNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0083] REGN5715 (H4H16822P)

[0084] VH-Cγ4 (SEQ ID NO 163):

[0085] QVQLVQSGAEVKKPGASVKVSCKASGYTFISYNIFWVRQATGQGLDWMGWMNPFRNNAGYAQKFQGRVTVTWDTSISTAYMELSSLSSEDTAIYYCAREHGSSWGFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0086] VL-Cκ (SEQ ID NO 166):

[0087] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0088] Epitope

[0089] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site, known as a complementary site, in the variable region of an antibody molecule. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on the antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen that elicits a response from B cells and / or T cells. It also refers to the antigenic region that is bound by an antibody. Epitopes can be linear or conformational. Linear epitopes are generated from adjacent amino acid residues in a polypeptide chain. Conformational epitopes are generated from spatially juxtaposed amino acids from different segments of a linear polypeptide chain. In some embodiments, epitopes may comprise determinants of chemically active surface groups of the molecule (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups), and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. Epitopes may also be defined as structural epitopes or functional epitopes. Functional epitopes are typically a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes formed from consecutive amino acids are typically retained when exposed to denaturing solvents, while epitopes formed from tertiary folds are typically lost when treated with denaturing solvents.

[0090] Full-length antibody

[0091] When used herein, the phrase “full-length antibody” refers to an antibody (e.g., a parental or variant antibody) that contains all constant and variable domains of the heavy and light chains, the constant and variable domains corresponding to those that are typically present in wild-type antibodies of the same type.

[0092] Human origin

[0093] The term "human" in this specification means that at least the CDR of an antibody is derived from a human antibody or a humanized transgenic animal, but a "human" antibody may contain other elements different from those derived from the human antibody or the humanized transgenic animal from which the CDR originates. Examples include partial or complete structures of the variable regions of the heavy and / or light chains and partial or complete structures of the constant regions of the heavy and / or light chains. For example, the variable regions, portions thereof, or the CDR may be derived from human IgE antibodies and may be grafted into a scaffold of IgG or IgA antibodies (such as human IgG or IgA antibodies). The peptide sequence of a human antibody may have 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% identity with the sequence of an antibody extracted from a human.

[0094] Preferably, the peptide sequence of the human antibody can have at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the sequence of an antibody extracted from a human.

[0095] IMGT

[0096] In the context of this invention, the term "IMGT" refers to a method for determining the CDR region. Here, the amino acid residues of the immunoglobulin monovariable domain are numbered using conserved amino acids, which always have the same position. For example, cysteine ​​23, tryptophan 41, leucine 89, cysteine ​​104. The FR1 of VHH contains amino acid residues at positions 1-26, the CDR1 of VHH contains amino acid residues at positions 27-38, the FR2 of VHH contains amino acid residues at positions 39-55, the CDR2 of VHH contains amino acid residues at positions 56-65, the FR3 of VHH contains amino acid residues at positions 66-104, the CDR3 of VHH contains amino acid residues at positions 105-117, and the FR4 of VHH contains amino acid residue at position 118 and the remainder of the sequence. The maximum length of the CDR is as defined above. For shorter CDRs, gaps are created (Lefranc et al., 2002).

[0097] Multispecific antibodies

[0098] The term "multispecific antibody or multispecific antigen-binding fragment" in this specification refers to an antibody or antigen-binding fragment capable of binding to different antigens or at least two different epitopes on the same antigen. Conventional monospecific antibodies typically have two identical epitope binding sites (complementary sites), one on each Fab region, and therefore are specific to only one epitope (two complementary sites and single specificity). Multispecific antibodies or multispecific antigen-binding fragments have at least two different types of complementary sites, meaning they can bind to at least two different types of epitopes.

[0099] Specifically, a multispecific antibody may contain two or more complementary sites, where one or more complementary sites may be identical, such that all complementary sites of the construct belong to at least two different types of complementary sites, thus the antibody has at least two specificities. For example, a multispecific antibody may contain four complementary sites, where every two complementary sites are identical, meaning they have the same specificity. Therefore, a multispecific antibody is both bispecific and tetravalent. Thus, a monospecific antibody refers to one or more complementary sites having the same specificity. Bispecific refers to an antibody having one, two, three, or other complementary sites but in total having two specificities. For example, a bispecific antibody may have one complementary site for each specificity. In another embodiment, a multispecific antibody may have two or more complementary sites for each specificity.

[0100] Therefore, in one embodiment, the multispecific antibody may be bispecific, trispecific, or tetraspecific.

[0101] complementary position

[0102] The term "complementary site" refers to the antigen-binding (or epitope-binding) site of an antibody.

[0103] Recombinant host cells

[0104] As used herein, the phrase “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 such terms are intended to refer not only to the specific test cell but also to the progeny of such cells. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may actually differ from the parent cells but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfected tumors such as CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytes, as well as prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.

[0105] Therapeutic effective dose

[0106] The phrase “therapeutic effective amount” means the amount that defines the desired effect produced by its application. The exact amount will depend on the purpose of treatment and will be determined by a person skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0107] Tree pollen allergens.

[0108] The phrase “tree pollen allergen” in this document refers to allergens that include at least the pollen from birch, alder, hazel, and / or oak trees. Among these tree pollen allergens, the most clinically relevant allergens are the major allergens Bet v1, Aln g 1, Cor a 1, and Que a 1. The prefix “n” before the allergen name (e.g., nBet v1) indicates that the allergen exists in its naturally occurring form, including isotypes and various naturally occurring post-translational derivatives (e.g., glycosylation). Natural allergens may be indicated by the prefix (n) to distinguish them from recombinant allergens, which are indicated by the prefix (r) before the allergen name (e.g., nBet v1 vs. rBet v1). The term “natural allergen” should be used to refer to any allergen purified from materials of natural origin.

[0109] Typically, as used in this article, the phrase “tree pollen allergen” is intended to refer to a group of allergens including Bet v1, Aln g1, Cor a1, and Que a1.

[0110] carrier

[0111] As used herein, the phrase “vector” is intended to refer to a nucleic acid molecule capable of inducing transcription of a nucleic acid segment linked to the 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 a nucleic acid segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (such as non-attached mammalian vectors) can be integrated into the host cell’s genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are effectively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Typically, expression vectors useful in recombinant DNA technology are in plasmid form. In this specification, “plasmid” and “vector” are used interchangeably because plasmids are the most commonly used form of vector. However, this invention is intended to include other forms of expression vectors, such as viral vectors (such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

[0112] Short sequences not listed in the sequence list conforming to WIPO Standard 26 form part of this specification.

[0113] The following sequences are not included in the sequence listing because of their short length:

[0114]

[0115] Specific embodiments of the present invention

[0116] Antibody

[0117] According to a first aspect of the invention, antibodies or antigen-binding fragments thereof are provided capable of binding 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 can be specific for one, two, three, or four of the allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1, and Que a 1. Each of the antibodies described herein can be used alone or in combination of two, three, four, or more antibodies to promote the clearance 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 to 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 to pre-formed IgE in the subject, thereby leading to reduced activation of mast cells and basophils. In one embodiment of interest, the antibody or its antigen-binding fragment, or a combination of antibodies or their antigen-binding fragments, may be able to alleviate 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.

[0118] In some preferred embodiments, at least the CDR region is derived from a human antibody, such as a human IgE antibody, IgG1, or IgG4 antibody. Thus, a CDR region signifies a 100% amino acid sequence alignment with the human antibody in which the CDR region was initially detected. In a further preferred embodiment, the antibody disclosed herein preferably has a full-length human IgG scaffold, such as IgG1 or IgG4, but may be equipped with any Fc portion of the human antibody, such as IgE-Fc, IgG-Fc, or IgA-Fc. The hinge region may contain the mutant S228P to avoid Fab arm exchange. Therefore, the antibody disclosed herein may be a fully human antibody, but optionally contains small amounts, such as one, two, or three amino acid substitutions, deletions, or additions, to obtain an antibody with advantageous properties (e.g., extended half-life). In some alternative embodiments, the antibody may contain only the antigen-binding portion, such as Fab.

[0119] 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 v1, Aln g 1, Cor a 1, and Que a 1, such as binding to each of the allergens Bet v1 and Aln g 1. Preferably, the antibody or its antigen-binding fragment is capable of binding to at least three allergens selected from the group consisting of Bet v1, Aln g 1, Cor a 1, and Que a 1, such as binding to each of the allergens Bet v1, Aln g 1, and Cor a 1. More preferably, the antibody or its antigen-binding fragment is capable of binding to each of the allergens Bet v1, Aln g 1, Cor a 1, and Que a 1.

[0120] In another embodiment, the antibody or its antigen-binding fragment is capable of:

[0121] Binds to allergen Bet v1 with an affinity of 1 nM or less; and / or

[0122] Binds to allergen Aln g 1 with an affinity of 1 nM or less; and / or

[0123] Binds to allergen Cor a 1 with an affinity of 300 nM or less; and / or with

[0124] It binds to the allergen Que a 1 with an affinity of 300 nM or less.

[0125] Affinity can be determined by biolayer interferometry (BLI).

[0126] In a preferred embodiment, the Fc portion is IgG4-Fc.

[0127] The affinity of each antibody or its antigen-binding fragment is used to describe the strength of binding to the target (here used for binding to tree pollen allergens described herein), and in a preferred embodiment may be less than 1000 nM, such as 300 nM, such as 100 nM, such as 10 nM, preferably 1 nM, more preferably 0.1 nM, and even more preferably 0.01 nM.

[0128] This invention relates to antibodies or antigen-binding fragments thereof capable of binding to allergens selected from the group consisting of Bet v 1, Aln g 1, Cor a 1 and Que a 1.

[0129] 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 v1, Alng 1, Cor a 1, and Que a 1, such as binding to each of the allergens Bet v1 and Alng 1. Preferably, the antibody or its antigen-binding fragment is capable of binding to at least three allergens selected from the group consisting of Bet v1, Alng 1, Cor a 1, and Que a 1, such as binding to each of the allergens Bet v1, Alng 1, and Cor a 1. More preferably, the antibody or its antigen-binding fragment is capable of binding to each of the allergens Bet v1, Alng 1, Cor a 1, and Que a 1. In another embodiment, the antibody or its antigen-binding fragment is capable of...

[0130] Binds to allergen Bet v1 with an affinity of 1 nM or less; and / or

[0131] Binds to allergen Aln g 1 with an affinity of 1 nM or less; and / or

[0132] Binds to allergen Cor a1 with an affinity of 300 nM or less; and / or

[0133] Que a 1 binds to the allergen with an affinity of 300 nM or less.

[0134] Affinity can be determined by BLI assay.

[0135] In another embodiment, the antibody or its antigen-binding fragment is capable of binding to the allergen Bet v1 with an affinity of 1000 nM or less, such as 300 nM or less, such as 100 nM or less, such as 10 nM or less.

[0136] In another embodiment, the antibody or its antigen-binding fragment is capable of binding allergen Aln g 1 with an affinity of 1000 nM or less, such as 300 nM or less, such as 100 nM or less, such as 10 nM or less.

[0137] In another embodiment, the antibody or its antigen-binding fragment is capable of binding to the allergen Cor a1 with an affinity of 1000 nM or less, such as 300 nM or less, such as 100 nM or less, such as 10 nM or less.

[0138] In another embodiment, the antibody or its antigen-binding fragment is capable of binding to the allergen Que a 1 with an affinity of 1000 nM or less, such as 300 nM or less, such as 100 nM or less, such as 10 nM or less.

[0139] In yet another embodiment, the antibody or its antigen-binding fragment is capable of binding 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, such as 300 nM or less, such as 100 nM or less, such as 10 nM or less.

[0140] In one embodiment, the antibody or its antigen-binding fragment is capable of binding to the allergen Bet v1 with an affinity of 1 nM or less.

[0141] In one embodiment, the antibody or its antigen-binding fragment is capable of binding allergen Aln g 1 with an affinity of 1 nM or less.

[0142] In another embodiment, the antibody or its antigen-binding fragment is capable of binding the allergen Cor a1 with an affinity of 300 nM or less.

[0143] In another embodiment, the antibody or its antigen-binding fragment is capable of binding the allergen Que a 1 with an affinity of 300 nM or less.

[0144] As mentioned above, affinity can be determined by BLI.

[0145] The antibodies or antigen-binding fragments thereof described herein may be obtained from humans. The individuals from whom the antibodies or antigen-binding fragments thereof 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, optionally without 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.

[0146] Therefore, in a preferred embodiment, the antibody of the present invention is a human antibody.

[0147] Therefore, in one implementation, the antibody is human-derived (such as a human antibody) and its antigen-binding fragment is derived from a human amino acid sequence.

[0148] In another embodiment, the antibody is a human antibody whose antigen-binding fragment contains a human amino acid sequence.

[0149] In some embodiments, the antibody is derived from a person 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 person sensitized to Bet v 1. In another embodiment, the antibody is derived from a person sensitized to Cor a 1. In another embodiment, the antibody is derived from a person sensitized to Aln g 1. In another embodiment, the antibody is derived from a person sensitized to Que a 1.

[0150] Each antibody of the present invention comprises six separate CDR regions in its allergen-binding portion: three in the heavy chain variable region (HCVR) and three in the light chain variable region (LCVR), which, when combined, are referred to as the "fab" domain. Furthermore, the fab domain can be coupled to a constant FC region.

[0151] Table 1 shows non-limiting exemplary antibodies defined by their fab regions. Table 1 provides the full-length amino acid sequences of the individual HCVR and LCVR for each antibody, which combine to form the fab region.

[0152] Table 1 shows examples of the antibodies of the present invention. Each antibody (mAb) is defined by HCVR and LCVR sequences.

[0153]

[0154] Therefore, one embodiment of the present invention relates to an antibody or antigen-binding fragment thereof 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).

[0155] In an embodiment of the first aspect of the invention, the antibody or its antigen-binding fragment has a combination of HCVR and LCVR with amino acid sequences of SEQ ID NO:1 and 2 (HCVR and LCVR of antibody 2E02, respectively), SEQ ID NO:21 and 22 (HCVR and LCVR of antibody 2C10, respectively), SEQ ID NO:41 and 42 (HCVR and LCVR of antibody 2B04, respectively), SEQ ID NO:61 and 62 (HCVR and LCVR of antibody 2E07, respectively), SEQ ID NO:81 and 82 (HCVR and LCVR of antibody A07, respectively), SEQ ID NO:101 and 102 (HCVR and LCVR of antibody B10, respectively) or SEQ ID NO:121 and 122 (HCVR and LCVR of antibody 2_10, respectively), wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions.

[0156] In another embodiment, each combination of HCVR and LCVR as shown in Table 1 is linked to an FC domain (preferably an IgG-Fc domain, more preferably an IgG4-Fc domain).

[0157] Because this antibody may undergo affinity maturation, humanization, or other amino acid changes in its amino acid sequence, alternative embodiments involve having a component selected from SEQ ID NO: Antibodies with amino acid sequences that have at least 80% (such as at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) identity in groups consisting of 1 and 2, 21 and 22, 41 and 42, 61 and 62, 81 and 82, 101 and 102 or 121 and 122.

[0158] Therefore, another embodiment of the first aspect of the invention relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises HCVR and LCVR, wherein,

[0159] 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 (HCVR and LCVR of antibody 2E02, respectively), wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or

[0160] 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 (HCVR and LCVR of antibody 2C10, respectively), wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions; or

[0161] The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence SEQ ID NO:41 and an LCVR having the amino acid sequence SEQ ID NO:42 (HCVR and LCVR of antibody 2B04, respectively), wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or

[0162] The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence SEQ ID NO:61 and an LCVR having the amino acid sequence SEQ ID NO:62 (HCVR and LCVR of antibody 2E07, respectively), wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or

[0163] The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence SEQ ID NO:81 and an LCVR having the amino acid sequence SEQ ID NO:82 (HCVR and LCVR of antibody A07, respectively), wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or

[0164] The antibody or its antigen-binding fragment comprises an HCVR having the amino acid sequence SEQ ID NO:101 and an LCVR having the amino acid sequence SEQ ID NO:102 (HCVR and LCVR of antibody B10, respectively), wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions; or

[0165] 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 (HCVR and LCVR of antibody 2_10, respectively), wherein each amino acid sequence may have 1, 2 or 3 amino acid substitutions.

[0166] In a preferred embodiment, the antibody or its antigen-binding fragment comprises HCVR and LCVR as defined for antibodies selected from the group consisting of 2E02, 2C10, 2B04, 2E07, and A07.

[0167] Antibodies defined by CDR regions

[0168] Those skilled in the art will know that each variable domain (including HCVR and LCVR in the antibody or its antigen-binding region) contains three complementarity-determining regions (CDRs), which are distributed by four framework regions (FR1-4) spaced apart.

[0169] In this article, the antibody binding region means that it includes one or more or all six complementarity-determining regions, three of which are on the HCVR (HCDR1, HCDR2, and HCDR3) and three of which are on the LCVR (HCDR1, HCDR2, and HCDR3).

[0170] The primary purpose of the CDR region is to define epitope-specific regions. However, not all residues in the CDR are responsible for epitope binding; they only determine structural elements (Wilton E. et al. 2018, Reference 6). Furthermore, the framework region is not specifically responsible for structure. Residues in the FR adjacent to the CDR can influence binding.

[0171] Different methods can be used to determine different antibody frameworks and the location of CDR regions.

[0172] One approach is the “Kabat numbering scheme” or “Kabat”, first described by American scientist Elvis Kabat. Here, the amino acid residues of the immunoglobulin single variable domain can be numbered according to the general numbering of the VH domain given by Kabat et al. (Sequence of proteins of immunological interest, U.S. Public Health Service, NIH Bethesda, MD, Publication No. 91).

[0173] It should be noted that, as is well known in the art, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number. That is, one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number. This means that, generally, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. The total number of amino acid residues in the HCVR domain is typically in the range of 110 to 120, usually between 112 and 115. However, it should be noted that smaller and longer sequences are also suitable for the purposes described herein.

[0174] Another method for identifying CDR regions in HCVR and LCVR is through the Chothia numbering scheme. Here, the amino acid residues of the immunoglobulin monovariable domain are numbered using conserved amino acids that always have the same position (Dondelinger M et al., frontiers in immunology 2018).

[0175] Another method for determining the CDR region in HCVR and LCVR is the IMGT method. Here, the amino acid residues of the immunoglobulin monovariable domain are numbered using conserved amino acids that always have the same position. For example, cysteine ​​23, tryptophan 41, leucine 89, cysteine ​​104. FR1 of HCVR and LCVR contains amino acid residues at positions 1-26, CDR1 of HCVR and LCVR contains amino acid residues at positions 27-38, FR2 of HCVR and LCVR contains amino acid residues at positions 39-55, CDR2 of HCVR and LCVR contains amino acid residues at positions 56-65, FR3 of HCVR and LCVR contains amino acid residues at positions 66-104, CDR3 of HCVR and LCVR contains amino acid residues at positions 105-117, and FR4 of HCVR and LCVR contains amino acid residue at position 118 and the remainder of the sequence. The maximum length of the CDR is as defined above. For shorter CDRs, gaps are created (Lefranc et al. 2002 – Developmental and Comparative Immunology).

[0176] Another method for identifying the CDR region in an antibody is by using the Aho numbering scheme (Mitchelle & Colwell, Proteins, 2017 and Honegger & Pluckthun et al., 2001).

[0177] Other definitions of CDRs that have received attention include, but are not limited to, those published in the following literature: Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001; 309: 657-670; Ofran et al., “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008; 181: 6230-6235; Almagro, “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 Padlan et al., “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of the references cited is explicitly incorporated into this paper by reference.

[0178] As previously mentioned, the combination of CDR regions in an antibody determines its specificity. The six independent CDRs (three in each of the HCVR and LCVR regions) can be determined by different methods (including IMGT, Kabat, or Chothia as previously mentioned).

[0179] Tables 2a-2c provide the amino acid sequences (determined by IMGT, Kabat, and Chothia methods) of the six CDR regions for each exemplary antibody disclosed in Table 1.

[0180] Table 2a shows the amino acid sequences of HCDR1, HCRD2, HCDR3, LCDR1, LCDR2, and LCDR3 for each fab region determined by IMGT.

[0181]

[0182] Table 2b shows the amino acid sequences of HCDR1, HCRD2, HCDR3, LCDR1, LCDR2, and LCDR3 for each fab region determined by Kabat.

[0183]

[0184] Table 2c shows the amino acid sequences of HCDR1, HCRD2, HCDR3, LCDR1, LCDR2, and LCDR3 for each fab region determined by Chothia.

[0185]

[0186] Therefore, a first aspect of the invention also relates to antibodies defined by their CDR regions (e.g., as determined by IMGT). One aspect relates to an antibody or an antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein;

[0187] 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, wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody 2E02); or

[0188] 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, wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody 2C10); or

[0189] 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, wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody 2B04); or

[0190] 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, wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody 2E07); or

[0191] 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, wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody A07); or

[0192] 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, wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody B10); or

[0193] 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, wherein each amino acid sequence may have 1, 2, or 3 amino acid substitutions (CDR of antibody 2_10).

[0194] 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) as defined for antibodies selected from the group consisting of 2E02, 2C10, 2B04, 2E07, and A07.

[0195] CDR as determined by HCVR and LCVR

[0196] Alternatively, a single CDR region may originate from a larger sequence and be identified by different numbering schemes (such as, but not limited to, Kabat, Chothia, or IMTG). Thus, in one embodiment, the antibody or antigen-binding fragment of the present invention comprises a combination of complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) in the HCVR and LCDR1, LCDR2, and LCDR3 in the LCVR, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise, or consist of, an amino acid sequence as determined by Kabat, Chothia, or IMGT in any of the amino acid sequences selected from SEQ ID NO: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121, and 122. Because such sdAbs may undergo affinity maturation, humanization, or other amino acid changes in the amino acid sequence, alternative embodiments involve monomeric sdAbs having an amino acid sequence that is at least 80% (such as at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121, and 122.

[0197] In another embodiment, the antibody or antigen-binding fragment of the present invention comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the CDR has an amino acid sequence as determined by Kabat, Chothia, IMTG, or Aho in any of the amino acid sequences selected from SEQ ID NO: 1, 2, 21, 22, 41, 42, 61, 62, 81, 82, 101, 102, 121, and 122. Preferably, one, two, or three amino acids in each CDR region determined by Kabat, Chothia, IMTG, or Aho may be substituted to obtain desired effects, such as altered affinity and / or lower immunogenicity.

[0198] Therefore, in other words, the first aspect of the present invention relates to an antibody or an antigen-binding fragment thereof 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.

[0199] Therefore, the first aspect also relates to antibodies or antigen-binding fragments thereof comprising three heavy CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein;

[0200] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence, the HCDR2 amino acid sequence, and the HCDR3 amino acid sequence contained in SEQ ID NO:1, the LCDR1 amino acid sequence, the LCDR2 amino acid sequence, and the LCDR3 amino acid sequence contained in SEQ ID NO:2 (antibody 2E02); or

[0201] The antibody or its antigen-binding fragment comprises 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

[0202] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence, the HCDR2 amino acid sequence, and the HCDR3 amino acid sequence contained in SEQ ID NO:41, the LCDR1 amino acid sequence, the LCDR2 amino acid sequence, and the LCDR3 amino acid sequence contained in SEQ ID NO:42 (antibody 2B04); or

[0203] The antibody or its antigen-binding fragment comprises 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

[0204] The antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence, the HCDR2 amino acid sequence, and the HCDR3 amino acid sequence contained in SEQ ID NO:81, the LCDR1 amino acid sequence, the LCDR2 amino acid sequence, and the LCDR3 amino acid sequence contained in SEQ ID NO:82 (antibody A07); or

[0205] The antibody or its antigen-binding fragment comprises 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 LCDR2 amino acid sequence contained in SEQ ID NO:102, and the LCDR3 amino acid sequence contained in SEQ ID NO:102 (antibody B10); or

[0206] 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).

[0207] Each CDR (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3) as described above can be determined according to the IMGT, kabat or Chothia method.

[0208] Optionally, one, two, or three amino acids may be substituted in each CDR region determined by the IMGT, Kabat, or Chothia method, such as 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.

[0209] 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), wherein the CDRs are derived from antibodies selected from the group consisting of 2E02, 2C10, 2B04, 2E0, A07, B10 and 2_10.

[0210] Modification of the amino acid sequence of antibodies and their antigen-binding fragments.

[0211] For the purpose of modifying biological activity, expression levels, stability, or other functional properties, any amino acid sequence (including the fab and Fc regions) in the CDR or external framework regions may be substituted, inserted, or deleted. In a preferred embodiment, amino acid changes are integrated only outside the CDR region.

[0212] In some embodiments, the antibodies of the present invention consist of affinity-matured human or humanized amino acid sequences. In other embodiments, the amino acid sequence of the CDR region may not change after affinity maturation or humanization of the antibody. In such embodiments, the antibody consists of affinity-matured human or humanized amino acid sequences, but not in the CDR region involved in target binding.

[0213] In some embodiments, one or more CDRs (i.e., CDR1, CDR2, and / or CDR3) of an HCVR or LCVR can be independently substituted with amino acids, such as substituted with one, two, three, or more amino acid residues. Amino acid substitutions in CDRs can be conserved amino acid substitutions. A “conserved” amino acid substitution is typically an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a similar chemical structure and / or charge, said substitution having little or no effect on the function, activity, or other biological properties of the resulting binding region of the antibody described herein. Such conserved amino acid substitutions are well known in the art. For example, conservative substitutions are preferred where one amino acid residue from the following groups (a)-(e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar residues with negative charges and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar residues with positive charges: His, Arg, and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0214] The following are particularly preferred conserved amino acid substitutions: Ala becomes Gly; Ala becomes Ser; Arg becomes Lys; Asn becomes Gln; Asn becomes His; Asp becomes Glu; Cys becomes Ser; Gln becomes Asn; Glu becomes Asp; Gly becomes Ala; Gly becomes Pro; His becomes Asn; His becomes Gln; Ile becomes Leu; Ile becomes Val; Leu becomes Ile; Leu becomes Val; Lys becomes Arg; Lys becomes Gln; Lys becomes Glu; Met becomes Leu; Met becomes Tyr; Met becomes Ile; Phe becomes Met; Phe becomes Leu; Phe becomes Tyr; Ser becomes Thr; Thr becomes Ser; Trp becomes Tyr; Tyr becomes Trp; Phe becomes Val; Phe becomes Ile and / or Phe or becomes Leu.

[0215] Conserved amino acid substitutions can also be performed in non-CDR amino acid sequences.

[0216] In other embodiments, amino acid substitutions may be made in the amino acid sequence of the CDR to provide an antibody or antigen-binding fragment thereof with altered (e.g., enhanced) affinity for the target. The amino acid sequence of the CDR is generally more determinant of target affinity than the amino acid sequence outside the CDR. Therefore, the antibodies or antigen-binding fragments thereof described herein may also undergo affinity maturation by introducing one or more alterations into the amino acid sequence of one or more CDRs, resulting in an altered, for example, enhanced, affinity for the target compared to a first (parental) antibody or antigen-binding fragment thereof. Methods for affinity maturation of antibodies or antigen-binding fragments thereof are known in the art and are routine for those skilled in the art.

[0217] Amino acid substitutions, insertions, or deletions may also occur in one or more framework regions outside the CDR.

[0218] Compared to substitutions within the CDR, any amino acid substitutions in the amino acid sequence outside the CDR generally provide less modified biological activity for the antibody or its antigen-binding fragment. However, depending on the host organism used to express the antibody or its antigen-binding fragment described herein, any alterations (e.g., deletions, insertions, and / or substitutions) in the amino acid sequence of the antibody or its antigen-binding fragment can also be designed to increase expression levels. For example, alterations can be designed to remove one or more post-translational modification sites (such as one or more glycosylation sites), which is within the capabilities of those skilled in the art. Alternatively, substitutions or insertions can be designed to introduce one or more sites for attaching functional groups, for example, to allow the insertion of an affinity tag (His tag) or site-specific PEGylation. The possibility of N-terminal post-translational modification can be eliminated by changing the N-terminal glutamic acid (E) to aspartic acid (D). Thus, the amino acid difference could be that glutamic acid (Glu) at position 1 (as determined by the Kabat number) is changed to aspartic acid (Asp).

[0219] As mentioned above, sequence variations may occur in the parental CDR region, such as the substitution, deletion, or addition of 1, 2, or 3 amino acids. This variant may have the same, enhanced, or strengthened allergen-binding activity.

[0220] mutation

[0221] As disclosed herein, fully human monoclonal antibodies that specifically bind to Bet v1, Que a1, Aln g1, and / or Cor a1 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 from, for example, public antibody sequence databases. This invention includes antibodies and antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated to one or more corresponding residues of the germline sequence from which the antibody is derived, or mutated to one or more corresponding residues of another human germline sequence, or mutated to conserved amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily generate numerous antibody and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein.

[0222] FC region of antibody

[0223] As described above, the antibody fragments (e.g., Fc regions) of the antibodies disclosed herein can be fragments of 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 with altered Fc portions that optionally provide reduced or enhanced effector cell activity or biodistribution, serum half-life, or excretion rate. The antibody fragments can be derived from any animal species, but are preferably human-derived Fc. Exemplary effector functions include C1-q binding; GDC; Fc-receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions typically require interaction between the Fc region and the receptor, such as through FcyR, IFcyRIIA, FcyRIIBI, FcyRIIB2, FcyRIIIA, FcyRIIIB receptors, and / or low-affinity FcRn receptors. Furthermore, the Fc region can be a "dead" Fc, which is an Fc that has been mutagenized to maintain, for example, prolonged serum half-life, but does not activate high-affinity Fc receptors. The Fc may also have reduced complement binding.

[0224] When Fc interaction with receptors (e.g., FcyRIIB) is undesirable, antibody fragments can be CH1 immunoglobulin domains (such as IgG1-CH1 or IgG4-CH1 domains). Unlike the commonly used IgG-FC domains, they do not bind to inhibitory FcγRIIb receptors, 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).

[0225] In some implementations, the antibody fragment is derived from IgG4, such as IgG4-Fcm, such as human IgG4-Fc.

[0226] Therefore, the Fc portion of the antibody of the present invention can be selected from the IgG4 antibody fragment.

[0227] IgG4 is unstable in vivo because the "half-antibody exchange" phenomenon means that it becomes bispecific (or, in most cases, a functional monomer). Therefore, when used for therapeutic purposes, a single amino acid mutation can be introduced into the hinge region to prevent this dissociation—the so-called S228P mutation.

[0228] Another way to prevent IgG4 from becoming unstable in vivo is to use IgG4-Fc scaffolds in the form of knobs-into-holes (KIH), which can prevent such dissociation.

[0229] Therefore, in one implementation, the Fc portion is IgG4 Fc.

[0230] In another implementation, IgG4 Fc contains the mutation S228P.

[0231] Furthermore, the Fc region can be a natural sequence Fc region containing the same amino acid sequence as a naturally occurring Fc region. Natural sequence human Fc regions include the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as its naturally occurring variants. Alternatively, the Fc region can be a variant Fc region containing an amino acid sequence that differs from the natural sequence Fc region by at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the natural sequence Fc region or the Fc region of the parent peptide, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the natural sequence Fc region or the Fc region of the parent peptide. The variant Fc region described herein 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 parental antibody fragment, most preferably at least about 90% homology, and more preferably at least about 95% homology.

[0232] In one implementation, the variant Fc sequence may include three amino acid substitutions in the CH2 region to reduce FcyRI 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)). Substituting the lgG2 residues at positions 233-236 and the lgG4 residues at positions 327, 330, and 331 into human lgG1 significantly reduces ADCC and CDC (see, for example, Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 2001, J Biol Chem. 276(9):6591-604). Other Fc variants are also possible, including, but not limited to, variants in which a region capable of forming disulfide bonds is deleted, or in which certain amino acid residues are eliminated at the N-terminus of the native Fc form or methionine residues are added. Thus, one or more Fc portions of the molecule may contain one or more mutations in the hinge region to eliminate disulfide bonding. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, the molecule may contain Fc variants.

[0233] Furthermore, Fc variants can be constructed by substituting, deleting, or adding amino acid residues to achieve complement binding or Fc receptor binding. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications Nos. WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0234] Antibody fragments, such as the Fc moiety, can also be modified by substituting, deleting, or inserting amino acid residues for constructive reasons (e.g., expression yield). Non-limiting examples of mutations introduced to improve expression yield are described below.

[0235] Antibody fragments can be modified to have native glycans, an increased number of glycans compared to the native form, or a reduced number of glycans compared to the native form, or they can be aglycosylated or deglycosylated. The increase, decrease, removal, or other modification of glycans can be achieved by methods commonly used in the art (such as chemical methods, enzymatic methods) or by expression in genetically engineered cell lines. Such cell lines can include microorganisms that naturally express glycosylation enzymes, such as Pichia pastoris, and mammalian cell lines, such as CHO cells. Furthermore, microorganisms or cells can be engineered to express glycosylation enzymes, or to prevent them from expressing glycosylation enzymes. As an example of cells engineered to have altered sialylation activity, the α-2,6-sialyltransferase 1 gene has been engineered into Chinese hamster ovary cells and Sf9 cells. The construct expressed by these engineered cells is thus sialylated with the exogenous gene product. Another method for obtaining Fc molecules with altered amounts of sugar residues compared to a variety of natural molecules involves, for example, separating the various molecules into glycosylated and non-glycosylated fractions using lectin affinity chromatography. The presence of specific glycosylated moieties has been shown to alter immunoglobulin function. For example, removal of sugar chains from Fc molecules results in a sharp decrease in binding affinity to the C1-q portion of the first complement component C1, leading to a reduction or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thus preventing the induction of unwanted immune responses in vivo. Other important modifications include sialylation and fucosylation: the presence of sialic acid in IgG is associated with anti-inflammatory activity, while removal of fucose from IgG results in enhanced ADCC activity. Constructs can, for example, possess Fc sequences with enhanced effector functions by enhancing their binding affinity to FcyRI and enhancing ADCC activity. For example, fucose attached to the / V-linked glycan at Asn-297 of Fc spatially hinders the interaction between Fc and FcyRIIIA, and removing fucose through glycoengineering increases binding to FcyRIIIA, translating into ADCC activity more than 50-fold higher than the wild-type IgG1 control. Protein engineering has generated several variants that increase the affinity of Fc for FcyRIIIA binding through amino acid mutations in the Fc moiety of IgG1. Notably, the triple alanine mutant S298A / E333A / K334A exhibits a 2-fold increase in FcyRIIIA binding and ADCC function. The S239D / I332E (2X) and S239D / I332E / A330L (3X) variants show significantly increased affinity for FcyRIIIA binding and enhanced ADCC capacity both in vitro and in vivo. Other Fc variants identified by yeast display have also shown enhanced FcyRIIIA binding.See, for example, Liu et al. (2014) JBC 289(6):3571-90, which is explicitly incorporated herein by reference.

[0236] As described above, antibodies can be engineered to provide multispecificity against 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 those disclosed herein.

[0237] The second aspect is the composition / combination of antibodies.

[0238] According to a second aspect of the invention, the antibodies or antigen-binding fragments thereof disclosed herein may be combined in a composition comprising two or more antibodies.

[0239] In one embodiment of the invention, the composition comprises an antibody, wherein each antibody or antigen-binding fragment is capable of simultaneously binding to a Bet v1 molecule.

[0240] When combining different antibodies, the key is to select antibodies that can bind simultaneously to the target allergen (such as Bet v1 and / or any of the other three tree pollen allergens). For example, BLI can be used to verify long-distance epitope binding through epitope binning studies. Furthermore, antibodies can be selected based on their binding affinity to all target allergens (such as all four tree pollen allergens mentioned in this article).

[0241] In one embodiment of the invention, the composition comprises an antibody or antigen-binding fragment capable of simultaneously binding to the same Bet v 1 molecule. In one embodiment, the composition comprises an antibody or antigen-binding fragment capable of simultaneously binding to the same Que a 1 molecule. In one embodiment of the invention, the composition comprises an antibody or antigen-binding fragment capable of simultaneously binding to the same Aln g 1 molecule. In one embodiment, the composition comprises an antibody or antigen-binding fragment capable of simultaneously binding to the same Cor a 1 molecule.

[0242] In a preferred embodiment of the invention, the composition comprises an antibody or antigen-binding fragment capable of simultaneously binding to the same Bet v 1 and / or Cor a1 and / or Aln g 1 and / or Que a 1.

[0243] like Figure 2 As can be seen, the antibodies in Table 1 can be grouped according to the epitopes they bind to on allergens. When a group of antibodies binds to a distant epitope on the same target allergen (meaning...), the antibodies can be grouped together. Figure 2 When non-overlapping circles are formed, they may simultaneously bind to the target allergen and then block the allergen from binding to IgE, thus reducing allergic reactions.

[0244] In the example, antibodies 2B04, A07, and 2E02 (in) Figure 2 (Marked in bold and dashed circles) does not combine any overlapping epitopes, such as Figure 2 As shown in the circle that is split in the middle.

[0245] Furthermore, the same applies to antibodies 2B04, A07, and 2C10. Figure 2 In the diagram, they are shown as completely separated by thick dashed circles. Further description is given in Example 3.

[0246] The number of antibodies capable of blocking the binding of one or more target allergens to IgE can be tested in a BLI assay. Typically, a combination of three antibodies is sufficient to achieve effective IgE blockade. However, occasionally two different antibodies may provide the desired effect.

[0247] Therefore, a second aspect of the invention relates to compositions comprising two, such as at least two, antibodies or antigen-binding fragments thereof. Typically, the composition comprises three, such as at least three, antibodies or antigen-binding fragments thereof, wherein the antibodies are selected from antibodies or antigen-binding fragments thereof as defined herein.

[0248] In one embodiment, the composition comprises four different antibodies or antigen-binding fragments thereof, such as the five different antibodies or antigen-binding fragments thereof as defined herein.

[0249] In a preferred embodiment, the composition comprises two or three antibodies. As described above, the combination of antibodies should be able to prevent IgE in a person suffering from tree pollen allergy from binding to tree pollen allergens. Preferably, the combination should provide at least satisfactory blocking activity against Bet v1. More preferably, the combination should provide blocking activity against one or more, preferably all, tree pollen allergens selected from the group consisting of Bet v1, Aln g1, Cor a1, and Que a1. Typically, blocking activity can be determined by a basophil activation test using basophils from an individual with tree pollen allergy, showing a reduction in IgE binding to one or all of the four tree pollen allergens.

[0250] In some embodiments, the composition comprises two antibodies or antigen-binding fragments thereof having antigen-binding regions (such as CDRs contained in antibodies selected from the group consisting of antibodies 2B04, A07, and 2E02 or the group consisting of antibodies 2B04, A07, and 2C10). In a preferred embodiment, the composition comprises two antibodies or antigen-binding fragments thereof with antigen-binding regions, such as CDRs contained in antibodies selected from the group consisting of antibodies 2B04, A07, and 2E02 or the group consisting of antibodies 2B04, A07, and 2C10.

[0251] Furthermore, as seen in Example 5, the combination of the two antibodies provides a high degree of inhibition against the binding of allergens to IgE, thereby inhibiting basophil activation. It was also revealed that antibody A07 provides high blocking activity in the basophil activation assay. Therefore, the compositions described herein may at least comprise an antibody or an antigen-binding fragment thereof having an antigen-binding region (such as the CDR contained in antibody A07).

[0252] Therefore, in one embodiment, the composition comprises a combination of a first antibody and a second antibody, wherein,

[0253] The first 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, and 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; or

[0254] The first 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, and the second 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; or

[0255] The first 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, and 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; or

[0256] The first 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, and the second 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; or

[0257] The first 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 second 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.

[0258] As previously mentioned, antibodies can be defined based on their CDR regions. In the following implementation, the HCDR and LCDR regions have been determined by IMGT.

[0259] Therefore, in other embodiments, the composition comprises a combination of a first antibody and a second antibody, each antibody comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein,

[0260] The first antibody comprises 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; and the second antibody comprises 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

[0261] The first antibody comprises 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; and the second antibody comprises 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

[0262] The first antibody comprises 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; and the second antibody comprises 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

[0263] The first antibody comprises 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; and the second antibody comprises 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

[0264] The first antibody comprises 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, and the second antibody comprises 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.

[0265] In another embodiment, the composition comprises a combination of three antibodies, such as a composition comprising a combination of a first antibody, a second antibody, and a third antibody, wherein,

[0266] 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;

[0267] 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

[0268] 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.

[0269] In another embodiment, the composition comprises a triantibody, wherein,

[0270] 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;

[0271] 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

[0272] 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.

[0273] In yet another embodiment, the composition comprises three antibodies, wherein,

[0274] 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;

[0275] 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

[0276] 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.

[0277] In yet another embodiment, the composition comprises three antibodies, wherein,

[0278] The first 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;

[0279] 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

[0280] 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.

[0281] In yet another embodiment, the composition comprises three antibodies, wherein,

[0282] The first 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;

[0283] 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

[0284] 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.

[0285] In yet another embodiment, the composition comprises three antibodies, wherein,

[0286] The first 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;

[0287] 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

[0288] 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.

[0289] As previously mentioned, antibodies can be defined based on their CDR regions. Therefore, in one embodiment, the composition comprises three antibodies, wherein...

[0290] The first antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:83, 84 and 85 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:86, 87 and 88 respectively.

[0291] The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NO:43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:46, 47, and 48, respectively; and

[0292] The third antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:3, 4 and 5 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:6, 7 and 8 respectively.

[0293] In another embodiment, the composition comprises a triantibody, wherein,

[0294] The first antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:83, 84 and 85 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:86, 87 and 88 respectively.

[0295] The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NO:43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:46, 47, and 48, respectively; and

[0296] The third antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:123, 124 and 125 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:126, 127 and 128 respectively.

[0297] In yet another embodiment, the composition comprises three antibodies, wherein

[0298] The first antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:83, 84 and 85 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:86, 87 and 88 respectively.

[0299] The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NO:43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:46, 47, and 48, respectively; and

[0300] The third antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:23, 24 and 25, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:26, 27 and 28, respectively.

[0301] In yet another embodiment, the composition comprises three antibodies, wherein

[0302] The first antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:103, 104 and 105 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:106, 107 and 108 respectively.

[0303] The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NO:43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:46, 47, and 48, respectively; and

[0304] The third antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:3, 4 and 5 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:6, 7 and 8 respectively.

[0305] In yet another embodiment, the composition comprises three antibodies, wherein,

[0306] The first antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:103, 104 and 105 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:106, 107 and 108 respectively.

[0307] The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NO:43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:46, 47, and 48, respectively; and

[0308] The third antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:123, 124 and 125 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:126, 127 and 128 respectively.

[0309] In yet another embodiment, the composition comprises three antibodies, wherein,

[0310] The first antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:103, 104 and 105 respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:106, 107 and 108 respectively.

[0311] The second antibody comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences of SEQ ID NO:43, 44, and 45, respectively, and LCDR1, LCDR2, and LCDR3 having amino acid sequences of SEQ ID NO:46, 47, and 48, respectively; and

[0312] The third antibody comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences of SEQ ID NO:23, 24 and 25, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences of SEQ ID NO:26, 27 and 28, respectively.

[0313] Optionally defined, the compositions herein comprise a first antibody, a second antibody, and a third antibody, each antibody comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein,

[0314] The first antibody comprises 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 comprises 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; and the third antibody comprises 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

[0315] The first antibody comprises each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO:1 and LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO:2; the second antibody comprises 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; and the third antibody comprises 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

[0316] The first antibody comprises each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO:21 and LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO:22; the second antibody comprises 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; and the third antibody comprises 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

[0317] The first antibody comprises 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 comprises 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; and the third antibody comprises 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

[0318] The first antibody comprises 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 comprises 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 comprises 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.

[0319] In another embodiment, the composition comprises:

[0320] An antibody or its antigen-binding fragment comprising 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; and

[0321] One or more isolated human monoclonal antibodies or antigen-binding fragments thereof, which are capable of binding to one or more of the allergens Bet v1, Aln g 1, Cor a 1, and Que a 1.

[0322] In another embodiment, the composition comprises:

[0323] An antibody or its antigen-binding fragment comprising 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; and

[0324] An antibody or its antigen-binding fragment comprising 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.

[0325] In another embodiment, the composition comprises:

[0326] An antibody or its antigen-binding fragment comprising 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;

[0327] An antibody or its antigen-binding fragment comprising 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; and

[0328] An antibody or its antigen-binding fragment comprising 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.

[0329] Pharmaceutical Composition

[0330] A third aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of one or more antibodies or antigen-binding fragments thereof as defined herein, and one or more pharmaceutically acceptable excipients. Thus, the antibodies of the first aspect of the invention or the antibody combinations of the second aspect of the invention can be formulated into pharmaceutical compositions comprising antibodies (including the multispecific antibodies of the first aspect of the invention or the antibody combinations of the second aspect of the invention) and one or more pharmaceutically acceptable excipients.

[0331] Typically, pharmaceutical compositions are formulated for administration by injection, such as subcutaneous (sc) or intramuscular (im) injection.

[0332] Therapeutic applications

[0333] As described herein, the antibodies of the present invention are capable of binding to any different allergen. Furthermore, different antibodies can bind to a single allergen simultaneously because they bind to non-overlapping epitopes, as described in the Examples section. Therefore, by binding to different allergens, a single antibody can block the binding of IgE to that specific allergen. By combining the binding of different antibodies, more allergens can be covered, resulting in increased IgE blocking.

[0334] Therefore, in one embodiment, the antibody or its antigen-binding fragment according to the invention can reduce or inhibit the binding of IgE antibody to Bet v 1 and / or Cor a 1 and / or Aln g 1 and / or Que a 1.

[0335] Since IgE is the direct link between allergens and allergic reactions, inhibiting the binding of IgE to allergens through the activation of mast cells and basophils is an effective way to prevent cell activation, which can be used to treat allergies.

[0336] Therefore, a fourth aspect of the invention relates to a method for preventing or reducing mast cell degranulation associated with sensitization to Bet v 1 and / or Cor a 1 and / or Aln g 1 and / or Que a 1 and / or blocking basophil activation associated with sensitization to Bet v 1 and / or Cor a 1 and / or Aln g 1 and / or Que a 1, the method comprising administering to a subject in need an antibody or antigen-binding fragment thereof according to the first aspect, an antibody combination according to the second aspect, or a pharmaceutical composition according to the third aspect.

[0337] Furthermore, a fourth aspect of the invention relates to a method for treating tree pollen allergy, the method comprising administering to a subject in need an antibody or antigen-binding fragment thereof according to the first aspect, an antibody combination according to the second aspect, or a pharmaceutical composition according to the third aspect.

[0338] Furthermore, a fourth aspect of the invention relates to a method for treating tree pollen allergy in a subject in need of an antibody or antigen-binding fragment thereof according to the first aspect, an antibody combination according to the second aspect, or a pharmaceutical composition according to the third aspect.

[0339] Furthermore, a fourth aspect of the invention relates to the use of an antibody or antigen-binding fragment thereof according to the first aspect, an antibody combination according to the second aspect, or a pharmaceutical composition according to the third aspect for preparing a medicament for treating tree pollen allergy in a subject in need.

[0340] It should be understood that tree pollen allergy can be selected from the group consisting of allergies to birch pollen, alder pollen, hazel pollen, and oak pollen. Furthermore, it is understood that tree pollen allergy may be associated with allergy or sensitization to one or more of the allergens Bet v 1, Cor a 1, Aln g 1, and Que a 1.

[0341] Subjects who are in need may be sensitive to Bet v1. That is, patients may have detectable IgE antibodies against Bet v1, such as IgE levels above 0.7 kU in the blood.

[0342] Another embodiment relates to a method for treating a subject exhibiting sensitivity to Aln g 1, the method comprising administering one or more antibodies according to the invention, antibody compositions described herein, or pharmaceutical compositions.

[0343] One embodiment relates to a method for treating a subject exhibiting sensitivity to Cor a1, the method comprising administering one or more antibodies according to the invention, antibody compositions described herein, or pharmaceutical compositions.

[0344] One embodiment relates to a method for treating a subject exhibiting sensitivity to Que a 1, the method comprising administering one or more antibodies according to the invention, antibody compositions described herein, or pharmaceutical compositions.

[0345] In the preferred embodiment, the subject is a human being.

[0346] Expression System

[0347] The antibodies or antigen-binding fragments described herein can be obtained in several different ways. One option is to use a host cell with an expression vector containing a nucleic acid molecule encoding the antibody or its antigen-binding fragment described herein.

[0348] In this regard, those skilled in the art will readily understand that a polynucleotide encoding a variable domain of at least the light chain and / or the heavy chain can encode a variable domain of two immunoglobulin chains or only one immunoglobulin chain. Similarly, the polynucleotide can be under the control of the same promoter or can be expressed separately.

[0349] Furthermore, this invention relates to vectors, particularly plasmids, granules, viruses, and bacteriophages commonly used in genetic engineering, which contain antigens encoding the present invention.

[0350] Alternatively, the vector may be a polynucleotide of a variable domain of an immunoglobulin chain of an antibody; said polynucleotide may optionally be combined with a polynucleotide of the present invention encoding a variable region of another immunoglobulin chain of the present invention. Preferably, the vector is an expression vector and / or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses can be used to deliver the polynucleotides or vectors of the present invention into a target cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors.

[0351] This invention also relates to host cells transformed with the polynucleotides or vectors of the invention. The host cells can be prokaryotic or eukaryotic cells. The polynucleotides or vectors of the invention present in the host cells can be integrated into the genome of the host cells or can be retained extrachromosomally. Host cells can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant, animal, or human cells. Preferred fungal cells are, for example, those of the genus *Saccharomyces*, particularly those of *Saccharomyces cerevisiae*. The term "prokaryotic" means all bacteria that can be transformed or transfected with DNA or RNA molecules to express the antibodies or corresponding immunoglobulin chains of the invention. Prokaryotic hosts can include Gram-negative and Gram-positive bacteria, such as *Escherichia coli*, *Salmonella typhimurium*, *Serratia marcescens*, and *Bacillus subtilis*. The term "eukaryotic cell" means cells including yeast, higher plants, insects, and preferably mammalian cells, most preferably HEK293, NSO, and CHO cells.

[0352] On the other hand, methods for preparing anti-allergen antibodies or allergen-binding fragments thereof include culturing cells containing polynucleotides encoding antibodies or binding fragments thereof as described herein, and isolating antibodies or allergen-binding fragments thereof from cells or cell culture media.

[0353] In another embodiment, the present invention relates to a method for producing an antibody or a binding fragment thereof, the method comprising:

[0354] (a) Cell culture as described herein; and

[0355] (b) Isolate the antibody or its binding fragment from the culture.

[0356] The transformed host can be grown in a fermenter and cultured according to techniques known in the art to achieve optimal cell growth. Once expressed, the complete antibody of the present invention, its dimer, single light and heavy chains, or other immunoglobulin forms can be purified according to standard methods in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, gel electrophoresis, etc.; see Scopes, "Protein Purification", Springer Verlag, NY (1982). The antibody of the present invention or one or more of its corresponding immunoglobulin chains can then be isolated from growth medium, cell lysates, or cell membrane fractions. The isolation and purification of the antibody or immunoglobulin chains of the present invention, for example, recombinantly expressed, can be carried out by any conventional method, such as preparative chromatographic separation and immunoassay (e.g., those involving the use of monoclonal or polyclonal antibodies, for example, targeting the constant region of the antibody of the present invention). It will be apparent to those skilled in the art that the antibody of the present invention can be further conjugated to other parts for, for example, drug targeting and imaging applications. Such conjugation can be carried out chemically after the antibody or antigen is expressed to the attachment site, or the conjugation product can be engineered into the antibody or antigen of the present invention at the DNA level. The DNA is then expressed in a suitable host system, and if necessary, the expressed proteins are collected and renatured.

[0357] 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.

[0358] One implementation involves an expression vector comprising a nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof as described herein.

[0359] Another implementation involves a host cell containing the expression vector described herein.

[0360] On the other hand, a method for preparing anti-allergen antibodies or allergen-binding fragments thereof is involved, which consists of culturing cells containing polynucleotides encoding the antibodies or their binding fragments described herein, and isolating the antibodies or their allergen-binding fragments from the cells or the cell culture medium. Example 1

[0361] Production of human antibodies

[0362] Target

[0363] The aim of this study was to generate fully human Bet v1-specific antibodies to prevent effector cells from binding to Bet v1 and triggering an immediate response.

[0364] Materials and methods

[0365] Biotinylation of nBet v1, nQue a1, and OVA: To allow for the isolation of Bet v1 and Que a1-specific memory B cells via FACS, Bet v1 and Que a1 were biotinylated and complexed with streptavidin-Biotin (SA PE). nBet v1 and nQue a1 were purified from pollen extract to a protein concentration >1 mg / mL. EZ-Link™ Sulfo-NHS-LC-LC-Biotin (Thermo Scientific) was diluted in water and immediately added to nBet v1, nQue a1, and OVA at biotin:allergen ratios of 2:1, 1:2, and 1:2, respectively, and incubated at room temperature with stirring for 30 min in the absence of light.

[0366] Generation of fluorescent nBet v1, nQue a1, and OVA (decoy) polymers: To generate biotinylated nBet v1 and Que a1 SA PE polymers, SA-PE (BioLegend) was added at ratios of 1:2 and 1:4, respectively. To generate biotinylated OVA SA PE polymers, SA-PE (BioLegend) was added at a ratio of 1:4 (1 part biotinylated OVA to 4 parts SA APC).

[0367] Isolation and freezing of PBMCs from blood donors: Peripheral blood samples were drawn from individuals with birch pollen allergy. PBMCs were isolated from plasma and erythrocytes using leucosep™. Cells were diluted to approximately 5 x 10⁻⁶ cells in RPMI 1640 + GlutaMAX™ (Gibco). 7 Cells / mL were counted and viability was checked using a NucleoCounter NC-200 (ChemoMetec) with a Via1 box (ChemoMetec).

[0368] Single-cell sorting of Bet v1 and Que a1 specific memory B cells: approximately 5-10.10 were used. 7Single-celled organisms (isolated as described above) were added to the cells. OVA decoy polymers were added to the cells at a final concentration of 5 nM, and the corresponding antigens nBet v1 or nQue a 1 fluorescent polymers were added to a final concentration of 1 nM. Cells were enriched according to the protocol of lyophilized anti-PE microbeads (Miltenyi Biotec), and then control cells were stained with IgDFITC (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) in Brilliant Stain Buffer (BD Horizon).

[0369] 150 single antigen-specific B cells (CD3) - CD14 - CD19 + IgD+IgM - Allergen - PE + Lure - AF647 - Single cells were sorted into 96-well plates.

[0370] Single-cell RNA sequencing: cDNA and libraries were generated from single cells (obtained as described above) according to the SMART-Seq® Single-Cell Kit User Manual (version 101619). The cDNA was then amplified and purified, quantified using a Qubit 4 (Invitrogen), and quality assessed using a TapeStation 4150 (Agilent). Library preparation for Illumina sequencing was performed using the NovaSeq 6000 kit (Illumina; 20028400). Prior to sequencing, the cDNA was fragmented, indexed, amplified, and merged. Sequencing was then performed on an Illumina NovaSeq 6000. Sequencing results from tetramer-sorted memory B cells were analyzed using the RNA-sequence aligner STAR (v. 2.7.10a). The B cell receptor sequence was reconstructed using an improved version of the BraCeR pipeline originally created by Lindemant et al. (2018) (Dockerpull nielsphk / bracer:1.3).

[0371] 137 pairs of heavy and light chain paired B cell receptor sequences were generated from B cells sorted by single cells.

[0372] Plasmid design: Human VH and VL sequences derived from selected B cells were cloned into a mammalian expression vector.

[0373] Heavy chain expression vector: The synthetic gene consisting of a signal peptide followed by VH + human IgHG4 (with the so-called S228P mutation) was cloned as the HindIII / BamHI fragment into the expression vector pcDNA3.1(+).

[0374] Light chain expression vector: The synthetic gene consisting of a signal peptide followed by VL + human IgK or IgL was cloned as the HindIII / BamHI fragment into the expression vector pcDNA3.1(+).

[0375] The sequence was codon-optimized for mammalian expression. Transfection-grade plasmid formulations were obtained from Genscript (NJ, USA).

[0376] mAb expression: The antibody was expressed using a 1:1 heavy chain:light chain encoding plasmid according to the "Expi293™ Expression System User Guide" (ThermoFisher, Publication No.: MAN0019402, Revision No.: B.0). Six days post-transfection, the supernatant was harvested by centrifugation at 2700g for 1 hour at 4°C and filtered through a 0.45 µm filter.

[0377] result

[0378] Single-cell sorting and scRNA-seq were performed on 150 B cells to obtain 76 pairs of paired heavy and light chain sequences, which expressed 70 antibodies, resulting in 36 Bet v1-specific antibodies.

[0379] Example 2

[0380] Affinity assay for antibody binding to tree pollen allergens

[0381] Target

[0382] The purpose of this embodiment is to determine 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.

[0383] Materials and methods

[0384] On an Octet RED96e instrument, the equilibrium dissociation constant (KD) of different associated allergens bound to monoclonal antibodies was determined using biolayer interferometry (BLI). All binding studies were performed at 25°C and 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 expressing IgG4, the Octet® ProA biosensor was loaded with the monoclonal antibody before allergen binding. For antibodies expressing IgE, the Octet® SAX biosensor was loaded with biotinylated anti-IgE VHH, and then the monoclonal antibody was captured before allergen binding. Binding studies were conducted on the following allergens: birch (nBet v 1 and rBet v 1.0112), alder (Aln g 1), hazel (Cor a 1), and white oak (Quea 1). Fixed monoclonal antibodies were immersed in serially diluted 2-fold allergens for 5 minutes to allow association, while dissociation of the monoclonal antibodies bound to the allergens was monitored for 30 minutes in kinetic buffer. Double reference subtraction was performed. Real-time binding sensor maps were fitted to a 1:1 model using Data Analysis HT 11.1 software (Sartorius) to determine maximum allergen capture levels and overall binding rate (kJ / kJ). a ), dissociation (k d ) rate and dissociation equilibrium constant (K D Antibodies REGN5713, REGN5714, and REGN5715 from Regeneron were added for comparison.

[0385] result

[0386] Tables 3 to 8 show the binding kinetic parameters of natural and recombinant Bet v 1, natural Aln g 1, natural Cor a 1 and natural Que a 1 with the different monoclonal antibodies of the present invention at 25°C.

[0387] The affinity (Kd) of the antibodies of this invention is lower than that of the control antibody. Antibodies 2E02, 2C10, 2B04, 2E07, A07, B10, and 2_10 all showed higher affinity for Bet v1, Aln g1, Cor a1, and Que a1 allergens than REGN5713, REGN5714, and REGN5715.

[0388] Table 3 – Affinity (Kd) of rBet v1, nAln g 1, nCor a 1, and nQue a 1 to the monoclonal antibody of the present invention at 25 °C.

[0389]

[0390] As shown in Table 3, 7 of the 36 antibodies tested were able to bind to all four of the tested allergens. In addition, only one of the three control antibodies (REGN5713, REGN5714, and REGN5715) (REGN5715) was able to bind to all four allergens, indicating that the binding affinity for Bet v1 does not always exhibit cross-reactivity with the other three allergens.

[0392] Table 4 - Binding kinetic parameters of recombinant Bet v1 to monoclonal antibody at 25℃

[0393]

[0394] As shown in Table 4, all 36 monoclonal antibodies of this invention exhibited measurable binding to recombinant Bet v1, of which K D The values ​​ranged from 249 pM to 468 nM. Furthermore, antibodies 2B04, 2C10, 2e02, and A07 all exhibited stronger affinity compared to REGN5713, REGN5714, and REGN5715.

[0395] Table 5 - Binding kinetic parameters of natural Bet v1 and monoclonal antibody at 25℃

[0396]

[0397] "ND" indicates that the affinity is undetermined.

[0398] As shown in the table, all 34 of the 36 monoclonal antibodies of this invention exhibited measurable binding to natural Betv 1, of which K D The values ​​ranged from 219 pM to 506 nM. Furthermore, antibodies 2E02, 2B04, A07, and B10 all exhibited greater affinity for natural Bet V1 compared to control antibodies REGN5713, REGN5714, and REGN5715.

[0400] Table 6 - Binding kinetic parameters of natural Aln g 1 and monoclonal antibody at 25℃

[0401]

[0402] "NB" indicates that no binding was observed under the current experimental conditions (no data displayed).

[0403] As shown in the table, 15 antibodies in the monoclonal antibodies of this invention exhibit measurable binding to natural Aln g 1, of which K D The values ​​ranged from 626 pM to 4.34 uM. The other 21 antibodies did not show any measurable binding to Aln g 1 under the test conditions. Furthermore, compared to the control antibodies REGN5713, REGN5714, and REGN5715, 2_9, 2A03, 2A09, 2B04, 2C10, 2E02, 2E07, A07, and B10 all showed higher affinity.

[0405] Table 7 - Binding kinetic parameters of natural Cor a1 and monoclonal antibodies at 25℃

[0406]

[0407] NB indicates that no binding was observed under the current experimental conditions (no data shown).

[0408] As shown in the table, 14 of the monoclonal antibodies in this invention exhibit measurable binding to natural Cor a1, of which K D The values ​​ranged from 2.69 nM to 2.18 uM. The other 22 antibodies did not show any measurable binding to Cor a1 under the test conditions. Compared to REGN5713, REGN5714, and REGN5715, 2_20, 2B04, 2C10, 2E02, 2E07, A07, and B10 all showed greater affinity for Cor a1.

[0410] Table 8 - Binding kinetic parameters of natural Que a1 and monoclonal antibodies at 25℃

[0411]

[0412] NB indicates that no binding was observed under the current experimental conditions (data is now displayed).

[0413] As shown in the table, seven antibodies in the monoclonal antibodies of this invention exhibit measurable binding to natural Que a 1, among which K D The values ​​ranged from 660 pM to 158 nM. The other 29 antibodies did not show any measurable binding to Que a 1 under the test conditions. Furthermore, antibodies 2_10, 2E02, 2E07, 2C10, 2B04, and A07 all showed greater affinity for Que a 1 compared to REGN5713, REGN5714, and REGN5715.

[0414] in conclusion

[0415] Here, we show 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 therefore exhibit greater cross-reactivity compared to control antibodies REGN5713, REGN5714, and REGN5715.

[0416] Example 3

[0417] Epitope binning on Bet v1

[0418] Target

[0419] The goal of this study is to perform epitope binning on a subgroup of mAbs in Bet v1.

[0420] Materials and methods

[0421]

[0422] result

[0423] like Figure 3 As you can see, the different antibodies can be grouped into different groups based on their epitope specificity. Each ring represents one of the antibodies in Table 4. Overlapping rings indicate that the antibody binds to an overlapping epitope on Bet v1, while non-overlapping rings indicate that the antibody binds to a different non-overlapping epitope.

[0424] Antibodies can be grouped based on their binding to individual epitopes, and antibodies in the same group can bind to Bet v1 simultaneously.

[0425] Examples of this type of group include:

[0426] A07, 2B04 and 2E02 or

[0427] A07, 2B04 and 2_10 or

[0428] A07, 2B04 and 2C10 or

[0429] B10, 2B04 and 2E02 or

[0430] B10, 2B04 and 2_10 or

[0431] B10, 2B04 and 2C10.

[0432] in conclusion

[0433] In summary, we were able to group the antibodies based on their epitope specificity to Bet v1. Furthermore, this grouping allows for the definition of antibody groups that simultaneously bind to Bet v1.

[0434] Example 4

[0435] Assay for the simultaneous binding of three anti-Bet v1 monoclonal antibodies to Bet v1

[0436] Target

[0437] The aim of this experiment was to use predictions from Example 3 and to demonstrate that the Bet v 1 epitopes bound by the three selected Bet v 1 monoclonal antibodies are unique and that no steric hindrance is exhibited when the three antibodies bind simultaneously, regardless of the order in which they bind. The sequence-dependent binding of the three Bet v 1 monoclonal antibodies was also evaluated.

[0438] Materials and methods

[0439] The simultaneous binding of three anti-Bet v1 monoclonal antibodies to Bet v1 was determined using real-time label-free biolayer interferometry (BLI) based on Octet RED96e (Sartorius). Experiments were performed at 25 °C in kinetic buffer (8 mM NaH2PO4, 3 mM KCl, 2 mM KH2PO4, 0.1% BSA, and 0.02% Tween-20).

[0440] In three independent experiments, each of the three IgG4 mAbs (A07, 2B04, and 2E02) analyzed in this example was initially captured to a level of 2.5–3.3 nm by the protein A sensor tip (Table 5). These three mAbs had previously been shown to bind Bet v1 with high affinity and to exhibit high cross-reactivity to PR-10 allergens from related tree species (Example 2) and to bind Bet v1 simultaneously in pairs (Example 3). The remaining protein A binding sites were then blocked with a high concentration (more than 10-fold) of an unrelated non-Bet v1 IgG4 mAb antibody. The sensor tip was then immersed in a well containing 4.25 µg / ml Bet v1 (for 5 minutes) and captured to a level of 0.3–0.5 nm by the protein A-bound anti-Bet v1 antibody (Table 5). The sensor tip is then sequentially immersed in wells containing either the same anti-Bet v 1 mAb (negative control) as the antibody directly captured on the tip of protein A, or two other mAb clones (mAb-1-3, Table 5) (each step lasts 5 minutes). See exemplary sensing diagram Figure 4.

[0441] A baseline step is included between all the above steps, which involves immersing the sensor tip in a kinetic buffer.

[0442] result

[0443] Binding levels (nm) were measured using a data analysis HT 11.1.3.50 (Molecular Devices), and the results are shown in Table 9. Signals less than 0.05 nm indicate no binding, while signals greater than 0.1 nm indicate binding, meaning the mAb did not compete for Bet v1 binding. All three Bet v1 monoclonal antibodies included in this embodiment were able to bind to Bet v1 simultaneously, and the binding reaction was not affected by the order in which the antibodies were added.

[0444] Table 9:

[0445]

[0446] NB = No binding (<0.05 nm)

[0447] in conclusion

[0448] This indicates that a set of three antibodies with different epitope specificities can bind to Bet v1 simultaneously. Regardless of the order in which each of the three mAbs binds to Bet v1, there is no competition preventing all three antibodies from binding simultaneously, meaning that mAb clones A07, 2B04, and 2E02 bind to different, non-overlapping epitopes without steric hindrance, as predicted in Example 4.

[0449] Example 5

[0450] Inhibition of allergen-specific IgE binding to tree pollen allergens

[0451] Target

[0452] The aim of this study is to determine whether the monoclonal antibody of the present invention can block the binding of IgE to nBet v1, nQue a1, nAln g1, or nCor a1 and inhibit the activation of basophils from individuals with tree pollen allergies when challenged with nBet v1, nQue a1, nAln g1, or nCor a1.

[0453] Materials and methods

[0454] The mAbs of this invention were tested for blocking of IgE epitopes and inhibition of basophil activation. As previously described (Orengo et al., 2018; Atanasio et al., 2022), blocking of IgE epitopes and inhibition of basophil activation are key in vitro assays for evaluating the efficacy of antibody mixtures in suppressing allergic reactions.

[0455] PBMCs were isolated from freshly drawn blood from tree pollen-allergic donors. Cells were pre-incubated with IL-3 and then mixed with a single tree pollen allergen pre-incubated for 1 h with various mAbs (mixed mAbs or single mAbs). Basophil activation was performed at 37°C for 1 h. Cells were then washed and stained with antibodies targeting specific surface cell proteins (CD123, CD203c, and CD63) for analysis by flow cytometry. Basophil activation was measured by upregulation of CD63 on basophils identified in PBMCs based on CD123 and CD203c.

[0456] result

[0457] Different mAb mixtures were tested (Table 10) and their effects were compared with those of single mAbs.

[0458] Table 10:

[0459]

[0460] The experiment was conducted using basophils isolated from six pollen-allergic donors. The basophils were inhibited by a mixture of mAbs, each at a concentration of 4 nM (Figures 5-8). Allergen nBet v1 was used. Figures 5A-5F ), nAln g 1( Figures 6A-6F ), nCor a 1( Figures 7A-7F ) and nQue a 1( Figure 8A-Figure 8F) to stimulate with allergens.

[0461] like Figures 5A-5F As shown, the antibody mixture strongly inhibited activation compared to the buffer control in all six donors.

[0462] 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 the activation, while control-1 showed no effect on activation or only a very limited effect in all six donors (Figure 6-). Figure 8A -Figure 8F).

[0463] Next, as the concentration of each mAb increased (from 5 x 10e-4 nM to 200 nM), basophil activation was inhibited (Figures 9–10 and Tables 11–18). The concentrations of the allergens used to stimulate basophils (nBet v1, nAln g1, nCor a1, and nQue a1) were individually selected for each donor AF (due to differences in sensitivity among donors). Figures 9–10A–10D show two representative individual donors (donors D and F from Tables 11–18) stimulated (one after another) with all four allergens.

[0464] like Figure 9A As shown in Figure 10A, ALK-1, ALK-2, and control-1 were able to inhibit nBet v1-induced activation to a greater extent than the antibodies alone.

[0465] When basophils were stimulated with any of nAln g 1, nCor a 1, and nQue a 1, ALK-1 and ALK-2 showed the strongest inhibitory effects in all tested donors compared with control-1 and monoclonal antibodies. Figures 9B-9D (Figures 10B-10D and Tables 11-14).

[0466] Table 11: Percentage of inhibition of basophil activation produced in 6 individual donors using different mAb combinations after activation with nBet v1.

[0467]

[0468] Table 12: Percentage of inhibition of basophil activation produced in 6 individual donors after activation with nAln g 1 using different mAb combinations.

[0469]

[0470] Table 13: Percentage of inhibition of basophil activation produced in 6 individual donors using different mAb combinations after activation with nCor a 1.

[0471]

[0472] Table 14: Percentage of inhibition of basophil activation produced in 6 individual donors using different mAb combinations after activation with nQue a 1.

[0473]

[0474] The inhibitory effects of ALK-3 and ALK-4 on basophil activation were tested after stimulation with nAln g 1, nCor a 1, and nQue (Tables 15-18). In all six donors, ALK-3 and ALK-4 showed stronger inhibition of basophil activation compared to individual antibodies.

[0475] Table 15: Percentage of inhibition of basophil activation produced in 6 individual donors using different mAb combinations after activation with nBet v1.

[0476]

[0477] Table 16: Percentage of inhibition against basophil activation produced in 6 individual donors using different mAb combinations after activation with nAln g 1.

[0478]

[0479] Table 17: Percentage of inhibition of basophil activation produced in 6 individual donors using different mAb combinations after activation with nCor a 1.

[0480]

[0481] Table 18: Percentage of inhibition of basophil activation produced in 6 individual donors using different mAb combinations after activation with nQue a 1.

[0482]

[0483] in conclusion

[0484] This section demonstrates that ALK-1 and ALK-2 can block the binding of IgE to allergens (nBet v1, nAln g1, nCor a1, and nCor a1), thereby inhibiting basophil activation. The inhibitory effect of ALK-1 and ALK-2 on nBet v1-induced basophil activation is comparable to that of the reference mixture "Control-1". Figures 5A-5F , Figure 9A (and Figure 10A). In contrast, compared with "Control-1", ALK-1 and ALK-2 showed greater inhibition of basophil activation induced by nAln g 1, nCor a 1 and nQue a 1 (Figure 6-). Figure 8B -F、 Figures 9B-9D (and Figures 10B-10D).

[0485] Furthermore, the mixture of the two antibodies was shown to inhibit basophil activation induced by all four allergens to a greater extent than the individual antibodies (AB).

[0486] References

[0487] Almagro JC (2004), 'Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size:implications for the rational design of antibody repertoires', Journal ofMolecular Recognition, doi.org / 10.1002 / jmr.659

[0488] Atanasio et al., (2022) 'Targeting immunodominant Bet v 1 epitopeswith monoclonal antibodies prevents the birch allergic response', Journal ofAllergy and Clinical Immunology.DOI:10.1016 / j.jaci.2021.05.038

[0489] Armour KL, Clark MR, Handly AG, Williamson LM.(1999) ‘Recombinanthuman IgG molecules lacking Fcgamma receptor I binding and monocytetriggering activities’ Euro J Immunol.DOI:10.1002 / (SICI)1521-4141(199908)29:08<2613::AID-IMMU2613>3.0.CO;2-J

[0490] Asam et al., (2015) 'Tree pollen allergens—an update from amolecular perspective' European JournalofAllergy and clinical immunology'.DOI: 10.1111 / all.12696

[0491] Canfield SM, and Morrison SL.(1991) ‘The binding affinity of humanIgG for its high affinity Fc receptor is determined by multiple amino acidsin the CH2 domain and is modulated by the hinge region’ J. Exp.Med.DOI:10.1084 / jem.173.6.1483

[0492] Canonica et al., (2014) ‘sublingual immunotherapy:World AllergyOrganization position paper 2013 update’, World Allergy Organixation Journal,DOI: doi.org / 10.1186 / 1939-4551-7-6

[0493] D’Amato et al., (2007) ‘Allergenic pollen and pollen allergy inEurope’, Allergy, DOI:10.1111 / j.1398-9995.2007.01393.x

[0494] Dondelinger M, Filee P, Sauvage E, Quinting B, Muydermans S, GalleniM and Vandevenne M (2018) ‘Understanding the Significance and Implications ofAntibody Numbering and Antigen-Binding Surface / Residue Definition’frontiersin immunology. doi.org / 10.3389 / fimmu.2018.02278

[0495] EA, Oadkan, C Abergel and JP Tipper (1995), ‘Identification ofspecificity-determining residues in antibodies’, The FASEB journal, DOI:10.1096 / fasebj.9.1.7821752

[0496] Lefranc MP, (2002) ‘IMGT, the international ImMunoGeneTics database:a high-quality information system for comparative immunogenetics andimmunology’, Developmental and Comparative Immunology. doi.org / 10.1016 / S0145-305X(02)00026-5

[0497] VA Lloyd (1999) ‘The Art, Science and Technology of PharmaceuticalCompounding’, Amer Pharmaceutical Assn.ISBN-10:0917330889

[0498] Mitchelle LS & Colwell LJ, (2018) ‘Comparative analysis of nanobodysequence and structure data’ Proteins,DOI:10.1002 / prot.25497.

[0499] Ofran Y, Schlessinger and Rost B, (2008) ‘Automated identification ofcomplementarity determining regions (CDRs) reveals peculiar characteristicsof CDRs and B cell epitopes’, J Immunol, DOI:10.4049 / jimmunol.181.9.6230

[0500] Orengo et al., (2018), ‘Treating cat allergy with monoclonal IgGantibodies that binds allergen and prevents IgE engagement’, NatureCommunication.DOI:10.1038 / s41467-018-03636-8

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Claims

1. 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; the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained in SEQ ID NO: 1, a HCDR2 amino acid sequence contained in SEQ ID NO: 1, a HCDR3 amino acid sequence contained in SEQ ID NO: 1, a LCDR1 amino acid sequence contained in SEQ ID NO: 2, a LCDR2 amino acid sequence contained in SEQ ID NO: 2, and a LCDR3 amino acid sequence contained in SEQ ID NO: 2; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained in SEQ ID NO: 21, a HCDR2 amino acid sequence contained in SEQ ID NO: 21, a HCDR3 amino acid sequence contained in SEQ ID NO: 21, a LCDR1 amino acid sequence contained in SEQ ID NO: 22, a LCDR2 amino acid sequence contained in SEQ ID NO: 22, and a LCDR3 amino acid sequence contained in SEQ ID NO: 22; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained in SEQ ID NO: 41, a HCDR2 amino acid sequence contained in SEQ ID NO: 41, a HCDR3 amino acid sequence contained in SEQ ID NO: 41, a LCDR1 amino acid sequence contained in SEQ ID NO: 42, a LCDR2 amino acid sequence contained in SEQ ID NO: 42, and a LCDR3 amino acid sequence contained in SEQ ID NO: 42; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained in SEQ ID NO: 61, a HCDR2 amino acid sequence contained in SEQ ID NO: 61, a HCDR3 amino acid sequence contained in SEQ ID NO: 61, a LCDR1 amino acid sequence contained in SEQ ID NO: 62, a LCDR2 amino acid sequence contained in SEQ ID NO: 62, and a LCDR3 amino acid sequence contained in SEQ ID NO: 62; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained in SEQ ID NO: 81, a HCDR2 amino acid sequence contained in SEQ ID NO: 81, a HCDR3 amino acid sequence contained in SEQ ID NO: 81, a LCDR1 amino acid sequence contained in SEQ ID NO: 82, a LCDR2 amino acid sequence contained in SEQ ID NO: 82, and a LCDR3 amino acid sequence contained in SEQ ID NO: 82; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained in SEQ ID NO: 101, a HCDR2 amino acid sequence contained in SEQ ID NO: 101, a HCDR3 amino acid sequence contained in SEQ ID NO: 101, a LCDR1 amino acid sequence contained in SEQ ID NO: 102, a LCDR2 amino acid sequence contained in SEQ ID NO: 102, and a LCDR3 amino acid sequence contained in SEQ ID NO: 102; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence contained in SEQ ID NO: 121, a HCDR2 amino acid sequence contained in SEQ ID NO: 121, a HCDR3 amino acid sequence contained in SEQ ID NO: 121, a LCDR1 amino acid sequence contained in SEQ ID NO: 122, a LCDR2 amino acid sequence contained in SEQ ID NO: 122, and a LCDR3 amino acid sequence contained in SEQ ID NO: 122, wherein each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 can be determined by IMGT, Kabat, or Chothia, and optionally wherein each CDR region determined by IMGT, Kabat, or Chothia can be subject to 1, 2, or 3 amino acid substitutions, such that each HCDR1 can contain 1, 2, or 3 amino acid substitutions, each HCDR2 can contain 1, 2, or 3 amino acid substitutions, each HCDR3 can contain 1, 2, or 3 amino acid substitutions, each LCDR1 can contain 1, 2, or 3 amino acid substitutions, each LCDR2 can contain 1, 2, or 3 amino acid substitutions, and / or each LCDR3 can contain 1, 2, or 3 amino acid substitutions.

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

3. An antibody or antigen-binding fragment thereof, wherein; the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 3, a HCDR2 amino acid sequence of SEQ ID NO: 4, a HCDR3 amino acid sequence of SEQ ID NO: 5, a LCDR1 amino acid sequence of SEQ ID NO: 6, a LCDR2 amino acid sequence of SEQ ID NO: 7, and a LCDR3 amino acid sequence of SEQ ID NO: 8; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 23, a HCDR2 amino acid sequence of SEQ ID NO: 24, a HCDR3 amino acid sequence of SEQ ID NO: 25, a LCDR1 amino acid sequence of SEQ ID NO: 26, a LCDR2 amino acid sequence of SEQ ID NO: 27, and a LCDR3 amino acid sequence of SEQ ID NO: 28; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 43, a HCDR2 amino acid sequence of SEQ ID NO: 44, a HCDR3 amino acid sequence of SEQ ID NO: 45, a LCDR1 amino acid sequence of SEQ ID NO: 46, a LCDR2 amino acid sequence of SEQ ID NO: 47, and a LCDR3 amino acid sequence of SEQ ID NO: 48; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 63, a HCDR2 amino acid sequence of SEQ ID NO: 64, a HCDR3 amino acid sequence of SEQ ID NO: 65, a LCDR1 amino acid sequence of SEQ ID NO: 66, a LCDR2 amino acid sequence of SEQ ID NO: 67, and a LCDR3 amino acid sequence of SEQ ID NO: 68; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 83, a HCDR2 amino acid sequence of SEQ ID NO: 84, a HCDR3 amino acid sequence of SEQ ID NO: 85, a LCDR1 amino acid sequence of SEQ ID NO: 86, a LCDR2 amino acid sequence of SEQ ID NO: 87, and a LCDR3 amino acid sequence of SEQ ID NO: 88; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 103, a HCDR2 amino acid sequence of SEQ ID NO: 104, a HCDR3 amino acid sequence of SEQ ID NO: 105, a LCDR1 amino acid sequence of SEQ ID NO: 106, a LCDR2 amino acid sequence of SEQ ID NO: 107, and a LCDR3 amino acid sequence of SEQ ID NO: 108; or the antibody or antigen-binding fragment thereof comprises a HCDR1 amino acid sequence of SEQ ID NO: 123, a HCDR2 amino acid sequence of SEQ ID NO: 124, a HCDR3 amino acid sequence of SEQ ID NO: 125, a LCDR1 amino acid sequence of SEQ ID NO: 126, a LCDR2 amino acid sequence of SEQ ID NO: 127, and a LCDR3 amino acid sequence of SEQ ID NO: 128, wherein each HCDR1 can contain 1, 2, or 3 amino acid substitutions, each HCDR2 can contain 1, 2, or 3 amino acid substitutions, each HCDR3 can contain 1, 2, or 3 amino acid substitutions, each LCDR1 can contain 1, 2, or 3 amino acid substitutions, each LCDR2 can contain 1, 2, or 3 amino acid substitutions, and / or each LCDR3 can contain 1, 2, or 3 amino acid substitutions.

4. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO: 1 and a LCVR having an amino acid sequence of SEQ ID NO: 2; or the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO: 21 and a LCVR having an amino acid sequence of SEQ ID NO: 22; or the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO: 41 and a LCVR having an amino acid sequence of SEQ ID NO: 42; or the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO: 61 and a LCVR having an amino acid sequence of SEQ ID NO: 62; or the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO: 81 and a LCVR having an amino acid sequence of SEQ ID NO: 82; or the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO: 101 and a LCVR having an amino acid sequence of SEQ ID NO: 102; or the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence of SEQ ID NO: 121 and a LCVR having an amino acid sequence of SEQ ID NO: 122; or wherein each HCVR can comprise 1, 2, or 3 amino acid substitutions, and / or each LCVR can comprise 1, 2, or 3 amino acid substitutions.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is capable of reducing or inhibiting the binding of IgE antibodies 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 antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody comprises an Fc region of human origin.

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

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

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

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

11. A multispecific antibody comprising two or more antigen binding fragments selected from the antigen binding fragment according to any one of claims 1-2 and claims 5-10 dependent on claims 1-2, or selected from the antibody or antigen binding fragment thereof according to claim 3 and claims 5-10 dependent on claim 3, or selected from the antibody or antigen binding fragment thereof according to claim 4 and claims 5-10 dependent on claim 4.

12. A composition comprising two antibodies or antigen binding fragments thereof selected from the antibody or antigen binding fragment thereof according to any one of claims 1-2 and claims 5-10 dependent on claims 1-2, or selected from the antibody or antigen binding fragment thereof according to claim 3 and claims 5-10 dependent on claim 3, or selected from the antibody or antigen binding fragment thereof according to claim 4 and claims 5-10 dependent on claim 4.

13. The composition according to claim 12, wherein the composition comprises 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 wherein 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, wherein the composition comprises 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 wherein each of LCDR1, LCDR2 and LCDR3 has the amino acid sequence contained in SEQ ID NO:

82.

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

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

17. The composition according to claim 16, comprising 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 comprises 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 comprises 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, and the third antibody comprises 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 comprises each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 1 and LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 2, and the second antibody comprises 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, and the third antibody comprises 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 comprises each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22, and the second antibody comprises 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, and the third antibody comprises 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 comprises each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 21 and comprises each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 22, and the second antibody comprises each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 41 and comprises each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 42, and the third antibody comprises each of HCDR1, HCDR2, and HCDR3 having the amino acid sequence contained in SEQ ID NO: 101 and comprises each of LCDR1, LCDR2, and LCDR3 having the amino acid sequence contained in SEQ ID NO: 102; or the first antibody comprises 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, and the second antibody comprises 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, and the third antibody comprises 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.

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

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

20. The antibody or antigen-binding fragment thereof according to any one of claims 1-10, the multispecific antibody according to claim 11 or the composition according to any one of claims 12-17 for use in a method of treating tree pollen allergy, wherein the tree pollen allergy is associated with allergy to one or more of the allergens Bet v 1, Cor a 1, Aln g 1 and Que a 1.

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

22. An expression vector comprising the nucleic acid molecule encoding the human monoclonal antibody or fragment thereof according to claim 21.

23. A host cell comprising the expression vector of claim 22.

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