Antibody or antigen binding fragment thereof aiming at IgE (Immunoglobulin E) and Fc variant

Abstract

The invention discloses an antibody or an antigen binding fragment thereof specifically binding to IgE under a neutral or alkaline condition, and a pharmaceutical composition and a kit containing the antibody or the antigen binding fragment thereof, and also relates to application of the antibody or the antigen binding fragment in reducing or inhibiting the IgE level or preventing and / or treating IgE-related diseases of a subject. The invention also relates to an Fc variant of IgG Fc, a nucleic acid molecule and a carrier which encode a nucleotide sequence of the Fc variant, an antibody or an antigen binding fragment of the antibody containing the Fc variant of IgG Fc, and application of the antibody or the antigen binding fragment in reducing or inhibiting IgE level or preventing and / or treating IgE-related diseases of subjects.

Description

Technical Field

[0001] The present application relates to an antibody or an antigen-binding fragment thereof that specifically binds to IgE, a pharmaceutical composition and a kit comprising the same, and also relates to their use in reducing or inhibiting IgE levels or preventing and / or treating IgE-related diseases in a subject. The present application also relates to an Fc variant of IgG Fc, a nucleic acid molecule and a vector comprising a nucleotide sequence encoding the same, and also relates to an antibody or an antigen-binding fragment thereof comprising the Fc variant, and their use in reducing or inhibiting IgE levels or preventing and / or treating IgE-related diseases in a subject. Background Art

[0002] IgE (immunoglobulin E) is an immune molecule that plays a major role in the pathogenesis of type I hypersensitivity diseases, such as allergic rhinitis, allergic cough, asthma, atopic dermatitis, eczema, acute and chronic urticaria, etc. IgE interacts with two main receptors, FcεRI and FcεRII (CD23), and is involved in different immune processes respectively. Among them, FcεRI is a high-affinity receptor for IgE and exists on the surface of immune cells such as mast cells and basophils. Crosslinking of this receptor induced by IgE triggers activation and degranulation of mast cells and basophils, etc., releasing bioactive mediators such as histamine, prostaglandins, and leukotrienes, causing physiological function disorders such as local or systemic inflammatory reactions, that is, type I hypersensitivity reactions. The interaction between IgE and the low-affinity receptor CD23 is involved in immune processes such as antigen presentation, antigen transport across the airway and intestinal epithelium, and regulation of IgE synthesis.

[0003] Omalizumab, a humanized anti-IgE monoclonal antibody, is currently the only clinically approved IgE-targeted therapeutic drug for the treatment of patients with severe allergic asthma and high IgE levels in the blood. The binding of omalizumab to IgE can simultaneously inhibit the interaction of IgE with two receptors, playing a neutralizing role on IgE, thereby treating allergies. However, in clinical use, omalizumab significantly increases the total IgE content in patients' blood while reducing free IgE. This is because omalizumab, as an IgG-type antibody, has a relatively long half-life. During the clinical treatment process, IgE is neutralized by omalizumab and also protected by it. When IgE bound to omalizumab encounters endocytosis, it will instead be recycled and released extracellularly through FcRn together with omalizumab and not be degraded by lysosomes, thus greatly prolonging the half-life of IgE and causing a large accumulation of IgE in the blood. As a neutralizing antibody, to achieve the drug effect, the dosage of omalizumab cannot be lower than the total amount of IgE in the patient's body, otherwise there will definitely be some IgE that is not neutralized. Therefore, to cope with the continuous secretion of IgE in the patient's body, a large amount of omalizumab needs to be provided to ensure the neutralizing effect on IgE, which is clinically manifested as the need to inject omalizumab into patients in multiple large doses. This brings a relatively large economic burden to patients and may also cause adverse reactions such as anemia. Moreover, the upper limit of each clinical dose of omalizumab is 600 mg. If patients are exposed to allergens for a long time and a large amount of IgE is secreted in the body for a long time, then omalizumab will also face the risk of failure.

[0004] Therefore, it is necessary to develop a new generation of anti-allergy or anti-asthma drugs with high efficiency and low dosage. Summary of the Invention

[0005] To solve the above problems, the present application provides the following three aspects of antibodies.

[0006] In the first aspect, the present application provides an antibody or its antigen-binding fragment, which has one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residue substitutions and / or additions in the VH complementarity-determining regions 1-3 (CDR1-3) contained in the heavy-chain variable region (VH) of omalizumab monoclonal antibody and / or the VL complementarity-determining regions 1-3 (CDR1-3) contained in the light-chain variable region (VL).

[0007] Among them, the omalizumab monoclonal antibody comprises: VH CDR1, VH CDR2, and VH CDR3 contained in the heavy-chain variable region shown in SEQ ID NO: 14; and VL CDR1, VL CDR2, and VL CDR3 contained in the light-chain variable region shown in SEQ ID NO: 15.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residue substitutions and / or additions in VL CDR1 and / or VL CDR2 contained in the light chain variable region of omalizumab monoclonal antibody; preferably, the substitution is a conservative substitution.

[0009] In certain embodiments, VH CDR1-3 and / or VL CDR1-3 of the omalizumab monoclonal antibody are defined by the Kabat, IMGT or Chothia numbering system.

[0010] In certain embodiments, VH CDR1-3 and / or VL CDR1-3 of the omalizumab monoclonal antibody are defined by the Kabat numbering system.

[0011] In certain embodiments, the heavy chain variable region (VH) of the omalizumab monoclonal antibody comprises VH CDR1 having the sequence of SEQ ID NO:16, VH CDR2 having the sequence of SEQ ID NO:17, and VH CDR3 having the sequence of SEQ ID NO:18; the light chain variable region (VL) comprises VL CDR1 having the sequence of SEQ ID NO:19, VL CDR2 having the sequence of SEQ ID NO:20, and VL CDR3 having the sequence of SEQ ID NO:21.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof as described above has one or more (e.g., 2, 3, 4, 5, 6) histidine residue substitutions and / or additions in VL CDR1 contained in the light chain variable region of omalizumab monoclonal antibody.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof has one or more (e.g., 2, 3, 4, 5, 6) histidine residue substitutions among the amino acid residues at positions 30, 31, 32, 33, 35, 36 in the light chain variable region of omalizumab monoclonal antibody.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at the amino acid residues at positions 30, 31, 32, 35 and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at the amino acid residues at positions 30, 31, 32, 33, 35 and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 33, and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, and 33 in the light chain variable region as shown in SEQ ID NO:15.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, and 32 in the light chain variable region as shown in SEQ ID NO:15.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof as described above has a substitution and / or addition of one or more (e.g., 2, 3) histidine residues in the VL CDR2 contained in the light chain variable region of omalizumab monoclonal antibody.

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution and / or addition of one or more (e.g., 2, 3) histidine residues at amino acid residues 55, 56, and 57 in the light chain variable region of omalizumab monoclonal antibody.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 56 and 57 in the light chain variable region as shown in SEQ ID NO:15.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 55, 56, and 57 in the light chain variable region as shown in SEQ ID NO:15.

[0024] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 55 and 56 in the light chain variable region as shown in SEQ ID NO:15.

[0025] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 56 and 57 in the light chain variable region as shown in SEQ ID NO:15 and an addition of 1 histidine residue.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof, as described above, independently has one or several (e.g., 2, 3, 4, 5, 6) histidine residue substitutions and / or additions in each of VL CDR1 and VL CDR2 contained in the light chain variable region of omalizumab monoclonal antibody.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof has 3, 4, 5 or 6 histidine residue substitutions in VL CDR1 contained in the light chain variable region of omalizumab monoclonal antibody, and 2 or 3 histidine residue substitutions in VL CDR2.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at amino acid residues 30, 31, 32, 35, 36 in the light chain variable region as shown in SEQ ID NO:15, and has histidine residue substitutions and 1 histidine residue addition at amino acid residues 56 and 57.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at amino acid residues 30, 31, 32, 33, 35, 36, 56 and 57 in the light chain variable region as shown in SEQ ID NO:15.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at amino acid residues 30, 31, 32, 36, 56 and 57 in the light chain variable region as shown in SEQ ID NO:15.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at amino acid residues 30, 31, 32, 33, 36, 56 and 57 in the light chain variable region as shown in SEQ ID NO:15.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at amino acid residues 30, 31, 32, 35, 36, 55, 56 and 57 in the light chain variable region as shown in SEQ ID NO:15.

[0033] In certain embodiments, the antibody or antigen-binding fragment thereof has histidine residue substitutions at amino acid residues 30, 31, 33, 56 and 57 in the light chain variable region as shown in SEQ ID NO:15.

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 35, 36, 55, and 56 of the light chain variable region as shown in SEQ ID NO:15.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 34, 36, 55, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0036] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 33, 35, 36, 55, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof as described above has a substitution and / or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residues in VH CDR1 and / or VH CDR2 contained in the heavy chain variable region of omalizumab monoclonal antibody.

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of one or more (e.g., 2, 3, 4, 5) histidine residues at amino acid residues 26, 28, 30, 31, and 54 of the heavy chain variable region of omalizumab monoclonal antibody.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:22; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:23.

[0040] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:30; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:31.

[0041] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:38; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:39.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 46; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 47.

[0043] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 54; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 55.

[0044] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 62; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 63.

[0045] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 70; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 71.

[0046] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 78; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 79.

[0047] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 86; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 87.

[0048] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 94; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 95.

[0049] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 102; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 103.

[0050] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 110; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 111.

[0051] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 24, VH CDR2 having the sequence of SEQ ID NO: 25, VH CDR3 having the sequence of SEQ ID NO: 26; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 27, VL CDR2 having the sequence of SEQ ID NO: 28, VL CDR3 having the sequence of SEQ ID NO: 29.

[0052] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 32, VH CDR2 having the sequence of SEQ ID NO: 33, VH CDR3 having the sequence of SEQ ID NO: 34; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 35, VL CDR2 having the sequence of SEQ ID NO: 36, VL CDR3 having the sequence of SEQ ID NO: 37.

[0053] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 40, VH CDR2 having the sequence of SEQ ID NO: 41, VH CDR3 having the sequence of SEQ ID NO: 42; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 43, VL CDR2 having the sequence of SEQ ID NO: 44, VL CDR3 having the sequence of SEQ ID NO: 45.

[0054] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:48, VH CDR2 having the sequence of SEQ ID NO:49, VH CDR3 having the sequence of SEQ ID NO:50; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:51, VL CDR2 having the sequence of SEQ ID NO:52, VL CDR3 having the sequence of SEQ ID NO:53.

[0055] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:56, VH CDR2 having the sequence of SEQ ID NO:57, VH CDR3 having the sequence of SEQ ID NO:58; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:59, VL CDR2 having the sequence of SEQ ID NO:60, VL CDR3 having the sequence of SEQ ID NO:61.

[0056] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:64, VH CDR2 having the sequence of SEQ ID NO:65, VH CDR3 having the sequence of SEQ ID NO:66; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:67, VL CDR2 having the sequence of SEQ ID NO:68, VL CDR3 having the sequence of SEQ ID NO:69.

[0057] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: a heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:72, VH CDR2 having the sequence of SEQ ID NO:73, VH CDR3 having the sequence of SEQ ID NO:74; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:75, VL CDR2 having the sequence of SEQ ID NO:76, VL CDR3 having the sequence of SEQ ID NO:77.

[0058] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:80, VH CDR2 having the sequence of SEQ ID NO:81, VH CDR3 having the sequence of SEQ ID NO:82; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:83, VL CDR2 having the sequence of SEQ ID NO:84, VL CDR3 having the sequence of SEQ ID NO:85.

[0059] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:88, VH CDR2 having the sequence of SEQ ID NO:89, VH CDR3 having the sequence of SEQ ID NO:90; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:91, VL CDR2 having the sequence of SEQ ID NO:92, VL CDR3 having the sequence of SEQ ID NO:93.

[0060] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:96, VH CDR2 having the sequence of SEQ ID NO:97, VH CDR3 having the sequence of SEQ ID NO:98; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:99, VL CDR2 having the sequence of SEQ ID NO:100, VL CDR3 having the sequence of SEQ ID NO:101.

[0061] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:104, VH CDR2 having the sequence of SEQ ID NO:105, VH CDR3 having the sequence of SEQ ID NO:106; and / or, a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:107, VL CDR2 having the sequence of SEQ ID NO:108, VL CDR3 having the sequence of SEQ ID NO:109.

[0062] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:112, VH CDR2 having the sequence of SEQ ID NO:113, VH CDR3 having the sequence of SEQ ID NO:114; and / or, a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:115, VL CDR2 having the sequence of SEQ ID NO:116, VL CDR3 having the sequence of SEQ ID NO:117.

[0063] In certain embodiments, the antibody or antigen-binding fragment thereof as described above further comprises a constant region.

[0064] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises an IgG heavy-chain constant region.

[0065] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light-chain constant region that is a κ light-chain constant region or a λ light-chain constant region.

[0066] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy-chain constant region as shown in SEQ ID NO:12.

[0067] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light-chain constant region as shown in SEQ ID NO:13.

[0068] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises an Fc region.

[0069] In certain embodiments, the Fc region is an Fc of IgG or an Fc variant; wherein, compared with the Fc of IgG, the Fc variant has an increased affinity for the Fcγ receptor (FcγR).

[0070] In a second aspect, the present application provides an Fc variant of IgG Fc, as well as an antibody or an antigen-binding fragment thereof comprising said Fc variant.

[0071] The present application provides an Fc variant of IgG Fc, which has three amino acid residue substitutions and one amino acid residue deletion at amino acid residues 327, 328, 330 and 331 of the amino acid sequence of IgG Fc.

[0072] In certain embodiments, the Fc variant has amino acid residue substitutions at amino acid residues 327, 328 and 330 of the amino acid sequence of IgG Fc and an amino acid residue deletion at amino acid residue 331; wherein, compared with IgG Fc, the Fc variant has an increased affinity for Fc gamma receptor (FcγR).

[0073] In certain embodiments, the Fc variant has a substitution of a glutamic acid residue at amino acid residue 327 of IgG Fc.

[0074] In certain embodiments, the Fc variant has a substitution of a tryptophan residue at amino acid residue 328 of IgG Fc.

[0075] In certain embodiments, the Fc variant has a substitution of a serine residue at amino acid residue 330 of IgG Fc.

[0076] In certain embodiments, the amino acid positions are obtained by the Eu numbering system.

[0077] In certain embodiments, the Fc variant has the sequence as shown in SEQ ID NO:7.

[0078] In certain embodiments, the Fc variant also has amino acid residue substitutions at amino acid residues 252, 254 and 256 of the amino acid sequence of IgG Fc.

[0079] In certain embodiments, the Fc variant has one or more of the following characteristics:

[0080] (1) The Fc variant has a substitution of a tyrosine residue at amino acid residue 252 of IgG Fc;

[0081] (2) The Fc variant has a substitution of a threonine residue at amino acid residue 254 of IgG Fc;

[0082] (3) The Fc variant has a substitution of a glutamic acid residue at amino acid residue 256 of IgG Fc.

[0083] In certain embodiments, the amino acid site is obtained by the Eu numbering system.

[0084] In certain embodiments, the Fc variant also has substitutions of amino acid residues at positions 37, 39, and 41 of the amino acid residues corresponding to SEQ ID NO:7.

[0085] In certain embodiments, the Fc variant has one or more of the following characteristics:

[0086] (1) The Fc variant has a substitution of a tyrosine residue at the 37th amino acid residue of the IgG Fc;

[0087] (2) The Fc variant has a substitution of a threonine residue at the 39th amino acid residue of the IgG Fc;

[0088] (3) The Fc variant has a substitution of a glutamic acid residue at the 41st amino acid residue of the IgG Fc.

[0089] In certain embodiments, the Fc variant has the sequence shown in SEQ ID NO:118.

[0090] On the other hand, the present application provides another Fc variant of IgG Fc.

[0091] In certain embodiments, the 267th amino acid residue of the amino acid sequence of the Fc variant in IgG Fc is replaced with glutamic acid and the 328th amino acid residue is replaced with phenylalanine;

[0092] Or,

[0093] The 52nd amino acid residue corresponding to SEQ ID NO:6 of the Fc variant is replaced with glutamic acid, and the 113th amino acid residue corresponding to SEQ ID NO:6 is replaced with phenylalanine.

[0094] In certain embodiments, the Fc variant has the sequence shown in SEQ ID NO:8.

[0095] In certain embodiments, the 267th amino acid residue of the amino acid sequence of the Fc variant in IgG Fc is replaced with glutamic acid and the 328th amino acid residue is replaced with phenylalanine; and, the 252nd, 254th, and 256th amino acid residues of the amino acid sequence of the Fc variant in IgG Fc are also replaced with tyrosine, threonine, and glutamic acid, respectively;

[0096] Or,

[0097] The Fc variant has a substitution of glutamic acid at the 52nd amino acid residue corresponding to SEQ ID NO: 6 and a substitution of phenylalanine at the 113th amino acid residue corresponding to SEQ ID NO: 6; and the Fc variant also has substitutions of tyrosine, threonine, and glutamic acid at the 37th, 39th, and 41st amino acid residues corresponding to SEQ ID NO: 6, respectively.

[0098] In certain embodiments, the Fc variant has the sequence as shown in SEQ ID NO: 119.

[0099] In certain embodiments, the Fc variant as described above, wherein the FcγR is selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, or any combination thereof.

[0100] In certain embodiments, the FcγR is derived from a mammal (e.g., mouse, human).

[0101] In certain embodiments, the FcγR is human FcγRIIb.

[0102] The present application also provides a fusion protein comprising the Fc variant as described above.

[0103] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the Fc variant or the fusion protein as described above.

[0104] The present application also provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0105] The present application also provides a host cell comprising the isolated nucleic acid molecule or the vector as described above.

[0106] The present application also provides a method for preparing the Fc variant or the fusion protein as described above, which includes culturing the host cell as described above under conditions allowing the expression of the polypeptide or protein, and recovering the Fc variant or the fusion protein from the cultured host cell culture.

[0107] The present application also provides an antibody or an antigen-binding fragment thereof comprising the Fc variant as described above.

[0108] In certain embodiments, the antibody is a humanized antibody, a chimeric antibody, a fully human antibody, a bispecific antibody, or a multispecific antibody.

[0109] In certain embodiments, the antibody of the first aspect comprises an Fc variant of the IgG Fc as described in the second aspect. In certain embodiments, the antibody of the second aspect comprises one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residue substitutions and / or additions in the VH CDR1-3 contained in the heavy chain variable region (VH) of the omalizumab monoclonal antibody and / or the VL CDR1-3 contained in the light chain variable region (VL). In certain embodiments, the antibodies described in the first and second aspects comprise the characteristics of the antibody or its antigen-binding fragment as described in the third aspect.

[0110] In a third aspect, the present application provides an antibody or its antigen-binding fragment that has one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residue substitutions and / or additions in the VH CDR1-3 contained in the heavy chain variable region (VH) of the omalizumab monoclonal antibody and / or the VL CDR1-3 contained in the light chain variable region (VL);

[0111] wherein the omalizumab monoclonal antibody comprises: VH CDR1, VH CDR2, and VH CDR3 contained in the heavy chain variable region as shown in SEQ ID NO:14; and VL CDR1, VL CDR2, and VL CDR3 contained in the light chain variable region as shown in SEQ ID NO:15;

[0112] and the antibody further comprises an Fc variant of IgG Fc.

[0113] In certain embodiments, the antibody or its antigen-binding fragment has one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residue substitutions and / or additions in VL CDR1 and / or VL CDR2 contained in the light chain variable region of the omalizumab monoclonal antibody; preferably, the substitution is a conservative substitution.

[0114] In certain embodiments, the VH CDR1-3 and / or VL CDR1-3 of the omalizumab monoclonal antibody are defined by the Kabat, IMGT, or Chothia numbering system.

[0115] In certain embodiments, the VH CDR1-3 and / or VL CDR1-3 of the omalizumab monoclonal antibody are defined by the Kabat numbering system.

[0116] In certain embodiments, the heavy chain variable region (VH) of the omalizumab monoclonal antibody comprises VH CDR1 having the sequence of SEQ ID NO:16, VH CDR2 having the sequence of SEQ ID NO:17, and VH CDR3 having the sequence of SEQ ID NO:18; the light chain variable region (VL) comprises VL CDR1 having the sequence of SEQ ID NO:19, VL CDR2 having the sequence of SEQ ID NO:20, and VL CDR3 having the sequence of SEQ ID NO:21.

[0117] In certain embodiments, the antibody or antigen-binding fragment thereof as described above has a substitution and / or addition of one or more (e.g., 2, 3, 4, 5, 6) histidine residues in VL CDR1 contained in the light chain variable region of the omalizumab monoclonal antibody.

[0118] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of one or more (e.g., 2, 3, 4, 5, 6) histidine residues at amino acid residues 30, 31, 32, 33, 35, and 36 in the light chain variable region of the omalizumab monoclonal antibody.

[0119] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues at amino acid residues 30, 31, 32, 35, and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0120] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues at amino acid residues 30, 31, 32, 33, 35, and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0121] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues at amino acid residues 30, 31, 32, and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0122] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues at amino acid residues 30, 31, 32, 33, and 36 in the light chain variable region as shown in SEQ ID NO:15.

[0123] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues at amino acid residues 30, 31, and 33 in the light chain variable region as shown in SEQ ID NO:15.

[0124] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, and 32 in the light chain variable region as shown in SEQ ID NO: 15.

[0125] In certain embodiments, the antibody or antigen-binding fragment thereof as described above has a substitution and / or addition of one or more (e.g., 2, 3) histidine residues in the VL CDR2 contained in the light chain variable region of omalizumab monoclonal antibody.

[0126] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution and / or addition of one or more (e.g., 2, 3) histidine residues at amino acid residues 55, 56, and 57 in the light chain variable region of omalizumab monoclonal antibody.

[0127] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 56 and 57 in the light chain variable region as shown in SEQ ID NO: 15.

[0128] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 55, 56, and 57 in the light chain variable region as shown in SEQ ID NO: 15.

[0129] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 55 and 56 in the light chain variable region as shown in SEQ ID NO: 15.

[0130] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 56 and 57 in the light chain variable region as shown in SEQ ID NO: 15 and an addition of 1 histidine residue.

[0131] In certain embodiments, the antibody or antigen-binding fragment thereof as described above independently has a substitution and / or addition of one or more (e.g., 2, 3, 4, 5, 6) histidine residues in each of the VL CDR1 and VL CDR2 contained in the light chain variable region of omalizumab monoclonal antibody.

[0132] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of 3, 4, 5, or 6 histidine residues in the VL CDR1 contained in the light chain variable region of omalizumab monoclonal antibody and a substitution of 2 or 3 histidine residues in the VL CDR2.

[0133] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 35, and 36 of the light chain variable region as shown in SEQ ID NO:15, and a substitution of a histidine residue and an addition of 1 histidine residue at amino acid residues 56 and 57.

[0134] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 33, 35, 36, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0135] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 36, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0136] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 33, 36, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 35, 36, 55, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0138] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 33, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0139] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 35, 36, 55, and 56 of the light chain variable region as shown in SEQ ID NO:15.

[0140] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 34, 36, 55, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0141] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of a histidine residue at amino acid residues 30, 31, 32, 33, 35, 36, 55, 56, and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0142] In certain embodiments, the antibody or antigen-binding fragment thereof as described above has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residue substitutions and / or additions in VH CDR1 and / or VH CDR2 contained in the heavy chain variable region of omalizumab monoclonal antibody.

[0143] In certain embodiments, the antibody or antigen-binding fragment thereof has one or more (e.g., 2, 3, 4, 5) histidine residue substitutions at amino acid residues 26, 28, 30, 31, 54 in the heavy chain variable region of omalizumab monoclonal antibody.

[0144] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:22; and / or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:23.

[0145] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:30; and / or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:31.

[0146] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:38; and / or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:39.

[0147] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:46; and / or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:47.

[0148] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:54; and / or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:55.

[0149] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 62; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 63.

[0150] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 70; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 71.

[0151] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 78; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 79.

[0152] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 86; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 87.

[0153] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 94; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 95.

[0154] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 102; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 103.

[0155] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises: three CDRs contained in the heavy-chain variable region (VH) as set forth in SEQ ID NO: 110; and / or three CDRs contained in the light-chain variable region (VL) as set forth in SEQ ID NO: 111.

[0156] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:24, VH CDR2 having the sequence of SEQ ID NO:25, VH CDR3 having the sequence of SEQ ID NO:26; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:27, VL CDR2 having the sequence of SEQ ID NO:28, VL CDR3 having the sequence of SEQ ID NO:29.

[0157] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:32, VH CDR2 having the sequence of SEQ ID NO:33, VH CDR3 having the sequence of SEQ ID NO:34; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:35, VL CDR2 having the sequence of SEQ ID NO:36, VL CDR3 having the sequence of SEQ ID NO:37.

[0158] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:40, VH CDR2 having the sequence of SEQ ID NO:41, VH CDR3 having the sequence of SEQ ID NO:42; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:43, VL CDR2 having the sequence of SEQ ID NO:44, VL CDR3 having the sequence of SEQ ID NO:45.

[0159] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:48, VH CDR2 having the sequence of SEQ ID NO:49, VH CDR3 having the sequence of SEQ ID NO:50; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:51, VL CDR2 having the sequence of SEQ ID NO:52, VL CDR3 having the sequence of SEQ ID NO:53.

[0160] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:56, VH CDR2 having the sequence of SEQ ID NO:57, VH CDR3 having the sequence of SEQ ID NO:58; and / or a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:59, VL CDR2 having the sequence of SEQ ID NO:60, VL CDR3 having the sequence of SEQ ID NO:61.

[0161] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:64, VH CDR2 having the sequence of SEQ ID NO:65, VH CDR3 having the sequence of SEQ ID NO:66; and / or a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:67, VL CDR2 having the sequence of SEQ ID NO:68, VL CDR3 having the sequence of SEQ ID NO:69.

[0162] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:72, VH CDR2 having the sequence of SEQ ID NO:73, VH CDR3 having the sequence of SEQ ID NO:74; and / or a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:75, VL CDR2 having the sequence of SEQ ID NO:76, VL CDR3 having the sequence of SEQ ID NO:77.

[0163] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:80, VH CDR2 having the sequence of SEQ ID NO:81, VH CDR3 having the sequence of SEQ ID NO:82; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:83, VL CDR2 having the sequence of SEQ ID NO:84, VL CDR3 having the sequence of SEQ ID NO:85.

[0164] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:88, VH CDR2 having the sequence of SEQ ID NO:89, VH CDR3 having the sequence of SEQ ID NO:90; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:91, VL CDR2 having the sequence of SEQ ID NO:92, VL CDR3 having the sequence of SEQ ID NO:93.

[0165] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy-chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:96, VH CDR2 having the sequence of SEQ ID NO:97, VH CDR3 having the sequence of SEQ ID NO:98; and / or a light-chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:99, VL CDR2 having the sequence of SEQ ID NO:100, VL CDR3 having the sequence of SEQ ID NO:101.

[0166] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:104, VH CDR2 having the sequence of SEQ ID NO:105, VH CDR3 having the sequence of SEQ ID NO:106; and / or a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:107, VL CDR2 having the sequence of SEQ ID NO:108, VL CDR3 having the sequence of SEQ ID NO:109.

[0167] In certain embodiments, the antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO:112, VH CDR2 having the sequence of SEQ ID NO:113, VH CDR3 having the sequence of SEQ ID NO:114; and / or a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO:115, VL CDR2 having the sequence of SEQ ID NO:116, VL CDR3 having the sequence of SEQ ID NO:117.

[0168] In certain embodiments, the Fc variant has three amino acid residue substitutions and one amino acid residue deletion at amino acid residues 327, 328, 330, and 331 of the amino acid sequence of IgG Fc.

[0169] In certain embodiments, the Fc variant has amino acid residue substitutions at amino acid residues 327, 328, and 330 of the amino acid sequence of IgG Fc and an amino acid residue deletion at amino acid residue 331; wherein the Fc variant has an increased affinity for Fcγ receptor (FcγR) compared to IgG Fc;

[0170] In certain embodiments, the Fc variant has amino acid residue substitutions at amino acid residues 327, 328, and 330 of the Fc of IgG and an amino acid residue deletion at amino acid residue 331.

[0171] In certain embodiments, the Fc variant has a substitution of a glutamic acid residue at amino acid residue 327 of the IgG Fc polypeptide.

[0172] In certain embodiments, the Fc variant has a substitution of a tryptophan residue at amino acid residue 328 of the IgG Fc polypeptide.

[0173] In certain embodiments, the Fc variant has a substitution of a serine residue at amino acid residue position 330 of the IgG Fc polypeptide.

[0174] In certain embodiments, the amino acid site is obtained by the Eu numbering system.

[0175] In certain embodiments, the Fc variant has the sequence as set forth in SEQ ID NO:7.

[0176] In certain embodiments, the Fc variant also has substitutions of amino acid residues at amino acid residue positions 252, 254, and 256 of the amino acid sequence of IgG Fc.

[0177] In certain embodiments, the Fc variant has one or more of the following characteristics:

[0178] (1) The Fc variant has a substitution of a tyrosine residue at amino acid residue position 252 of IgG Fc;

[0179] (2) The Fc variant has a substitution of a threonine residue at amino acid residue position 254 of IgG Fc;

[0180] (3) The Fc variant has a substitution of a glutamic acid residue at amino acid residue position 256 of IgG Fc.

[0181] In certain embodiments, the amino acid site is obtained by the Eu numbering system.

[0182] In certain embodiments, the Fc variant also has substitutions of amino acid residues at amino acid residue positions 37, 39, and 41 corresponding to SEQ ID NO:7.

[0183] In certain embodiments, the Fc variant has one or more of the following characteristics:

[0184] (1) The Fc variant has a substitution of a tyrosine residue at amino acid residue position 37 of IgG Fc;

[0185] (2) The Fc variant has a substitution of a threonine residue at amino acid residue position 39 of IgG Fc;

[0186] (3) The Fc variant has a substitution of a glutamic acid residue at amino acid residue position 41 of IgG Fc.

[0187] In certain embodiments, the Fc variant has the sequence as set forth in SEQ ID NO:118.

[0188] In certain embodiments, the Fc variant has a substitution of glutamic acid at the 267th amino acid residue and a substitution of phenylalanine at the 328th amino acid residue of the amino acid sequence of IgG Fc;

[0189] Alternatively,

[0190] the Fc variant has a substitution of glutamic acid at the 52nd amino acid residue corresponding to SEQ ID NO:6 and a substitution of phenylalanine at the 113th amino acid residue corresponding to SEQ ID NO:6.

[0191] In certain embodiments, the Fc variant has an increased affinity for Fcγ receptor (FcγR) compared to IgG Fc.

[0192] In certain embodiments, the Fc variant has the sequence shown in SEQ ID NO:8.

[0193] In certain embodiments, the Fc variant has a substitution of glutamic acid at the 267th amino acid residue and a substitution of phenylalanine at the 328th amino acid residue of the amino acid sequence of IgG Fc; and, the Fc variant also has substitutions of tyrosine, threonine, and glutamic acid at the 252nd, 254th, and 256th amino acid residues of the amino acid sequence of IgG Fc, respectively;

[0194] Alternatively,

[0195] the Fc variant has a substitution of glutamic acid at the 52nd amino acid residue corresponding to SEQ ID NO:6 and a substitution of phenylalanine at the 113th amino acid residue corresponding to SEQ ID NO:6; and, the Fc variant also has substitutions of tyrosine, threonine, and glutamic acid at the 37th, 39th, and 41st amino acid residues corresponding to SEQ ID NO:6, respectively.

[0196] In certain embodiments, the Fc variant has an increased affinity for Fcγ receptor (FcγR) compared to IgG Fc.

[0197] In certain embodiments, the Fc variant has the sequence shown in SEQ ID NO:119.

[0198] In certain embodiments, the FcγR is selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, or any combination thereof.

[0199] In certain embodiments, the FcγR is derived from a mammal (e.g., mouse, human).

[0200] In certain embodiments, the FcγR is human FcγRIIb.

[0201] In certain embodiments, the antibody or antigen-binding fragment thereof as described above further comprises a constant region.

[0202] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises an IgG heavy chain constant region.

[0203] In certain embodiments, the light chain constant region of the light chain of the antibody or antigen-binding fragment thereof is a κ light chain constant region or a λ light chain constant region.

[0204] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO: 12.

[0205] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region as shown in SEQ ID NO: 13.

[0206] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, a heavy chain constant region, the Fc or Fc variant of IgG.

[0207] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof has a sequence as shown in SEQ ID NO: 4, 9, 11, 120 or 121.

[0208] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof has a sequence as shown in SEQ ID NO: 4, 9, 11, 120 or 121, and the light chain of the antibody or antigen-binding fragment thereof has a sequence as shown in SEQ ID NO: 5.

[0209] In certain embodiments, the antibody is a humanized antibody, a chimeric antibody, a fully human antibody, a bispecific antibody or a multispecific antibody.

[0210] In certain embodiments, the antibody or antigen-binding fragment thereof as described above, wherein the antibody or antigen-binding fragment thereof is labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or biotin.

[0211] In a fourth aspect, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect.

[0212] In a fifth aspect, the present application provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0213] In a sixth aspect, the present application provides a host cell comprising the isolated nucleic acid molecule as described above or the vector as described above.

[0214] In a seventh aspect, the present application provides a method for preparing the antibody or its antigen-binding fragment as described in the first, second, or third aspect, which comprises culturing the host cell as described above under conditions allowing the expression of the antibody or its antigen-binding fragment, and recovering the antibody or its antigen-binding fragment from the cultured host cell culture.

[0215] In an eighth aspect, the present application provides a multispecific molecule comprising the antibody or its antigen-binding fragment as described in the first, second, or third aspect.

[0216] In certain embodiments, the multispecific molecule specifically binds IgE and additionally specifically binds one or more other targets.

[0217] In certain embodiments, the multispecific molecule further comprises at least one second specific binding molecule (such as a second antibody) having specificity for a second target.

[0218] In a ninth aspect, the present application provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment as described in the first, second, or third aspect or the multispecific molecule as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0219] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0220] In certain embodiments, the additional pharmaceutically active agent is a drug having anti-allergic or anti-asthmatic properties.

[0221] In a tenth aspect, the present application provides a kit containing the antibody or its antigen-binding fragment as described in the first, second, or third aspect.

[0222] In certain embodiments, the antibody or its antigen-binding fragment is labeled with a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.

[0223] In certain embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or its antigen-binding fragment as described above.

[0224] In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.

[0225] In an eleventh aspect, the present application provides the use of the antibody or its antigen-binding fragment as described in the first aspect, the second aspect, or the third aspect, the isolated nucleic acid molecule as described above, the vector as described above, the host cell as described above, the multispecific molecule as described above, or the pharmaceutical composition as described above for the preparation of a medicament for reducing or inhibiting IgE levels or for preventing and / or treating IgE-related diseases in a subject.

[0226] In certain embodiments, the IgE-related disease is a type I hypersensitivity disease.

[0227] In certain embodiments, the IgE-related diseases are selected from allergic reactions, allergic rhinitis, allergic cough, anaphylactic shock, food allergy, hypersensitivity, asthma (such as moderate to severe persistent allergic asthma), chronic urticaria (such as chronic spontaneous urticaria), acute urticaria, acute bronchospasm, atopic dermatitis, inflammatory skin diseases, eczema, laryngeal edema, angioneurotic edema, nasal polyps, sinus inflammation, and the like.

[0228] In certain embodiments, the subject is a mammal, such as a human, a monkey, or a mouse.

[0229] In certain embodiments, the antibody or its antigen-binding fragment is used alone or in combination with another pharmaceutically active agent.

[0230] In certain embodiments, the antibody or its antigen-binding fragment specifically binds to IgE under neutral or alkaline conditions; preferably, the neutral or alkaline condition is a condition with a pH greater than or equal to 7 (e.g., a pH equal to 7.4).

[0231] In certain embodiments, the antibody or its antigen-binding fragment does not specifically bind to IgE under a condition with a pH equal to 6.

[0232] In a twelfth aspect, the present application provides a method for reducing or inhibiting IgE levels or for preventing and / or treating IgE-related diseases in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or its antigen-binding fragment as described in the first aspect, the second aspect, or the third aspect, the isolated nucleic acid molecule as described above, the vector as described above, the host cell as described above, the multispecific molecule as described above, or the pharmaceutical composition as described above.

[0233] In certain embodiments, the IgE-related disease is a type I hypersensitivity disease.

[0234] In certain embodiments, the IgE-related disease is selected from the group consisting of anaphylaxis, allergic rhinitis, allergic cough, anaphylactic shock, food allergy, hypersensitivity, asthma (such as moderate to severe persistent allergic asthma), chronic urticaria (such as chronic spontaneous urticaria), acute urticaria, acute bronchospasm, atopic dermatitis, inflammatory skin disease, eczema, laryngeal edema, angioneurotic edema, nasal polyps, sinus inflammation, and the like.

[0235] In certain embodiments, the subject is a mammal, such as a human, a monkey, or a mouse.

[0236] In certain embodiments, the method further comprises administering an additional drug having anti-allergic or anti-asthmatic activity.

[0237] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to IgE under neutral or basic conditions; preferably, the neutral or basic condition is a condition with a pH greater than or equal to 7 (e.g., a pH equal to 7.4).

[0238] In certain embodiments, the antibody or antigen-binding fragment thereof does not specifically bind to IgE under a condition with a pH equal to 6.

[0239] In a thirteenth aspect, the present application provides a method for detecting the presence or amount of IgE in a sample, which comprises the following steps:

[0240] (1) contacting the sample with the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect, or the third aspect;

[0241] (2) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and IgE or detecting the amount of the complex.

[0242] In certain embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable label.

[0243] In certain embodiments, the IgE is human IgE.

[0244] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to IgE under neutral or basic conditions; preferably, the neutral or basic condition is a condition with a pH greater than or equal to 7 (e.g., a pH equal to 7.4).

[0245] In certain embodiments, the antibody or antigen-binding fragment thereof does not specifically bind to IgE under a condition with a pH equal to 6.

[0246] Term Definition

[0247] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the operation steps such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are all conventional steps widely used in the corresponding fields. At the same time, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0248] As used herein, the term "IgG" refers to a polypeptide encoded by the immunoglobulin gamma gene. In humans, IgG includes IgG1, IgG2, IgG3, and IgG4. In mice, IgG includes IgG1, IgG2a, IgG2b, and IgG3. The known Ig domains VH, Cγ1, Cγ2, Cγ3, VL, and CL in the IgG class of antibodies.

[0249] As used herein, the terms "Fc" and "Fc region" have the same meaning and can be used interchangeably. It refers to the constant region other than the first constant region of the immunoglobulin domain. In certain embodiments, Fc refers to the last two constant regions of the IgA, IgD, and IgG immunoglobulin domains. In certain embodiments, Fc refers to the last three constant regions of the IgE and IgM immunoglobulin domains. In certain embodiments, Fc contains an N-terminal flexible hinge. In certain embodiments, Fc contains the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. In certain embodiments, Fc is an antibody fragment formed by the disulfide bond binding of the second and third constant regions of the first heavy chain of the antibody and the second and third constant regions of the second heavy chain. In certain embodiments, the numbering of the human IgG heavy chain Fc region follows the EU indexing method of Kabat (see, for example, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991). In certain embodiments, Fc is an isolated Fc region, or an Fc region located in an antibody or its antigen-binding fragment, or a polypeptide containing the amino acid sequence of the Fc region.

[0250] As used herein, the terms "Fcγ receptor" or "FcγR" have the same meaning and are used interchangeably. It refers to a protein that can bind to the Fc region of IgG and is encoded by the FcγR gene. Fcγ receptors include, but are not limited to, FcγRI (CD64), including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isotypes FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isotypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); see, e.g., Jefferis et al., 2002, Immunol Lett 82:57-65. FcγR can be from any organism, including, but not limited to, humans, mice, rats, monkeys, and humans.

[0251] Among them, FcγRIIa is an activating receptor, which is related to the occurrence of inflammation and tissue damage and is one of the important receptors leading to autoimmune diseases. At present, studies have confirmed that reducing the binding of IgG to FcγRIIa is beneficial to reducing the occurrence of autoimmune diseases. The structure of FcγRIIb is relatively special. It mediates immunosuppressive signals and will down-regulate the corresponding functions of cells after activation. FcγRIIb is mainly expressed on the surface of myeloid cells and B cells (the only FcR on the surface of B cells) and plays an important role in the negative regulation of B cell function.

[0252] As used herein, the term "pH-dependent" means that the antigen-binding activity of an antibody at acidic pH is different from that at neutral or basic pH. In certain embodiments, pH-dependent means that the antigen-binding activity of an antibody at acidic pH is weaker than that at neutral or basic pH.

[0253] As used herein, the term “antibody” refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen, and the immunoglobulin-derived molecule binds to the target antigen through at least one antigen-binding site located in its variable region. When referring to the term “antibody”, unless the context clearly indicates otherwise, it includes not only intact antibodies, but also antigen-binding fragments capable of specifically binding to a target antigen. An “intact antibody” typically consists of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified into kappa (κ) and lambda (λ) light chains. Heavy chains can be classified into μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, and the heavy chain also contains a “D” region of about 3 or more amino acids. Each heavy chain consists of a heavy-chain variable region (VH) and a heavy-chain constant region (CH). The heavy-chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light-chain variable region (VL) and a light-chain constant region (CL). The light-chain constant region consists of one domain CL. The constant domains do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further subdivided into regions with high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy-chain / light-chain pair form the antigen-binding site. The assignment of amino acids to each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0254] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the variable regions of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0255] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined by the Kabat, Chothia, or IMGT numbering systems.

[0256] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues defined as above.

[0257] The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0258] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of a full-length antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fd, Fv, disulfide-linked Fv, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody (sdAb), chimeric antibody, linear antibody, nanobody (technology from Domantis), probody, and such polypeptides that comprise at least a portion of an antibody sufficient to confer upon the polypeptide the ability to specifically bind an antigen. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0259] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, a "full-length heavy chain" refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be from a single species, such as human; they can also be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize / bind the same antigen.

[0260] As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "(Fab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; the term "Fab' fragment" means the fragment obtained by reducing the disulfide bond linking the two heavy chain fragments in the (Fab')2 fragment and consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of VH and CH1 domains).

[0261] As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a single variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind an antigen, although its affinity may be lower than that of the complete binding site.

[0262] As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, disulfide bonds can also exist between the VH and VL of the scFv. In certain embodiments of the present invention, the scFv can form a di-scFv, which refers to two or more individual scFvs linked in series to form an antibody. In certain embodiments of the present invention, the scFv can form a (scFv)2, which refers to two or more individual scFvs linked in parallel to form an antibody.

[0263] The term "bispecific antibody" refers to an antibody that has binding specificity for two or more (e.g., three or four) different antigens (or epitopes). A bispecific antibody contains multiple antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain contained in the bispecific antibody can independently be selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a (Fab’)2 fragment, or an scFv). In some cases, the individual antigen-binding domains are linked by a peptide linker.

[0264] Each of the above antibody fragments retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen.

[0265] Antigen-binding fragments of an antibody (e.g., the above antibody fragments) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as for intact antibodies.

[0266] As used herein, the term "Chimeric antibody" refers to an antibody in which a portion of the light chain or / and heavy chain is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), but in any case, it still retains the binding activity to the target antigen (U.S.P4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In certain embodiments, the term "chimeric antibody" may include an antibody in which the variable regions of the heavy and light chains of the antibody are from a first antibody, and the constant regions of the heavy and light chains of the antibody are from a second antibody.

[0267] As used herein, the term "humanized antibody" may include an antibody in which the variable regions of the heavy and light chains of the antibody are from a fully human antibody, and the constant regions of the heavy and light chains of the antibody are from murine constant regions. As used herein, the term "fully human antibody" refers to an antibody in which the entire amino acid sequence of the antibody is from a human and can be obtained by techniques such as phage and yeast display, transgenic animals, single B cells, etc.

[0268] As used herein, the term "specifically bind" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0269] The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The KD, kon, and kdis values can be measured using any valid method. In certain embodiments, surface plasmon resonance (SPR) in a Biacore can be used to measure the dissociation constant. Additionally, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

[0270] As used herein, the detectable label of the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics, or chemical means. Such labels are well known in the art and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as tris(bipyridine)ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0271] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, enabling the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain multiple elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication.

[0272] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0273] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue with another amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0274] The writing of the twenty conventional amino acids involved in this article follows the conventional usage. See, for example, Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0275] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining reagents, absorption delaying reagents, preservatives. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Osmotic pressure maintaining reagents include but are not limited to sugars, NaCl and the like. Absorption delaying reagents include but are not limited to monostearates and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include but are not limited to various antibacterial and antifungal reagents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% NaCl), glucose solution (such as 5% glucose), solution containing a surfactant (such as 0.01% polysorbate 20), pH buffer solution (such as phosphate buffer solution), Ringer's solution and any combination thereof.

[0276] As used herein, the term "prevention" refers to methods implemented to prevent or delay the occurrence of a disease, disorder, or symptom in a subject. As used herein, the term "treatment" refers to methods implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., not worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether in part or in whole), whether detectable or not. In addition, "treatment" may also refer to prolonging the survival period compared to the expected survival period (if untreated).

[0277] As used herein, the term "subject" refers to a mammal, such as a human, monkey, or mouse. In certain embodiments, the subject (such as a human, monkey, or mouse) has a disease associated with α-Synuclein (such as Parkinson's disease, Lewy body dementia, multiple system atrophy, or a combination thereof), or is at risk of having such a disease.

[0278] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease refers to an amount sufficient to prevent, inhibit, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, the amount effective for a therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other treatments administered simultaneously, and so on.

[0279] As used herein, the term "EU" is a numbering system for antibodies, also known as the EU Numbering System or Scheme. Its origin was in the late 1960s (1968 - 1969), when Gerald M Edelman et al. isolated and purified the first human IgG1 immunoglobulin, named Eu, determined its amino acid sequence, and numbered it (Edelman GM et al, 1969, Proc Natl Acad USA, 63:78 - 85). The heavy chain constant regions of other immunoglobulins are aligned with Eu for amino acid sequence comparison, and the corresponding amino acid positions are the Eu numbers.

[0280] Advantages of the Invention

[0281] In a first aspect, the present application performs histidine mutations on the CDR sequences contained in omalizumab to obtain an antibody with pH-dependent binding to IgE. The pH-dependent antibody of the present application has the following advantages: after binding to IgE in the weakly alkaline environment of body fluids, it can frequently carry IgE and be endocytosed by cells. When the antibody-IgE complex is endocytosed into the weakly acidic environment of endosomes, the antibody separates from IgE. Thereafter, IgE is transported to lysosomes for degradation, while the antibody is recycled back to the extracellular space with FcRn and mediates the endocytic degradation of IgE again. Thus, the pH-dependent antibody of the present application is beneficial for reducing the dosage of the drug and has the potential for drug development.

[0282] In a second aspect, the present application mutates the Fc region of the IgG antibody to obtain an antibody containing an Fc variant with high affinity for FcγRIIb, and the antibody has similar high affinity for human FcγRIIb and murine FcγRIIb.

[0283] Furthermore, the present application has experimentally confirmed that compared with antibodies in the prior art (e.g., omalizumab), the antibody with the above two mutations has an extended serum half-life of the antibody and can rapidly clear IgE at a lower dosage of the drug. Moreover, it can not only reduce the total IgE content in mice but also reduce the free IgE content. Therefore, the present application has great application potential in diseases related to IgE (e.g., type I hypersensitivity diseases) and shows great potential for drug development.

[0284] The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and not to limit the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. Brief Description of the Drawings

[0285] Figure 1 Shows the phage display library construction strategy and the nucleic acid sequences of the antibody library regions of the screened antibodies. Among them, in the figure, the degenerate bases S in the "library construction sequence" represent C or G, R represents A or G, Y represents C or T, M represents A or C, and W represents A or T; the "screened sequences" listed are all nucleic acid sequences of the pH-dependent IgE antibodies that have been confirmed, and the bases different from the omalizumab nucleic acid sequence are marked with shading.

[0286] Figure 2 Shows the SPR curves of omalizumab and 12 screened antibodies binding to IgE under the conditions of pH 7.4 and pH 6.0.

[0287] Figure 3The screening results of FcγRIIb high affinity Fc were shown, among which, Figure 3 A shows the results of three rounds of flow cytometry screening of the mammalian display system. Figure 3 B shows the comparison of the amino acid sequences of wild-type IgG Fc and Fc-EWPS mutant. Figure 3 Panel C shows the results of a horizontal comparison of the affinity of Fc-EWPS mutants with other FcγR receptors detected using SPR technology.

[0288] Figure 4 The results of the IgE clearance experiment in mice are shown. Figure 4 A in the figure shows the experimental scheme, including the administration time and dose, the injection time and dose of IgE, and the blood collection time points. Figure 4 B in the figure shows the method for determining the total IgE content in serum. Figure 4 C in the figure shows the method for determining the free IgE content in serum. Figure 4 D in the figure shows the changes in the total IgE content in the serum of wild-type C57 mice over time. Figure 4 Panel E shows the changes in the free IgE content in the serum of wild-type C57 mice over time. Figure 4 Panel F shows the changes in the total IgE content in the serum of C57-derived FcγRIIb humanized mice over time. Figure 4 Panel G shows the change in free IgE content in the serum of C57-derived FcγRIIb humanized mice over time.

[0289] Figure 5 The effect of drug dosage on IgE clearance was shown. Figure 5 AC in Figure 2 shows the changes in total IgE content in mouse serum over time at different drug doses. Figure 5 DF in FIG. 5 shows the change of free IgE content in mouse serum over time at different drug doses. Figure 5 G in Figure 1 shows the comparison of the free IgE content curves at different omalizumab dosages. The free IgE curves corresponding to the dosages of 50 μg, 20 μg and 10 μg are taken from the corresponding curves in panel (DF), and the free IgE curve corresponding to the dosage of 100 μg omalizumab is taken from Figure 5 The corresponding curve in G. Figure 5 H in Figure 1 shows the comparison of the free IgE content curves at different NK-2-12-EWPS dosages. The free IgE curves corresponding to the dosages of 50 μg, 20 μg and 10 μg are taken from the corresponding curves in panels (DF), and the free IgE curve corresponding to the dosage of 100 μg NK-2-12-EWPS is taken fromFigure 5 The corresponding curve in G. Figure 5 I in shows the curve of the change in IgE content when omalizumab is administered at 100 μg (the curve is taken from Figure 5 G), a comparison with the curve of the change in IgE content when NK-2-12-EWPS is administered at 20 μg.

[0290] Figure 6 Shows the results of the IgE clearance experiment in mice with NK-2-12-SELF. Among them, Figure 6 A in shows the change in the total IgE content in the serum of wild-type C57 mice over time. Figure 6 B in shows the change in the free IgE content in the serum of wild-type C57 mice over time. Figure 6 C in shows the change in the total IgE content in the serum of C57-derived FcγRIIb humanized mice over time. Figure 6 D in shows the change in the free IgE content in the serum of C57-derived FcγRIIb humanized mice over time.

[0291] Figure 7 Shows the effects of the serum half-life and dosage of NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF on the IgE clearance effect in mice. Among them, Figure 7 A in is the pharmacokinetic curve of NK-2-12-YTE-EWPS and NK-2-12-EWPS in FcRn humanized mice; Figure 7 B in is the pharmacokinetic curve of NK-2-12-YTE-SELF and NK-2-12-SELF in FcRn humanized mice; Figure 7 C in is the change in the total IgE content in the serum of FcγRIIb humanized mice over time at different dosages of NK-2-12-YTE-EWPS; Figure 7 D in is the change in the free IgE content in the serum of FcγRIIb humanized mice over time at different dosages of NK-2-12-YTE-EWPS; Figure 7 E in is the change in the total IgE content in the serum of FcγRIIb humanized mice over time at different dosages of NK-2-12-YTE-SELF; Figure 7 F in is the change in the free IgE content in the serum of FcγRIIb humanized mice over time at different dosages of NK-2-12-YTE-SELF.

[0292] Sequence information

[0293] Information on some of the sequences involved in the present invention is provided in Table 1 below.

[0294] Table 1: Description of Sequences

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305] Note: The amino acid residues marked by the boxes in the sequence are the mutated amino acid residues, and the hinge region of IgG Fc is underlined. Detailed implementation manners

[0306] The present invention will now be described with reference to the following examples which are intended to illustrate the invention (but not to limit the invention).

[0307] Unless otherwise specified, the experiments and methods described in the examples are generally carried out according to the conventional methods well-known in the art and described in various reference documents. For example, for the conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention, reference can be made to Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual", 2nd Edition (1989); "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., (1987)); the "Methods in Enzymology" series (Academic Press): "PCR 2: A Practical Approach" (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor (1995)), and "Animal Cell Culture" (edited by R.I. Freshney (1987)).

[0308] In addition, for those not specified with specific conditions in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially. Those skilled in the art know that the examples describe the present invention by way of illustration and are not intended to limit the scope of the present invention claimed. All the published cases and other reference materials mentioned herein are incorporated herein by reference in their entirety.

[0309] Example 1. Screening and Obtaining of Antibodies

[0310] 1. Establishment of phage library

[0311] By analyzing the crystal structure of the omalizumab-IgE complex (PDB: 5HYS) available in the public domain, the inventors found that omalizumab binds to the CH3 region of the Fc segment of IgE mainly through the heavy chain CDR1 (HCDR1), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), and light chain CDR2 (LCDR2). Only Tyr54 in the heavy chain CDR2 (HCDR2) contacts IgE, while the light chain CDR3 (LCDR3) does not contact IgE at all. Therefore, the inventors synthesized the nucleotide sequence encoding omalizumab in the form of VH-VL (scFv) (SEQ ID NO: 1) and primers containing degenerate bases (both the gene and primers were synthesized by Genewiz, Suzhou). Using the method of Overlap PCR, libraries were constructed for the four regions of HCDR1, HCDR3, LCDR1, and LCDR2 in the omalizumab scFv gene, as well as the codon of heavy chain Tyr54. The sequences of the library construction regions are as shown in Figure 1 as follows.

[0312] Thereafter, the scFv gene library was constructed between the two SfiI restriction sites of the phage display plasmid pCGMT3 to form a plasmid library, which was then transformed into Escherichia coli XL1-Blue. Then, according to the standard method of phage display, the helper phage M13KO7 was used to infect Escherichia coli XL1-Blue containing the plasmid library to propagate a recombinant phage library for subsequent screening.

[0313] Based on the amino acid sequence of IgE Fc in the crystal structure of the omalizumab-IgE complex (PDB: 5HYS), the inventors also synthesized the IgE Fc gene with an AVI tag and a 6×His tag (synthesized by Genewiz, Suzhou) and constructed it into the mammalian cell expression vector pTT5 (NovoPro, product number: V001466). Protein expression was carried out using HEK293F cells (Thermo Fisher Scientific, product number R79007) and FreeStyle TM medium (Gibco TM , product number: 12338-018). Thereafter, the inventors used an AKTA protein purifier (GE Healthcare, model: pure) and a HisTrap HP chromatography column (GE Healthcare, product number: 17524701) to purify IgE Fc with 6×His and AVI tags according to the purification steps described in the equipment instruction manual. A part of the purified protein was biotinylated using a biotinylation kit (GeneCopoeia, product number: BI001), and biotin was linked to the AVI tag of IgE Fc for use in the subsequent panning step.

[0314] During panning, biotinylated IgE Fc was first bound to streptavidin magnetic beads (Invitrogen, catalog number: 11205D) as the stationary phase. Then, according to the standard method of phage display, the magnetic beads bound with IgE Fc were first co-incubated with the phage library dissolved in BSA-PBST pH7.4 (PBS pH7.4 supplemented with 5% BSA and 0.05% Tween 20) at room temperature for two hours, and then the magnetic beads were washed four times with PBST pH 7.4 (PBS pH7.4 supplemented with 0.05% Tween 20) to remove non-specifically adsorbed phages. Finally, elution was performed with PBS pH 6.0 to obtain phages that bind to IgE at pH7.4 and dissociate from IgE at pH 6.0. Thereafter, according to the standard method of phage display, the phages obtained by elution were used to infect Escherichia coli XL1-Blue, enabling the plasmid of the panned library to enter the bacteria. Then, the bacteria were infected with the helper phage M13KO7 to amplify and propagate the recombinant phages for the next round of panning.

[0315] After three rounds of panning, the recombinant phages were significantly enriched, with an enrichment factor reaching 581-fold, indicating that phages with pH-dependent binding ability to IgE Fc had been obtained.

[0316] 2. Obtaining antibodies

[0317] The plasmids of the phages obtained after three rounds of panning were sequenced. The results showed that among 150 randomly selected monoclonal clones, Figure 1 the NK-2-12 sequence shown in [Figure] was frequently present, with more than one-third of the scFv sequences being this sequence, while other sequences rarely repeated. The inventors constructed the sequences of NK-2-12 and 14 other randomly selected clones (a total of 15 sequences) into the pTT5 (NovoPro, catalog number: V001466) vector to form complete antibody genes. Then, expression was performed using the aforementioned method for expressing IgE Fc, and purification was carried out using an AKTA protein purifier (GE Healthcare, model: pure) and a HiTrap ProteinA HP chromatography column (GE Healthcare, catalog number: 29048576) according to the purification steps described in the equipment instruction manual to obtain these 15 antibodies. The sequences of these antibodies were obtained through company sequencing, as shown in Table 3 specifically. Among them, the heavy chain amino acid sequence of NK-2-12 is as shown in SEQ ID NO:4, and the light chain amino acid sequence is as shown in SEQ ID NO:5.

[0318] In addition, the inventors also synthesized the gene of human IgE-type anti-hen egg white lysozyme (anti-HEL) antibody with a 6×His tag (synthesized by Suzhou Genewiz Biotechnology Co., Ltd.), constructed it into the pTT5 plasmid (NovoPro, catalog number: V001466), and expressed and purified it using the aforementioned method for expressing and purifying IgE Fc for subsequent research. The amino acid sequence of the heavy chain of this human anti-HEL IgE is shown in SEQ ID NO:2, and the amino acid sequence of the light chain of this human anti-HEL IgE is shown in SEQ ID NO:3.

[0319] The inventors used surface plasmon resonance (SPR) technology to detect the affinity of the above 15 antibodies and omalizumab for IgE under PBS pH 7.4 and PBS pH 6.0 conditions. The specific method is as follows: First, the anti-6×His antibody was immobilized on a Series S CM5 chip (Cytiva, catalog number: 29-1049-88) through an amino coupling reagent (Cytiva, catalog number: BR-1000-50), and then the chip was installed on a surface plasmon resonance instrument (GE Healthcare, model: Biacore T200). The anti-HEL IgE with a 6×His tag was diluted to a concentration of 400 nM with PBS of a specific pH and flowed through the chip at a flow rate of 10 μl / min for 60 s for sample injection and capture. Then, the above 15 antibodies were dissolved in PBS of the same pH, and a 2-fold serial dilution solution gradient (such as 500 nM, 250 nM, 125 nM, down to 15.625 nM, etc.) was prepared with this PBS and injected at a flow rate of 30 μl / min for 120 s of binding, and then flowed through the chip with this PBS for 240 s of dissociation. Then, a 10 mM Glycine-HCl (pH 1.5) regeneration buffer was used to elute IgE and the antibody at a flow rate of 30 μl / min for 30 s to regenerate the chip. In the above IgE capture, sample injection, and dissociation steps, when measuring the affinity of the antibody for IgE under pH 7.4 conditions, the PBS used was PBS pH7.4; when measuring the affinity of the antibody for IgE under pH 6.0 conditions, the PBS used was PBS pH 6.0. After alternately performing the IgE capture, sample injection, dissociation, and chip regeneration cycles, the obtained data were fitted according to the steady-state binding model using the instrument-supplied software Biacore T200 Evaluation Software to obtain the corresponding association constant Ka and dissociation constant Kd, and then the binding dissociation constant KD (KD = Kd / Ka) was calculated.

[0320] The measurement results showed that 12 out of the 15 tested antibodies (numbered NK-2-12, NK-2-5, NK-177, NK-76, NK-1-6, NK-1-5, NK-174, NK-100, NK-2-4, NK-1-11, NK-77, and NK-181, the nucleic acid sequences of the antibody library construction area are as Figure 1 shown) exhibited pH-dependent binding to IgE (SPR curves are as Figure 2 shown, and the calculated KD values are shown in Table 2). All 12 of these antibodies bound to IgE at pH 7.4, and most of their binding dissociation constants KD were in the order of 10 -8 mol / L, with a few in the order of 10 -7 mol / L. However, no binding of the antibodies to IgE was detected at pH 6.0. As a control, omalizumab did not exhibit pH-dependent binding to IgE and showed high affinity for IgE at both pH 7.4 and pH 6.0 ( Figure 2 , Table 2).

[0321] Table 2. Binding dissociation constants of the selected antibodies to IgE

[0322]

[0323] Note: "ND" indicates that no binding activity was detected.

[0324] Table 3. Sequences of the antibodies (Kabat)

[0325]

[0326]

[0327] Example 2. Analysis of Mutation Sites Related to pH-Dependence

[0328] Comparative analysis of the nucleic acid sequences of the antibody library construction area of these 12 antibodies ( Figure 1 ) showed that almost no mutations occurred in the HCDR3 region, proving that HCDR3 is the core region for antibody binding to IgE and has extremely low tolerance to mutations. No unified mutations were shown in the HCDR1 region of the 12 antibodies ( Figure 1 ), and 5 antibodies including NK-2-12 did not contain any mutations in this region at all, proving that mutations in HCDR1 are not the key factor for achieving pH-dependent binding of the antibody to IgE. Among the 12 antibodies, the sequences of NK-2-5 and NK-177 differed only at the heavy chain Y54 locus ( Figure 1)。Both antibodies showed pH-dependent binding to IgE, but NK-2-5 with the heavy chain Y54H mutation showed slightly higher affinity than NK-177 without this mutation at pH 7.4 (Table 2), demonstrating that the heavy chain Y54H mutation contributed less to the pH-dependence of the antibody but had a certain positive effect on the affinity of the antibody for IgE in the humoral environment.

[0329] A large number of consistent histidine substitution mutations occurred in the LCDR1 of 12 antibodies ( Figure 1 , Table 4). The original amino acid sequence of omalizumab LCDR1 was 30-DYDGDSY-36. Except that D34 hardly had histidine substitution mutations, in 12 pH-dependent antibodies, histidine substitution mutations of the other 6 amino acid residues (D30H, Y31H, D32H, G33H, S35H, and Y36H based on the omalizumab light chain) occurred with a frequency of more than two-thirds. Among the 12 antibodies, 5 antibodies contained all of the above 6 histidine substitution mutations, and 4 antibodies (including NK-2-12) contained 5 of the above 6 histidine substitution mutations. Among the remaining 3 antibodies, 2 antibodies contained 4 of the above 6 histidine substitution mutations, and 1 antibody contained 3 of the above 6 histidine substitution mutations.

[0330] In the LCDR2 region, a highly consistent histidine substitution mutation also occurred in 12 antibodies ( Figure 1 , Table 4), mainly manifested as S56H and Y57H mutations of the original amino acid sequence 53-YAASY-57 of omalizumab LCDR2, and A55H mutation also occurred in some antibodies. Among the 12 pH-dependent antibodies, as many as 11 antibodies contained both S56H and Y57H mutations, and all 12 antibodies contained at least two of the A55H, S56H, and Y57H mutations, demonstrating that the combination of these three mutations played an important role in achieving pH-dependent binding to IgE. In addition, in addition to the above two point mutations of S56H and Y57H in the LCDR2 of NK-2-12, a histidine residue was unexpectedly inserted between Ala55 and the mutated amino acid of S56H (numbering this inserted amino acid residue as 55a, and naming this insertion mutation as H55a insertion mutation). Comparing the sequences of the two antibodies NK-2-12 and NK-100 showed that they differed only in the LCDR2 region. NK-100 contained three consecutive histidine substitution mutations of A55H, S56H, and Y57H in this region, while NK-2-12 retained Ala55, but the H55a insertion mutation and S56H, Y57H also constituted three consecutive histidine mutations ( Figure 1, (Table 4). Although both NK-2-12 and NK-100 showed pH-dependence for IgE, the affinity of NK-2-12 for IgE at pH 7.4 was much higher than that of NK-100 (Table 2), indicating that the insertion of H55a had an important positive effect. Moreover, as mentioned above, the sequence of NK-2-12 was the only sequence that frequently appeared in the phage display screening results, indicating that NK-2-12 must have some special advantages compared with other antibodies, such as being more conducive to expression or folding, etc.

[0331] Table 4. Amino acid sequences of LCDR1 and LCDR2 of the selected antibodies

[0332]

[0333] Note: "*" indicates the H55a insertion mutation.

[0334] In summary, data analysis showed that introducing D30H, Y31H, D32H, G33H, S35H, Y36H, A55H, H55a insertion mutation, S56H, Y57H mutations or combinations of these mutations on the basis of the omalizumab light chain sequence could prepare antibodies that could bind to IgE under physiological pH conditions in the human body (~pH 7.4) but not bind to IgE at pH 6.0.

[0335] The pH-dependent antibodies of the present application have the following advantages: after binding to IgE in the weakly alkaline environment of body fluids, they can frequently carry IgE and be endocytosed by cells. When the complex of the antibody and IgE is endocytosed into the weakly acidic environment of endosomes, the antibody dissociates from IgE. Thereafter, IgE is transported to lysosomes for degradation, while the antibody is recycled extracellularly with FcRn and mediates the endocytic degradation of IgE again. Thus, the pH-dependent antibodies of the present application are beneficial to reducing the dosage of drugs and have the potential for drug development.

[0336] Example 3. Association between Mutation Sites and pH-Dependence

[0337] To further verify the relationship between the above-mentioned mutations in the LCDR1 and LCDR2 regions and the pH-dependence of the antibody, the inventors reverted some of the mutated sites in the LCDR1 and LCDR2 regions to the corresponding sequences of omalizumab on the basis of NK-2-12, and tested the binding dissociation constants of the related reverted mutant antibodies with IgE at pH 7.4 and pH 6.0. The methods for antibody expression purification and determination of binding dissociation constants were as described in Example 1. Specifically, based on the NK-2-12 antibody in Table 4, the antibody NK-2-12-L1 was constructed by transformation. The specific CDR sequences for transformation are shown in Table 5, and the remaining CDR sequences are the same as those of the NK-2-12 antibody.

[0338] The results showed that compared with NK-2-12, restoring the LCDR2 sequence of NK-2-12 to the LCDR2 sequence of omalizumab (NK-2-12-L1) hardly changed the affinity of the antibody for IgE at pH 7.4, but the affinity of the antibody for IgE was increased under the condition of pH 6.0, and the dissociation constant of binding changed from undetectable (ND) to 10 -5 orders of magnitude (Table 5). This indicates that the mutations contained in the LCDR2 region contribute to the pH-dependent binding of the antibody to IgE. At the same time, it can also be seen that even without the mutations in the LCDR2 region, the antibody can still maintain a relatively high level of pH-dependence, and the dissociation constant of binding of the antibody to IgE at pH 7.4 and pH 6.0 still differs by more than 350-fold, proving that the mutations contained in LCDR1 can independently achieve the pH-dependent binding of the antibody to IgE to a large extent.

[0339] Therefore, the above experiments showed that on the basis of the CDR sequences contained in omalizumab, only histidine mutations were made at some amino acid sites of LCDR1, and the obtained antibody still had pH-dependence, that is, the dissociation constant of binding of the antibody to IgE at pH 7.4 and pH 6.0 had a large difference.

[0340] Table 5. Amino acid sequences of LCDR1 and LCDR2 of the revertant antibodies and the dissociation constants of binding to IgE

[0341]

[0342] *H55a insertion mutation, ND indicates undetectable.

[0343] Example 4. Screening and Obtaining of FcγRIIb-High Affinity Fc

[0344] 1. Screening of high-affinity Fc of FcγRIIb

[0345] The inventors designed a new library construction strategy, randomly constructed a library for the region of IgG Fc that contacts FcγRIIb, and on this basis, used the mammalian display screening system developed and published by the inventors' team previously (Chen et al., 2021, doi: 10.7150 / thno.51299) to screen out an IgG Fc mutant that can enhance the affinity for the receptor FcγRIIb.

[0346] Based on the complex structures of wild-type IgG Fc (wtFc, SEQ ID NO:6) with FcγRs such as FcγRIIa (PDB: 3RY6) and FcγRIIIa (PDB: 1E4K), the amino acids at positions 327 - 331 in the IgG Fc amino acid sequence (amino acid sequence: ALPAP) are one of the key sites for the interaction between Fc and receptors. The inventors of this application proposed to construct a library for the amino acids at positions 327 - 331 corresponding to the amino acid sequence in the IgG Fc nucleic acid sequence by means of Overlap PCR, changing the original five consecutive codons in this region to four consecutive NNK (N represents any base, and K represents G or T base) codons, so that the translation product of the gene has the sequence ALPAP in this region changed to four consecutive random amino acid residues. Thereafter, the inventors constructed a mammalian display library according to the method described in the mammalian display screening system previously published by the team, and screened the extracellular region of the receptor FcγRIIb to obtain an IgG Fc mutant with high specific affinity for FcγRIIb.

[0347] The screening results showed that after three rounds of screening, cells with higher affinity for FcγRIIb were enriched ( Figure 3 A). Twenty single-cell clones were randomly selected for sequencing, and it was found that the screened sequences were highly unified. Approximately 70% of the sequences replaced the ALPAP of the amino acids at positions 327 - 331 in the wild-type IgG Fc amino acid sequence with EWPS (this mutation was named the EWPS mutation, Figure 3 B). The IgG Fc amino acid sequence with the EWPS mutation is shown in SEQ ID NO:7 (the EWPS mutation is marked with a box, and the hinge region of IgG Fc is underlined).

[0348] 2. Affinity identification of the IgG Fc-EWPS mutant with the receptor FcγRIIb

[0349] The inventors purified the IgG Fc-EWPS mutant by the aforementioned method, and then used SPR technology to detect the binding dissociation constants of the IgG Fc-EWPS mutant with the extracellular region of human FcγRIIb with a 6×His tag (ACROBiosystems, product number: CDB-H5228) and the extracellular region of murine FcγRIIb with a 6×His tag (ACROBiosystems, product number: CDB-M52H7), respectively. The results showed that the KD of the IgG Fc-EWPS mutant for human FcγRIIb was 2.47×10 -7 mol / L, which was lower than the binding dissociation constant (KD = 2.41×10-6 ) One order of magnitude lower, demonstrating that the EWPS mutation indeed significantly enhances the affinity between human IgG Fc and human FcγRIIb. At the same time, the KD of the IgG Fc-EWPS mutant for murine FcγRIIb is 2.94×10 -7 mol / L, which is similar to the dissociation constant for human FcγRIIb.

[0350] The inventors further made a horizontal comparison of the IgG Fc-EWPS mutant with human FcγRIIIa F158 (ACROBiosystems, catalog number: CDB-H5220), FcγRIIIa V158 (ACROBiosystems, catalog number: CD8-H52H4), and FcγRIIa H131 (ACROBiosystems, catalog number: CD1-H5223) and other receptors. The results showed that compared with wild-type IgG Fc, in addition to a significant increase in the affinity of IgG Fc-EWPS for FcγRIIb, there was no obvious change in the affinity for FcγRIIIa F158 and FcγRIIa H131 ( Figure 3 C), while the affinity for FcγRIIIa V158 showed a significant decrease. These further demonstrated that the EWPS mutation can specifically enhance the affinity between IgG Fc and FcγRIIb.

[0351] In summary, the EWPS mutant of IgG Fc specifically has a high affinity for the receptor FcγRIIb. At the same time, the EWPS mutant of IgG Fc has a similar affinity for human and murine FcγRIIb. Therefore, the EWPS mutation has the advantage of being applicable in non-transgenic mouse models of FcγRIIb.

[0352] Example 5. Clearance Effect of NK-2-12-EWPS on Human IgE in Mice

[0353] Based on the aforementioned antibody NK-2-12, the inventors introduced the EWPS mutation that can enhance the affinity between the antibody and the receptor FcγRIIb into the Fc region of the antibody to construct the NK-2-12-EWPS antibody. The amino acid sequence of the heavy chain of NK-2-12-EWPS is shown in SEQ ID NO:9 (the introduced histidine mutation and EWPS mutation are marked with boxes), and the amino acid sequence of the light chain of NK-2-12-EWPS is shown in SEQ ID NO:10.

[0354] The purified NK-2-12-EWPS antibody was used to detect the clearance effect on human IgE in wild-type C57 mice (purchased from Vital River Laboratories) or C57-derived FcγRIIb humanized mice. During the experiment, the control group and the experimental group were randomly divided into groups of 5 mice each. The experimental method was as described in Figure 4 Figure A. The IgE injected into the peritoneal cavity of the mice was the human anti-HEL IgE described in Example 1. 30 minutes before injecting IgE into the peritoneal cavity of the mice, the antibody to be tested (NK-2-12 or NK-2-12-EWPS) was injected into the peritoneal cavity of the mice at a dose of 100 μg / mouse as each experimental group, or omalizumab was injected as the positive control group, or no injection was made as the negative control group. After injecting IgE into the peritoneal cavity of the mice at a dose of 50 μg / mouse, mouse tail vein blood (50 μl was taken from each mouse each time) was collected at five time points of 1, 2, 3, 8, and 22 hours. After standing at room temperature until it coagulated, the serum was obtained by centrifugation.

[0355] The detection method for the total human IgE content in the serum was as described in Figure 4 Figure B, which was a standard sandwich ELISA. The antibody used to coat the 96-well ELISA plate was the murine IgM anti-human IgE antibody HP6061 (Southern Biotech, catalog number: 9240-01), and the working concentration was the recommended concentration in the antibody instruction manual. As described in the instruction manual, this antibody did not conflict with omalizumab or FcεRI when binding to human IgE, that is, this antibody could bind both free IgE and IgE that had already bound to omalizumab or FcεRI. The antibody HP6029 (Southern Biotech, catalog number: 9250-05) was a murine IgG anti-human IgE HRP-conjugated antibody. As described in the instruction manual, this antibody did not conflict with HP6061 or omalizumab when binding to human IgE. Therefore, this HRP-conjugated antibody could be used to detect the total amount of IgE captured by HP6061 in accordance with the recommended concentration in the instruction manual, in combination with the substrate ABTS (Thermo Scientific, catalog number: 002024). When detecting the total IgE content in the serum, first, a standard curve was established using the above two antibodies and IgE with a known concentration to calculate the IgE concentration in the serum sample to be tested.

[0356] The detection method for the free human IgE content in the serum was an improved sandwich ELISA, as described in Figure 4As shown in C. The protein used to coat the 96-well ELISA plate is the receptor FcεRI of IgE (Sino Biological, product number: 13193-HNAH), and the usage concentration is 1 μg / ml. Only free IgE can be captured by its receptor FcεRI. Since there is an epitope conflict between the enzyme-linked antibody HP6029 and FcεRI, it cannot be used to detect the concentration of IgE captured by FcεRI. Therefore, HP6061 can be used to bind the IgE captured by FcεRI first, and then a goat anti-mouse IgM HRP enzyme-linked antibody, combined with the substrate ABTS, is used to detect the content of HP6061 to reflect the content of free IgE. When detecting the content of free IgE in serum, a standard curve also needs to be established first with known concentrations of IgE through the above method to calculate the concentration of free IgE in the serum sample to be tested.

[0357] The results of the IgE clearance experiment in wild-type C57 mice are as Figure 4 shown in D and 4E. Injecting omalizumab into mice will cause an extension of the IgE half-life, manifested as the IgE content in the serum of this group of mice always being higher than that of the negative control group of mice that only received IgE injection ( Figure 4 D). This is completely consistent with the clinical situation that injecting omalizumab will cause a further increase in the total IgE content in the patient's blood. On the contrary, as a marketed drug, omalizumab has a significant effect on reducing the concentration of free IgE in mouse serum ( Figure 4 E). The NK-2-12-EWPS antibody of the present application shows excellent effects in reducing total IgE in mice. In the mice injected with NK-2-12-EWPS, the IgE in the serum was basically completely cleared after 2 to 3 hours ( Figure 4 D), showing far better performance than omalizumab. Since NK-2-12-EWPS has cleared almost all IgE in the mouse serum in a short time, the content of free IgE in the mouse serum is also extremely low ( Figure 4 E), being lower than that of the mice injected with omalizumab throughout the process. While the NK-2-12 antibody without Fc-end modification did not show any effects in reducing total IgE and free IgE in mice ( Figure 4 D, E).

[0358] To be closer to the human environment, the inventor further verified the clearance effect of NK-2-12-EWPS on human IgE in C57-derived FcγRIIb humanized transgenic mice (Model Animal Research Center, product number: NM-HU-2000010). The experimental results are similar to those of the human IgE clearance experiment in wild-type C57 mice. Injecting omalizumab effectively reduced the free IgE in mice, but extended the IgE half-life, resulting in the accumulation of total IgE ( Figure 4F, G). The NK-2-12 antibody without Fc modification also failed to reduce the total IgE and free IgE in mice Figure 4 F, G). However, the IgE in the mice injected with NK-2-12-EWPS was rapidly cleared. Whether it was the total IgE content or the free IgE content in the mice, it was lower than that in the omalizumab injection group throughout the process Figure 4 F, G).

[0359] These in vivo experiments in mice all showed that NK-2-12-EWPS could reduce the IgE content in vivo through a mechanism different from that of omalizumab. In particular, NK-2-12-EWPS had the ability to reduce the total IgE in vivo, which omalizumab did not possess. Even in terms of reducing free IgE, NK-2-12-EWPS was much superior to omalizumab to a great extent, so it had great potential for drug development.

[0360] Example 6. Dosage Study of NK-2-12-EWPS in Mice

[0361] In principle, omalizumab reduces the free IgE content by neutralizing IgE. Therefore, to obtain sufficient neutralization effect, the dosage of omalizumab cannot be lower than the total amount of IgE in the patient's body, otherwise there will definitely be some IgE that is not neutralized. In fact, the clinical dosage of omalizumab usually needs to be as high as hundreds of times the total amount of IgE in the patient's body (calculated according to the publicly available "Chinese Dosage Table of Omalizumab"). The NK-2-12-EWPS antibody proposed by the inventor reduces the free IgE content in vivo by clearing all IgE. Therefore, theoretically, the dosage of NK-2-12-EWPS does not need to be higher than the total amount of IgE in the patient's body to fully reduce the free IgE content in the patient's body.

[0362] The inventor detected the changes in the total IgE and free IgE content in C57-derived FcγRIIb humanized mice over time under the premise that the intraperitoneal injection amount of human IgE per mouse was fixed at 50 μg according to the method described in Example 5, for the dosages of 50 μg, 20 μg, and 10 μg (corresponding to 100%, 40%, and 20% of the IgE injection amount).

[0363] The results showed that when using omalizumab, regardless of the dose size, it would cause an increase in the total IgE content in the serum, and the higher the omalizumab dose, the higher the total IgE content in the serum Figure 5 A-C). In terms of free IgE, when the dosage of omalizumab was not lower than the IgE injection amount, it showed a good neutralization effect, and the free IgE content decreased to a relatively low level from the beginning Figure 5D). However, when the dosage of omalizumab is lower than the IgE injection amount, IgE cannot be fully neutralized, and the content of free IgE only slowly decreases over time similar to that of the negative control group without injection of the therapeutic antibody, rather than being rapidly reduced. Figure 5 D - F). By longitudinally comparing the free IgE curves when using different dosages of omalizumab, the influence of the change in the dosage of omalizumab on the neutralization effect can be seen more clearly. Figure 5 G). Whether the dosage of omalizumab is higher than the IgE injection amount is the watershed for omalizumab to be able to sufficiently reduce serum free IgE, which also conforms to common sense. By comparing the change curves of the free IgE content when the dosage of omalizumab is 50 μg and the dosage of 100 μg described in Example 5 (corresponding to 100% and 200% of the IgE injection amount respectively), it is found that a higher dosage of omalizumab does not further reduce the IgE content in mice. Considering the measurement error factor, the two experimental curves are basically the same. Figure 5 G). This proves that the increase in the dosage of omalizumab has limited further gain on the neutralization effect, which conforms to the chemical thermodynamics theory. According to the binding - dissociation formula, to further reduce the content of free IgE, it is necessary to increase the dosage of omalizumab by an order of magnitude. In addition, due to the existence of the binding - dissociation constant of the antibody, a certain part of the antigen will definitely dissociate from the antibody and be in a free state. Therefore, although the dosage of omalizumab in the above two experiments is not lower than the total amount of IgE, the free IgE level in mice is always slightly higher than the baseline level. Figure 5 G, I).

[0364] Different from the case of omalizumab, when using NK - 2 - 12 - EWPS, even when the antibody dosage is lower than the IgE injection amount, it still has a good IgE clearance effect. The total IgE and free IgE contents in the mouse serum both rapidly decrease within the first few hours. Figure 5 A - E). When the dosage of NK - 2 - 12 - EWPS (20 μg) is only 40% of the IgE injection amount, the IgE content in the mouse serum can also be reduced to the baseline level within 3 hours. Figure 5 A - E). When the dosage of NK - 2 - 12 - EWPS is further reduced to 10 μg (only corresponding to 20% of the IgE injection amount), the IgE content in the mouse serum fails to be reduced to the baseline level within 3 hours. Figure 5 A - E). By longitudinally comparing the free IgE curves at different NK - 2 - 12 - EWPS dosages, it can be observed that when NK - 2 - 12 - EWPS is present, the reduction of its dosage mainly affects the IgE content at the first - hour time point, without changing the trend of the sharp decrease in IgE content. Figure 5 H). Only when the dosage of NK - 2 - 12 - EWPS is too low (10 μg antibody dosage, corresponding to 20% of the IgE injection amount), the IgE clearance rate will be slightly delayed.Figure 5 H), which is different from the situation when omalizumab is used.

[0365] Further comparing the serum free IgE curves when using 20 μg (corresponding to 40% of the IgE injection amount) of NK-2-12-EWPS and 100 μg (corresponding to 200% of the IgE injection amount) of omalizumab, it can be seen that starting from the second hour time point, 20 μg of NK-2-12-EWPS showed better free IgE control effect than 100 μg of omalizumab. Only at the first hour time point, the free IgE content corresponding to 100 μg of omalizumab was lower than the free IgE content corresponding to 20 μg of NK-2-12-EWPS ( Figure 5 I). This indicates that as a human-derived IgE-clearing antibody, NK-2-12-EWPS takes about one hour to accumulate its drug effect after being injected into the body. After that, even if the dosage of NK-2-12-EWPS is much lower than that of omalizumab, it can still exert a better free IgE control effect than omalizumab.

[0366] In summary, as a new concept of human-derived IgE-clearing antibody, NK-2-12-EWPS has the characteristics of low dosage and good effect compared with omalizumab, and has great potential for drug development. In addition, considering the actual clinical situation, after injecting a sufficient amount of omalizumab into the patient, almost all of the IgE in the patient's body is neutralized, so the allergic symptoms are alleviated or disappear. However, since the allergen has not disappeared, under its continuous stimulation, the patient's body is still continuously producing IgE. When the newly produced IgE in the patient's body exceeds the redundant amount of omalizumab, that is, when the total IgE content in the patient's body is higher than the total omalizumab content, the allergic symptoms will inevitably reappear. And the NK-2-12-EWPS proposed by the inventor plays a role in continuously clearing IgE. Therefore, even if the patient's body is still continuously producing IgE, NK-2-12-EWPS can maintain the free IgE in the patient's body at an extremely low level for a long time, thus controlling the allergic symptoms for a long time. From this perspective, NK-2-12-EWPS is especially suitable for chronic allergic patients who need long-term medication.

[0367] Example 7. Clearance Effect of NK-2-12-SELF on Human IgE in Mice

[0368] Another mutation protocol that can enhance the affinity between an antibody and the receptor FcγRIIb was described in the paper published by Chu et al. in 2008 (Chu et al., 2008, doi:10.1016 / j.molimm.2008.06.027), namely the S267E / L328F double-point mutation (SELF mutation) protocol. The inventors also introduced this SELF mutation into the Fc region of the antibody NK-2-12 described in Example 1, constituting the NK2-12-SELF antibody. The IgG Fc amino acid sequence with the SELF mutation is shown in SEQ ID NO:8, the heavy chain amino acid sequence of NK-2-12-SELF is shown in SEQ ID NO:11 (the introduced histidine mutation and SELF mutation are marked with boxes), and the light chain amino acid sequence of NK-2-12-SELF is shown in SEQ ID NO:5 (the introduced histidine mutation is marked with a box, and this sequence is the same as the light chain sequence of the aforementioned NK-2-12 antibody).

[0369] The purified and expressed NK-2-12-SELF antibody was also used for the clearance experiment of human IgE in wild-type C57 mice or FcγRIIb humanized mice derived from C57 according to the method described in Example 5. The experimental results showed that in wild-type C57 mice, NK-2-12-SELF showed a certain ability to reduce total IgE and free IgE ( Figure 6 A, B), but the effect was far less than that of NK-2-12-EWPS ( Figure 4 D, E). Especially, the concentration of free IgE in mice was higher than that in the omalizumab group for a relatively long period. However, this does not mean that NK-2-12-SELF does not have the potential to become a drug. Because the SELF mutation is different from the EWPS mutation. The latter has a high affinity for both human and murine FcγRIIb, while the SELF mutation only has a high affinity for human FcγRIIb. The inventors did not detect the affinity between the SELF mutation and murine FcγRIIb. Therefore, the ability to clear human IgE in wild-type C57 mice is poor. On the contrary, in FcγRIIb humanized mice derived from C57, the NK-2-12-SELF antibody showed excellent human IgE clearance ability. The total IgE content and free IgE content in mice were not only lower than those in the omalizumab group throughout the whole process, but even compared with NK-2-12-EWPS ( Figure 4 F, G), NK2-12-SELF was able to reduce the IgE in mice to the baseline level faster ( Figure 6 C, D).

[0370] Example 8. NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF Have the Ability to Clear Human IgE in Mice and a Longer Serum Half-Life

[0371] Based on the IgG Fc with SELF mutation and the IgG Fc with EWPS mutation synthesized in the foregoing embodiments, this embodiment further introduces mutations at three sites (i.e., M252Y / S254T / T256E), and the mutated fragments are respectively named: IgG Fc with YTE-EWPS mutation (the amino acid sequence thereof is shown in SEQ ID NO: 118) and IgG Fc with YTE-SELF mutation (the amino acid sequence thereof is shown in SEQ ID NO: 119).

[0372] Furthermore, two antibodies, NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF, were respectively constructed using the above two Fc fragments and the antibody NK-2-12 obtained in the foregoing embodiment. Among them, the amino acid sequence of the heavy chain of the NK-2-12-YTE-EWPS antibody is shown in SEQ ID NO: 120, and the amino acid sequence of the light chain of the NK-2-12-YTE-EWPS antibody is shown in SEQ ID NO: 5; the amino acid sequence of the heavy chain of the NK-2-12-YTE-SELF antibody is shown in SEQ ID NO: 121, and the amino acid sequence of the light chain of the NK-2-12-YTE-SELF is shown in SEQ ID NO: 5.

[0373] Express and purify four antibodies, NK-2-12-YTE-EWPS, NK-2-12-YTE-SELF, NK-2-12-EWPS and NK-2-12-SELF, and inject them into the peritoneal cavity of FcRn humanized mice (Model Animal, catalog number: NM-HU-00109) respectively for detecting serum half-life. During the experiment, each antibody was injected into the peritoneal cavity of 3 randomly grouped FcRn humanized mice at a dose of 10 mg / kg. Thereafter, mouse tail vein blood was collected at 14 time points, namely 45 minutes, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours and 240 hours. After standing at room temperature until coagulation, the serum was obtained by centrifugation. The detection method for the content of human antibodies in serum was standard sandwich ELISA. The antibody used for coating the 96-well ELISA plate was goat anti-human IgG Fc antibody (Jackson, catalog number: 109-006-098), and HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109-036-088) in combination with substrate ABTS (Thermo Scientific, catalog number: 002024) was used to detect the captured human IgG content. The use concentrations of both antibodies were the recommended concentrations in the antibody instruction manuals. When detecting the content of human IgG in mouse serum samples, first establish a standard curve using the above two antibodies and human IgG with known concentrations, and then calculate the concentration of human IgG in the serum samples using this standard curve.

[0374] The experimental results are as Figure 7 shown, Figure 7 A shows that the serum half-life of NK-2-12-YTE-EWPS was significantly prolonged compared with that of NK-2-12-EWPS. The calculated serum half-life of NK-2-12-EWPS was 39.6 hours, while that of NK-2-12-YTE-EWPS was 66.9 hours. Similarly, Figure 7 B shows that the calculated serum half-life of NK-2-12-SELF was 62.7 hours, while the serum half-life of NK-2-12-YTE-SELF was increased to 103.8 hours. The serum half-lives of both antibodies with YTE mutations were significantly prolonged respectively.

[0375] It should be noted that although the detection results show that the antibody with the SELF mutation has a relatively long half-life in mice, the real reason is that the antibody with the SELF mutation does not bind to murine FcγRIIb (as described in Example 7), and thus is not easily endocytosed by mouse cells. Therefore, it creates an illusion that the antibody with the SELF mutation has a long half-life in mice. However, since the antibodies with the EWPS mutation or the SELF mutation have similar high affinities for human FcγRIIb and are both easily endocytosed by human cells in the human body, the antibodies with the EWPS mutation or the SELF mutation should have similar serum half-lives in the human body.

[0376] Therefore, it is not appropriate to make a horizontal comparison of the serum half-lives of NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF measured in mice. Similarly, it is also not appropriate to make a horizontal comparison of the mouse serum half-lives of NK-2-12-EWPS and NK-2-12-SELF.

[0377] The inventors detected the clearance effects of NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF on human IgE at different dosages in humanized mice of FcγRIIb derived from C57 according to the method described in Example 5. The results are as Figure 7 shown in C-7F. Both NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF can reduce the concentrations of total human IgE and free human IgE in the humanized mice of FcγRIIb to the baseline level in a short time. Moreover, the experimental results also show that even if the dosage of the two antibodies is reduced to one-fifth of the original, that is, from 100 μg to 20 μg, the clearance effects of the two antibodies on human IgE are not significantly reduced. Compared with omalizumab, even when the dosage of NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF is only 20 μg, the total IgE content and free IgE content in the test mice can be almost always lower than those in the omalizumab group with a dosage of 100 μg. Therefore, both NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF antibodies have far better drug effects than omalizumab in terms of reducing both the total IgE content and free IgE content in the body.

[0378] In summary, IgG Fc with the YTE-EWPS mutation and IgG Fc with the YTE-SELF mutation not only prolong the serum half-life of the antibody, but also enhance the drug effect of the antibody.

[0379] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings that have been published, and such changes are within the scope of protection of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody against IgE or an antigen-binding fragment thereof, characterized in that, It comprises: (a) A heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO:32, VH CDR2 with the sequence of SEQ ID NO:33, VH CDR3 with the sequence of SEQ ID NO:34; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO:35, VL CDR2 with the sequence of SEQ ID NO:36, VL CDR3 with the sequence of SEQ ID NO:37; (b) A heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO:40, VH CDR2 with the sequence of SEQ ID NO:41, VH CDR3 with the sequence of SEQ ID NO:42; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO:43, VL CDR2 with the sequence of SEQ ID NO:44, VL CDR3 with the sequence of SEQ ID NO:45; (c) A heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO:48, VH CDR2 with the sequence of SEQ ID NO:49, VH CDR3 with the sequence of SEQ ID NO:50; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO:51, VL CDR2 with the sequence of SEQ ID NO:52, VL CDR3 with the sequence of SEQ ID NO:53; (d) A heavy-chain variable region (VH) containing the following 3 complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO:56, VH CDR2 with the sequence of SEQ ID NO:57, VH CDR3 with the sequence of SEQ ID NO:58; and / or, a light-chain variable region (VL) containing the following 3 complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO:59, VL CDR2 with the sequence of SEQ ID NO:60, VL CDR3 with the sequence of SEQ ID NO:61; (e) A heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO:64, VH CDR2 with the sequence of SEQ ID NO:65, and VH CDR3 with the sequence of SEQ ID NO:66; and / or, a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO:67, VL CDR2 with the sequence of SEQ ID NO:68, and VL CDR3 with the sequence of SEQ ID NO:69; (f) A heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO:88, VH CDR2 with the sequence of SEQ ID NO:89, and VH CDR3 with the sequence of SEQ ID NO:90; and / or, a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO:91, VL CDR2 with the sequence of SEQ ID NO:92, and VL CDR3 with the sequence of SEQ ID NO:93; (g) A heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO:96, VH CDR2 with the sequence of SEQ ID NO:97, and VH CDR3 with the sequence of SEQ ID NO:98; and / or, a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO:99, VL CDR2 with the sequence of SEQ ID NO:100, and VL CDR3 with the sequence of SEQ ID NO:101; (h) A heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO: 104, VH CDR2 with the sequence of SEQ ID NO: 105, VH CDR3 with the sequence of SEQ ID NO: 106; and / or, a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO: 107, VL CDR2 with the sequence of SEQ ID NO: 108, VL CDR3 with the sequence of SEQ ID NO: 109; or, (i) a heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): VH CDR1 with the sequence of SEQ ID NO: 112, VH CDR2 with the sequence of SEQ ID NO: 113, VH CDR3 with the sequence of SEQ ID NO: 114; and / or, a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): VL CDR1 with the sequence of SEQ ID NO: 115, VL CDR2 with the sequence of SEQ ID NO: 116, VL CDR3 with the sequence of SEQ ID NO:

117.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein It comprises: (a) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 30; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 31; (b) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 38; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 39; (c) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 46; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 47; (d) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 54; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 55; (e) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 62; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 63; (f) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 86; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 87; (g) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 94; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 95; (h) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 102; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 103; or, (i) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 110; and / or, three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:

111.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein It comprises a constant region; The heavy chain constant region contained in the heavy chain of the antibody or its antigen-binding fragment is an IgG heavy chain constant region; The light chain constant region contained in the light chain of the antibody or its antigen-binding fragment is a κ light chain constant region or a λ light chain constant region.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO: 12; The light chain of the antibody or its antigen-binding fragment comprises a light chain constant region as shown in SEQ ID NO:

13.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein It comprises an Fc region; Preferably, the Fc region is the Fc or Fc variant of IgG; wherein, compared with the Fc of IgG, the Fc variant has an increased affinity for the Fcγ receptor (FcγR).

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein The antigen-binding fragment is selected from Fab, Fab’, (Fab’)2, Fv, disulfide-linked Fv, scFv and diabody; and / or, the antibody is a humanized antibody, a chimeric antibody, a fully human antibody, a bispecific antibody or a multispecific antibody.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein The Fc of IgG has the sequence as shown in SEQ ID NO: 6; The Fc variant has the sequence as shown in SEQ ID NO: 7; The Fc variant has the sequence as shown in SEQ ID NO: 118; The Fc variant has the sequence as shown in SEQ ID NO: 8; The Fc variant has the sequence as shown in SEQ ID NO:

119.

8. The antibody or antigen-binding fragment thereof according to claim 5, wherein FcγR is selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, or any combination thereof; preferably FcγRIIb.

9. An Fc variant of IgG Fc, characterized in that, The Fc variant has three amino acid residue substitutions and one amino acid residue deletion at amino acid residues 327, 328, 330 and 331 of the amino acid sequence of IgG Fc; Preferably, the Fc variant has amino acid residue substitutions at amino acid residues 327, 328 and 330 of the amino acid sequence of IgG Fc, and an amino acid residue deletion at amino acid residue 331; wherein, compared with IgG Fc, the Fc variant has an increased affinity for the Fcγ receptor (FcγR); Preferably, the Fc variant has a substitution of a glutamic acid residue at amino acid residue 327 of IgG Fc; Preferably, the Fc variant has a substitution of a tryptophan residue at amino acid residue 328 of IgG Fc; Preferably, the Fc variant has a substitution of a serine residue at amino acid residue 330 of IgG Fc.

10. The Fc variant according to claim 9, characterized in that, The Fc variant has the sequence shown in SEQ ID NO:7.

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