An antibody or antigen-binding fragment thereof and Fc variant against IgE

Abstract

The present application relates to an antibody or an antigen-binding fragment thereof that specifically binds to IgE under neutral or alkaline conditions, and 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 subjects. The present application also relates to an Fc variant of IgG Fc, and 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 of IgG Fc, and their use in reducing or inhibiting IgE levels or preventing and / or treating IgE-related diseases in subjects.

Description

Technical Field

[0001] The present application relates to an antibody or an antigen-binding fragment thereof that specifically binds to IgE, and 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, and 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 participates in different immune processes. Among them, FcεRI is a high-affinity receptor for IgE, which exists on the surface of immune cells such as mast cells and basophils. Under the induction of IgE, this receptor cross-links, triggering activation and degranulation of mast cells and basophils, releasing bioactive mediators such as histamine, prostaglandins, and leukotrienes, causing physiological dysfunction such as local or systemic inflammatory reactions, that is, type I hypersensitivity reactions. The interaction between IgE and the low-affinity receptor CD23 involves immune processes such as antigen presentation, antigen transport across the airways and intestinal epithelium, and IgE synthesis regulation.

[0003] Omalizumab, as a humanized anti-IgE monoclonal antibody, is the only clinically approved IgE targeted therapy for the treatment of severe allergic asthma and patients with high IgE blood. Omalizumab's binding to IgE can simultaneously inhibit the interaction between IgE and two receptors, neutralize IgE, and thus treat allergies. However, in clinical use, while reducing free IgE, omalizumab significantly increases the total IgE content in the patient's blood. This is because omalizumab, as an IgG-type antibody, has a long half-life. During clinical treatment, IgE is neutralized by omalizumab and protected by omalizumab. When IgE bound by omalizumab encounters endocytosis, it will be recirculated and released outside the cell through FcRn along with omalizumab, and will not be degraded by lysosomes, thus greatly prolonging the half-life of IgE and causing a large amount of IgE to accumulate in the blood. As a neutralizing antibody, in order to achieve efficacy, the dosage of Omalizumab cannot be lower than the total amount of IgE in the patient's body, otherwise some IgE will not be neutralized. Therefore, in order 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 neutralization of IgE. The clinical manifestation is that the patient needs to be injected with multiple large doses of Omalizumab. This brings a large economic burden to the patient and may also cause adverse reactions such as anemia. Not only that, the upper limit of each clinical use of Omalizumab is 600 mg. If the patient is exposed to allergens for a long time and IgE is secreted in large quantities 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-allergic or anti-asthma drugs with high efficiency and low dosage. Summary of the invention

[0005] In order to solve the above problems, the present application provides the following three antibodies.

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

[0007] Among them, the Omalizumab monoclonal antibody comprises: VHCDR1, VH CDR2 and VH CDR3 contained in the heavy chain variable region as shown in SEQ ID NO:14; and VL CDR1, VLCDR2 and VL CDR3 contained in the light chain variable region as shown in SEQ ID NO:15.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution and / or addition of one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residues in the 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.

[0009] 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 systems.

[0010] In certain embodiments, the 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 a VH CDR1 with a sequence of SEQ ID NO: 16, a VH CDR2 with a sequence of SEQ ID NO: 17, and a VH CDR3 with a sequence of SEQ ID NO: 18; the light chain variable region (VL) comprises a VL CDR1 with a sequence of SEQ ID NO: 19, a VL CDR2 with a sequence of SEQ ID NO: 20, and a VL CDR3 with a sequence of SEQ ID NO: 21.

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

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

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

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues in amino acid residues 30, 31, 32, 33, 35 and 36 of 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 histidine residues in amino acid residues 30, 31, 32 and 36 of 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 histidine residues in amino acid residues 30, 31, 32, 33 and 36 of 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 histidine residues in amino acid residues 30, 31 and 33 of 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 histidine residues in amino acid residues 30, 31 and 32 of 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 one or several (eg, 2, 3) histidine residues substituted and / or added in the VL CDR2 contained in the light chain variable region of the omalizumab monoclonal antibody.

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

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues in amino acid residues 56 and 57 of 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 histidine residues in amino acid residues 55, 56 and 57 of 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 histidine residues in amino acid residues 55 and 56 of 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 and an addition of one histidine residue at amino acid residues 56 and 57 of the light chain variable region as shown in SEQ ID NO:15.

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

[0027] 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 the omalizumab monoclonal antibody, and a substitution of 2 or 3 histidine residues in the VL CDR2.

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

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues in 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.

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

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

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues in 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.

[0033] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues in amino acid residues 30, 31, 33, 56 and 57 of 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 histidine residues in 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 histidine residues in 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 histidine residues in 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 one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residues substituted and / or added in the VH CDR1 and / or VH CDR2 contained in the heavy chain variable region of the omalizumab monoclonal antibody.

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

[0043] 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.

[0044] 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:62; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:63.

[0045] 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:70; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:71.

[0046] 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:78; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:79.

[0047] 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:86; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:87.

[0048] 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:94; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:95.

[0049] 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:102; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:103.

[0050] 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:110; and / or, 3 CDRs contained in the light chain variable region (VL) as shown 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 24, VHCDR2 with a sequence of SEQ ID NO: 25, and VH CDR3 with a sequence of SEQ ID NO: 26; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 27, VL CDR2 with a sequence of SEQ ID NO: 28, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 32, VHCDR2 with a sequence of SEQ ID NO: 33, and VH CDR3 with a sequence of SEQ ID NO: 34; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 35, VL CDR2 with a sequence of SEQ ID NO: 36, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO:40, VHCDR2 with a sequence of SEQ ID NO:41, and VH CDR3 with a sequence of SEQ ID NO:42; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO:43, VL CDR2 with a sequence of SEQ ID NO:44, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO:48, VHCDR2 with a sequence of SEQ ID NO:49, and VH CDR3 with a sequence of SEQ ID NO:50; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO:51, VL CDR2 with a sequence of SEQ ID NO:52, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 56, VHCDR2 with a sequence of SEQ ID NO: 57, and VH CDR3 with a sequence of SEQ ID NO: 58; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 59, VL CDR2 with a sequence of SEQ ID NO: 60, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 64, VHCDR2 with a sequence of SEQ ID NO: 65, and VH CDR3 with a sequence of SEQ ID NO: 66; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 67, VL CDR2 with a sequence of SEQ ID NO: 68, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 72, VHCDR2 with a sequence of SEQ ID NO: 73, and VH CDR3 with a sequence of SEQ ID NO: 74; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 75, VL CDR2 with a sequence of SEQ ID NO: 76, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 80, VHCDR2 with a sequence of SEQ ID NO: 81, and VH CDR3 with a sequence of SEQ ID NO: 82; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 83, VL CDR2 with a sequence of SEQ ID NO: 84, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 88, VHCDR2 with a sequence of SEQ ID NO: 89, and VH CDR3 with a sequence of SEQ ID NO: 90; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 91, VL CDR2 with a sequence of SEQ ID NO: 92, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 96, VHCDR2 with a sequence of SEQ ID NO: 97, and VH CDR3 with a sequence of SEQ ID NO: 98; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 99, VL CDR2 with a sequence of SEQ ID NO: 100, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 104, VH CDR2 with a sequence of SEQ ID NO: 105, and VH CDR3 with a sequence of SEQ ID NO: 106; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 107, VL CDR2 with a sequence of SEQ ID NO: 108, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 112, VH CDR2 with a sequence of SEQ ID NO: 113, and VH CDR3 with a sequence of SEQ ID NO: 114; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 115, VL CDR2 with a sequence of SEQ ID NO: 116, and VL CDR3 with a sequence of SEQ ID NO: 117.

[0063] In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof 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 kappa light chain constant region or a lambda 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 Fc of IgG or an Fc variant; wherein the Fc variant has increased affinity for Fcγ receptor (FcγR) compared to Fc of IgG.

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

[0071] The present application provides an Fc variant of IgG Fc, wherein 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.

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

[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 shown in SEQ ID NO:7.

[0078] In certain embodiments, the Fc variant also has substitutions of amino acid residues 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 characteristics selected from the following:

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

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

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

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

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

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

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

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

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

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

[0090] In another aspect, the present application provides another Fc variant of IgG Fc.

[0091] In certain embodiments, the Fc variant has amino acid residue 267 of the amino acid sequence of IgG Fc substituted with glutamic acid and amino acid residue 328 substituted with phenylalanine;

[0092] or,

[0093] The Fc variant has the amino acid residue at position 52 corresponding to SEQ ID NO: 6 replaced by glutamic acid, and the amino acid residue at position 113 corresponding to SEQ ID NO: 6 replaced by phenylalanine.

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

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

[0096] or,

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

[0098] In certain embodiments, the Fc variant has the sequence 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 of mammalian (eg, murine, human) origin.

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

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

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

[0104] The present application also provides a vector, which comprises 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, which comprises 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 comprises culturing the host cell as described above under conditions allowing 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, which comprises the Fc variant as described above.

[0108] In certain embodiments, the antibody is a murinized 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 an IgG Fc as described in the second aspect. In certain embodiments, the antibody of the second aspect comprises a substitution and / or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residues 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) as described in the first aspect. In certain embodiments, the antibodies described in the first and second aspects comprise the features of the antibodies or their antigen-binding fragments as described in the third aspect.

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

[0111] Wherein, the Omalizumab monoclonal antibody comprises: VHCDR1, VH CDR2 and VH CDR3 contained in the heavy chain variable region as shown in SEQ ID NO: 14; and VL CDR1, VLCDR2 and VL CDR3 contained in the light chain variable region as shown in SEQ ID NO: 15;

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

[0113] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution and / or addition of one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residues in the 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 systems.

[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 a VH CDR1 with a sequence of SEQ ID NO: 16, a VH CDR2 with a sequence of SEQ ID NO: 17, and a VH CDR3 with a sequence of SEQ ID NO: 18; the light chain variable region (VL) comprises a VL CDR1 with a sequence of SEQ ID NO: 19, a VL CDR2 with a sequence of SEQ ID NO: 20, and a VL CDR3 with a sequence of SEQ ID NO: 21.

[0117] In certain embodiments, the antibody or antigen-binding fragment thereof as described above has one or several (e.g., 2, 3, 4, 5, 6) histidine residues replaced and / or added in the 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 one or several (e.g., 2, 3, 4, 5, 6) histidine residues replaced at amino acid residues 30, 31, 32, 33, 35, 36 of 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 in amino acid residues 30, 31, 32, 35 and 36 of 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 in amino acid residues 30, 31, 32, 33, 35 and 36 of 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 in amino acid residues 30, 31, 32 and 36 of 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 in amino acid residues 30, 31, 32, 33 and 36 of 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 in amino acid residues 30, 31 and 33 of 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 histidine residues in amino acid residues 30, 31 and 32 of 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 one or several (eg, 2, 3) histidine residues substituted and / or added in the VL CDR2 contained in the light chain variable region of the omalizumab monoclonal antibody.

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

[0127] In certain embodiments, the antibody or antigen-binding fragment thereof has a substitution of histidine residues in amino acid residues 56 and 57 of 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 histidine residues in amino acid residues 55, 56 and 57 of 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 histidine residues in amino acid residues 55 and 56 of 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 and an addition of one histidine residue at amino acid residues 56 and 57 of the light chain variable region as shown in SEQ ID NO:15.

[0131] In certain embodiments, the antibody or antigen-binding fragment thereof as described above, each independently has one or several (e.g., 2, 3, 4, 5, 6) histidine residues replaced and / or added in the VL CDR1 and VL CDR2 contained in the light chain variable region of the 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 the 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 replacement of histidine residues at amino acid residues 30, 31, 32, 35, and 36 of the light chain variable region as shown in SEQ ID NO:15, and has a replacement of histidine residues 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 histidine residues in 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 histidine residues in 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 histidine residues in 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 histidine residues in 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 histidine residues in 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 histidine residues in 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 histidine residues in 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 histidine residues in 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 several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) histidine residues substituted and / or added in the VH CDR1 and / or VH CDR2 contained in the heavy chain variable region of the omalizumab monoclonal antibody.

[0143] In certain embodiments, the antibody or antigen-binding fragment thereof has one or several (e.g., 2, 3, 4, 5) histidine residues substituted at amino acid residues 26, 28, 30, 31, 54 of the heavy chain variable region of the 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: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:62; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:63.

[0150] 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:70; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:71.

[0151] 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:78; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:79.

[0152] 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:86; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:87.

[0153] 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:94; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:95.

[0154] 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:102; and / or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:103.

[0155] 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:110; and / or, 3 CDRs contained in the light chain variable region (VL) as shown 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 24, VHCDR2 with a sequence of SEQ ID NO: 25, and VH CDR3 with a sequence of SEQ ID NO: 26; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 27, VL CDR2 with a sequence of SEQ ID NO: 28, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 32, VHCDR2 with a sequence of SEQ ID NO: 33, and VH CDR3 with a sequence of SEQ ID NO: 34; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 35, VL CDR2 with a sequence of SEQ ID NO: 36, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO:40, VHCDR2 with a sequence of SEQ ID NO:41, and VH CDR3 with a sequence of SEQ ID NO:42; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO:43, VL CDR2 with a sequence of SEQ ID NO:44, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO:48, VHCDR2 with a sequence of SEQ ID NO:49, and VH CDR3 with a sequence of SEQ ID NO:50; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO:51, VL CDR2 with a sequence of SEQ ID NO:52, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 56, VHCDR2 with a sequence of SEQ ID NO: 57, and VH CDR3 with a sequence of SEQ ID NO: 58; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 59, VL CDR2 with a sequence of SEQ ID NO: 60, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 64, VHCDR2 with a sequence of SEQ ID NO: 65, and VH CDR3 with a sequence of SEQ ID NO: 66; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 67, VL CDR2 with a sequence of SEQ ID NO: 68, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 72, VHCDR2 with a sequence of SEQ ID NO: 73, and VH CDR3 with a sequence of SEQ ID NO: 74; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 75, VL CDR2 with a sequence of SEQ ID NO: 76, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 80, VHCDR2 with a sequence of SEQ ID NO: 81, and VH CDR3 with a sequence of SEQ ID NO: 82; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 83, VL CDR2 with a sequence of SEQ ID NO: 84, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 88, VHCDR2 with a sequence of SEQ ID NO: 89, and VH CDR3 with a sequence of SEQ ID NO: 90; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 91, VL CDR2 with a sequence of SEQ ID NO: 92, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 96, VHCDR2 with a sequence of SEQ ID NO: 97, and VH CDR3 with a sequence of SEQ ID NO: 98; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 99, VL CDR2 with a sequence of SEQ ID NO: 100, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 104, VH CDR2 with a sequence of SEQ ID NO: 105, and VH CDR3 with a sequence of SEQ ID NO: 106; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 107, VL CDR2 with a sequence of SEQ ID NO: 108, and VL CDR3 with a 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) comprising the following three complementarity determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 112, VH CDR2 with a sequence of SEQ ID NO: 113, and VH CDR3 with a sequence of SEQ ID NO: 114; and / or a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 115, VL CDR2 with a sequence of SEQ ID NO: 116, and VL CDR3 with a sequence of SEQ ID NO: 117.

[0168] In certain embodiments, the Fc variant has three substitutions of amino acid residues and one deletion of amino acid residue 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 has an amino acid residue deletion at amino acid residue 331; wherein the Fc variant has an increased affinity for Fcγ receptors (FcγR) compared to IgG Fc;

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

[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 330 of the IgG Fc polypeptide.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0188] In certain embodiments, the Fc variant has amino acid residue 267 of the amino acid sequence of IgG Fc substituted with glutamic acid and amino acid residue 328 substituted with phenylalanine;

[0189] or,

[0190] The Fc variant has the amino acid residue at position 52 corresponding to SEQ ID NO: 6 replaced by glutamic acid, and the amino acid residue at position 113 corresponding to SEQ ID NO: 6 replaced by phenylalanine.

[0191] In certain embodiments, the Fc variants have increased affinity for Fc gamma receptors (FcγRs) 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 amino acid residue 267 of the amino acid sequence of IgG Fc replaced by glutamic acid and amino acid residue 328 replaced by phenylalanine; and amino acid residues 252, 254 and 256 of the amino acid sequence of IgG Fc of the Fc variant are also replaced by tyrosine, threonine and glutamic acid, respectively;

[0194] or,

[0195] The Fc variant has glutamic acid substituted at amino acid residue 52 corresponding to SEQ ID NO:6, and phenylalanine substituted at amino acid residue 113 corresponding to SEQ ID NO:6; and the Fc variant also has tyrosine, threonine and glutamic acid substituted at amino acid residues 37, 39 and 41 corresponding to SEQ ID NO:6, respectively.

[0196] In certain embodiments, the Fc variants have increased affinity for Fc gamma receptors (FcγRs) 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 of mammalian (eg, murine, human) origin.

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

[0201] In certain embodiments, the aforementioned antibody or antigen-binding fragment thereof 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 of the antibody or antigen-binding fragment thereof comprises a light chain constant region that is a kappa light chain constant region or a lambda 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, 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 murinized antibody, a chimeric antibody, a fully human antibody, a bispecific antibody, or a multispecific antibody.

[0210] In certain embodiments, the antibody or its antigen-binding fragment as described above, wherein the antibody or its antigen-binding fragment carries a label. In certain embodiments, the antibody or its antigen-binding fragment carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., 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 the seventh aspect, the present application provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, comprising culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0215] In an eighth aspect, the present application provides a multispecific molecule comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the 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 (eg, a second antibody) for a second target.

[0218] In a ninth aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the 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 effects.

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

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

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

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

[0225] In the eleventh aspect, the present application provides the use of the antibody or antigen-binding fragment thereof 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 preparing a drug, wherein the drug is used to reduce or inhibit IgE levels or to prevent and / or treat IgE-related diseases in a subject.

[0226] In certain embodiments, the IgE-related disease is a type 1 hypersensitivity disease.

[0227] In certain embodiments, the IgE-related disease is selected from allergic reactions, allergic rhinitis, allergic cough, anaphylactic shock, food allergy, hypersensitivity reaction, 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, angioedema, nasal polyps, sinusitis, etc.

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

[0229] In certain embodiments, the antibodies or antigen-binding fragments thereof are used alone or in combination with another pharmaceutically active agent.

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

[0231] In certain embodiments, the antibody or antigen-binding fragment thereof does not specifically bind to IgE at pH 6.

[0232] In the twelfth aspect, the present application provides a method for reducing or inhibiting IgE levels in a subject or preventing and / or treating an IgE-related disease, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof 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 1 hypersensitivity disease.

[0234] In certain embodiments, the IgE-related disease is selected from allergic reactions, allergic rhinitis, allergic cough, anaphylactic shock, food allergy, hypersensitivity reaction, 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, angioedema, nasal polyps, sinusitis, etc.

[0235] In certain embodiments, the subject is a mammal, such as a human, monkey, or 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 alkaline conditions; preferably, the neutral or alkaline conditions are conditions with a pH greater than or equal to 7 (eg, pH equal to 7.4).

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

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

[0240] (1) contacting the sample with the antibody or antigen-binding fragment thereof according to 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 detectably labeled.

[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 alkaline conditions; preferably, the neutral or alkaline conditions are conditions with a pH greater than or equal to 7 (eg, pH equal to 7.4).

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

[0246] Definition of terms

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

[0248] As used herein, the term "IgG" refers to a polypeptide encoded by an 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 in the IgG class of antibodies are VH, Cγ1, Cγ2, Cγ3, VL, and CL.

[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 outside the first constant region of the immunoglobulin domain. In certain embodiments, Fc refers to the last two constant regions of the immunoglobulin domains of IgA, IgD and IgG. In certain embodiments, Fc refers to the last three constant regions of the immunoglobulin domains of IgE and IgM. In certain embodiments, Fc comprises a flexible hinge at the N-terminus. In certain embodiments, Fc comprises a hinge between immunoglobulin domains Cγ2 and Cγ3 and Cγ1 and Cγ2. In certain embodiments, Fc is an antibody fragment formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. In certain embodiments, the numbering of the Fc region of the human IgG heavy chain is in accordance with the EU indexing method of Kabat (see, e.g., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991). In certain embodiments, the Fc is an isolated Fc region, or an Fc region located in an antibody or antigen-binding fragment thereof, or a polypeptide comprising an Fc region amino acid sequence.

[0250] As used herein, the terms "Fcγ receptor" or "FcγR" have the same meaning and can be used interchangeably. They refer to proteins that can bind to the Fc region of IgG and are encoded by FcγR genes. 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); for the above, see, for example, 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 activated receptor, which is related to inflammation and tissue damage, and is one of the important receptors that cause 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 downregulate the corresponding functions of cells after activation. FcγRIIb is mainly expressed on the surface of myeloid cells and B cells (it is 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 dependence" refers to the fact that the antigen-binding activity of an antibody at an acidic pH is different from the antigen-binding activity of an antibody at a neutral or alkaline pH. In certain embodiments, pH dependence refers to the fact that the antigen-binding activity of an antibody at an acidic pH is weaker than the antigen-binding activity of an antibody at a neutral or alkaline pH.

[0253] The term "antibody" as used herein refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen, which is derived from an immunoglobulin and 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, it includes not only complete antibodies, but also antigen binding fragments capable of specifically binding to a target antigen. "Complete antibodies" are typically composed of two pairs of polypeptide chains (each pair having a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes 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 domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of 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 subdivided into highly variable regions (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition 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 "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated as 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 easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0255] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments 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 antibodies or antigen-binding fragments 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 as defined above.

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

[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 to the same antigen bound by the full-length antibody 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 edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fd, Fv, disulfide-linked Fv, complementary determining region (CDR) fragments, scFv, diabodies, single domain antibodies (sdAb), chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. 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, "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 direction from N-terminal to C-terminal; preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N-terminal to C-terminal. 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 direction from N-terminal to C-terminal. Two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be from a single species, such as humans; it may also be a chimeric antibody or a murinized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to 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 at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in a (Fab')2 fragment, 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 that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0262] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected 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, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences 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, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0263] The term "multispecific antibody" refers to an antibody that has binding specificity for two or more (e.g., three or four) different antigens (or epitopes). A multispecific antibody comprises 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 multispecific antibody can be independently 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 a scFv). In some cases, each antigen binding domain is connected by a peptide linker.

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

[0265] Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity 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 derived 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 derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP4,816,567 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 heavy chain variable region and the light chain variable region of the antibody are derived from a first antibody, and the heavy chain constant region and the light chain constant region of the antibody are derived from a second antibody.

[0267] As used herein, the term "murinized antibody" may include antibodies in which the heavy chain variable region and light chain variable region of the antibody are from fully human antibodies, and the heavy chain constant region and light chain constant region of the antibody are from murine constant regions. As used herein, the term "fully human antibody" refers to an antibody composed of an antibody amino acid sequence that is entirely human and can be obtained by phage and yeast display, transgenic animals, single B cells, and other technologies.

[0268] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. 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 measured using methods known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites / antigen complexes. Both "association rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate 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). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, surface plasmon resonance (SPR) can be used to measure the dissociation constant in Biacore. In addition, 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 that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, 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 materials (e.g., chemiluminescent materials, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine 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 delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0272] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, 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 S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, 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 change the expected properties of the protein / polypeptide comprising 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 amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a 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. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0274] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR 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 single-letter and three-letter abbreviations 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 a subject and an active ingredient, which is 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, agents that maintain osmotic pressure, agents that delay absorption, 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. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which 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 hydrolysate), 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 solutions (e.g., 0.9% NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solutions, and any combinations thereof.

[0276] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

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

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

[0279] As used herein, the term "EU" is a numbering system for antibodies, also known as the EU Numbering System or Scheme. Its origin is that in the late 1960s (1968-1969), Gerald M Edelman et al. separated 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 region of other immunoglobulins is compared with Eu for amino acid sequence, and the corresponding amino acid position is the Eu numbering.

[0280] Advantageous Effects of the Invention

[0281] In the first aspect, the present application performs histidine mutation on the basis of the CDR sequence contained in omalizumab, and obtains an antibody with pH dependence on IgE. The pH-dependent antibody of the present application has the following advantages: after binding with IgE in the weakly alkaline environment of body fluids, it can frequently carry IgE to be endocytosed by cells. When the complex of antibody and IgE is endocytosed into the weakly acidic environment of the endosome, the antibody is separated from IgE, after which IgE is transported to the lysosome for degradation, and the antibody is recirculated to the outside of the cell with FcRn, mediating the endocytic degradation of IgE again. Therefore, the pH-dependent antibody of the present application is conducive to reducing the dosage of the drug and has the potential to become a drug.

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

[0283] Further, the present application has experimentally confirmed that the antibody with the above two mutations has a longer serum half-life than the antibody in the prior art (e.g., omalizumab), and can quickly clear IgE at a lower dosage. Moreover, it can not only reduce the total IgE content in mice, but also reduce the free IgE content. Therefore, the present application has a large application potential in diseases related to IgE (e.g., type 1 hypersensitivity disease), showing a huge potential for drug development.

[0284] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0285] Figure 1 The phage display library construction strategy and the library construction region sequences of the screened antibodies are shown, wherein the degenerate base S in the "library construction sequence" in the figure represents 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 listed "screened sequences" are all confirmed nucleic acid sequences of IgE antibodies with pH dependence, and the bases that are different from the nucleic acid sequence of omalizumab are marked with shadows.

[0286] Figure 2 The SPR curves of omalizumab and 12 screened antibodies with IgE at pH 7.4 and pH 6.0 are shown.

[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 4 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 from Figure 4 The corresponding curve in G. Figure 5 I in the figure shows the IgE content change curve when omalizumab was used at 100 μg (the curve is taken from Figure 4 G), Comparison of the IgE content change curve when NK-2-12-EWPS was administered at 20 μg.

[0290] Figure 6 The results of the IgE clearance experiment of NK-2-12-SELF in mice are shown. Figure 6 Panel A shows the changes in total IgE content in the serum of wild-type C57 mice over time. Figure 6 B in the figure shows the change of free IgE content in the serum of wild-type C57 mice over time. Figure 6 Panel C shows the changes in total IgE content in the serum of C57-derived FcγRIIb humanized mice over time. Figure 6 D in the figure shows the change over time of free IgE content in the serum of C57-derived FcγRIIb humanized mice.

[0291] Figure 7 The results show the effects of serum half-life and dosage of NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF on IgE clearance in mice. Figure 7 A in the figure is the pharmacokinetic curve of NK-2-12-YTE-EWPS and NK-2-12-EWPS in FcRn humanized mice; Figure 7 B in the figure is the pharmacokinetic curve of NK-2-12-YTE-SELF and NK-2-12-SELF in FcRn humanized mice; Figure 7 C in the figure shows the change of 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 the figure is the change of 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 the figure is the change of 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 the figure shows the change of free IgE content in the serum of FcγRIIb humanized mice over time under different dosages of NK-2-12-YTE-SELF.

[0292] Sequence information

[0293] The information of the partial 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] Note: The amino acid residues marked with boxes in the sequence are the mutated amino acid residues, and the underlined residues are the hinge region of IgG Fc. DETAILED DESCRIPTION

[0303] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0304] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE (Animal Cell Culture). CELL CULTURE) (RI Freshney, ed. (1987)).

[0305] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0306] Example 1. Screening and obtaining of antibodies

[0307] 1. Construction of phage library

[0308] By analyzing the publicly available complex structure of omalizumab and IgE (PDB: 5HYS), the inventors found that omalizumab mainly binds to the CH3 region of the Fc segment of IgE through heavy chain CDR1 (HCDR1), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1) and light chain CDR2 (LCDR2), while only Tyr54 in the heavy chain CDR2 (HCDR2) contacts IgE, and the light chain CDR3 (LCDR3) does not contact IgE at all. Therefore, the inventors synthesized the nucleotide sequence encoding the VH-VL (scFv) form of omalizumab (SEQ ID NO: 1) and primers containing degenerate bases (the gene and primers were commissioned to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis), and constructed a library of the four regions of HCDR1, HCDR3, LCDR1, LCDR2 and the codon of heavy chain Tyr54 in the omalizumab scFv gene by Overlap PCR (the sequence of the library region is shown in Figure 2). Figure 1 shown).

[0309] Afterwards, 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 transferred into E. coli XL1-Blue. Then, according to the standard method of phage display, the E. coli XL1-Blue containing the plasmid library was infected with the helper phage M13KO7 to breed the recombinant phage library for subsequent screening.

[0310] The inventors also synthesized the IgE Fc gene with AVI tag and 6×His tag according to the IgE Fc amino acid sequence in the complex structure of omalizumab and IgE (PDB: 5HYS) (commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis), and constructed it into the mammalian cell expression vector pTT5 (NovoPro, Catalog No.: V001466), and used HEK293F cells (Thermo Fisher Scientific, Catalog No. R79007) and FreeStyle TM Culture medium (Gibco TM , Catalog No.: 12338-018) for protein expression. Thereafter, the inventors used an AKTA protein purifier (GE Healthcare, Model: pure) and HisTrap HP chromatography column (GE Healthcare, catalog number: 17524701) according to the purification steps described in the equipment manual to purify IgE Fc with 6×His and AVI tags, and biotinylation kit (GeneCopoeia, catalog number: BI001) was used to biotinylate part of the purified protein, and biotin was attached to the AVI tag of IgE Fc for subsequent panning steps.

[0311] During the panning, the biotinylated IgE Fc was first bound to streptavidin magnetic beads (Invitrogen, Cat. No. 11205D) as a stationary phase. Then, according to the standard method of phage display, the magnetic beads bound to IgE Fc were first incubated with the phage library dissolved in BSA-PBST pH7.4 (PBS pH7.4 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 with 0.05% Tween 20) to remove non-specifically adsorbed phages, and finally eluted with PBS pH 6.0, thereby obtaining phages that bind to IgE at pH 7.4 and dissociate from IgE at pH 6.0. Thereafter, according to the standard method of phage display, the eluted phage was used to infect Escherichia coli XL1-Blue, so that the library plasmid after panning entered the bacteria, and then the bacteria were infected with helper phage M13KO7 to amplify and reproduce the recombinant phage for the next round of panning.

[0312] After three rounds of panning, the recombinant phages were significantly enriched, with the enrichment factor reaching 581 times, indicating that phages with pH-dependent binding ability to IgE Fc had been obtained.

[0313] 2. Acquisition of Antibodies

[0314] Plasmid sequencing of phages obtained after three rounds of panning showed that among 150 randomly selected single clones, Figure 1 The NK-2-12 sequence shown in the figure appears frequently, and more than one-third of the scFv sequences are this sequence, while other sequences rarely appear repeatedly. The inventors constructed NK-2-12 and 14 other randomly selected cloned sequences (a total of 15 sequences) into the pTT5 (NovoPro, catalog number: V001466) vector to form a complete antibody gene. Then, the IgE Fc expression method was used as described above, and the protein was purified using an AKTA protein purifier (GE Healthcare, model: The 15 antibodies were purified using a 4% 4% 4% 5% 5% 2% 5% 2% 1% 2% 2% 3% 3% 4% 5% 2% 3% 5% 4% 2% 5% 2% 3 ...

[0315] In addition, the inventors also synthesized the gene of human IgE anti-hen egg white lysozyme (anti-HEL) antibody with a 6×His tag (commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis), constructed it into the pTT5 (NovoPro, catalog number: V001466) plasmid, and expressed and purified it using the aforementioned method for expressing and purifying IgE Fc for subsequent research. The amino acid sequence of the human anti-HEL IgE heavy chain is shown in SEQ ID NO: 2, and the amino acid sequence of the human anti-HEL IgE light chain is shown in SEQ ID NO: 3.

[0316] The inventors used surface plasmon resonance (SPR) technology to detect the affinity of the above 15 antibodies and omalizumab to IgE under PBS pH 7.4 and PBS pH 6.0. The specific method is to first fix the anti-6×His antibody 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 install the chip 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 400nM with PBS of a specific pH, and flowed through the chip at a flow rate of 10μl / min for 60s for sample capture. The above 15 antibodies were dissolved in PBS of the same pH, and the PBS was used to prepare a 2-fold serial dilution solution gradient (such as 500nM, 250nM, 125nM, to 15.625nM, etc.), injected at a flow rate of 30μl / min, combined for 120s, and then the PBS was flowed through the chip for dissociation for 240s. Then, 10mM Glycine-HCl (pH 1.5) regeneration buffer was used to elute IgE and antibodies for 30s at a flow rate of 30μl / min to regenerate the chip. In the above IgE capture, injection and dissociation steps, when measuring the affinity of antibodies to IgE under pH 7.4, the PBS used was PBS pH7.4; when measuring the affinity of antibodies to IgE under pH 6.0, the PBS used was PBS pH 6.0. After alternating cycles of IgE capture, injection, dissociation and chip regeneration, the data obtained were fitted according to the steady-state binding model using the instrument's supporting software, Biacore T200 Evaluation Software, to obtain the corresponding binding constant Ka and dissociation constant Kd, and then the binding dissociation constant KD was calculated (KD = Kd / Ka).

[0317] The measurement results showed that 12 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 library construction regions were Figure 1 ) showed pH dependence of binding to IgE (SPR curves as shown in Figure 2 The calculated KD is shown in Table 2). All of these 12 antibodies bind to IgE at pH 7.4, and the binding dissociation constants KD are mostly within 10 -8 mol / L order of magnitude, a few are 10 -7 mol / L, while the binding of the antibody to IgE was completely undetectable at pH 6.0. As a control, omalizumab showed no pH dependence on IgE binding and showed a high affinity for IgE regardless of pH 7.4 or pH 6.0 ( Figure 2 , Table 2).

[0318] Table 2. Binding dissociation constants of the screened antibodies to IgE

[0319]

[0320] Note: “ND” means no binding activity was detected.

[0321] Table 3. Antibody sequences (Kabat)

[0322]

[0323]

[0324] Example 2. Analysis of mutation sites associated with pH dependence

[0325] Comparative analysis of the library sequences of these 12 antibodies ( Figure 1 ) showed that there were almost no mutations in the HCDR3 region, proving that HCDR3 is the core region for antibody-IgE binding and has very low tolerance to mutations. The 12 antibodies in the HCDR1 region did not show relatively uniform mutations ( Figure 1 ), and 5 antibodies including NK-2-12 did not contain mutations in this region, proving that mutations in HCDR1 are not the key factor in achieving pH-dependent binding of antibodies to IgE. Among the 12 antibodies, the sequences of NK-2-5 and NK-177 differ only in the heavy chain Y54 site ( Figure 1). Both antibodies showed pH dependence in binding to IgE, but NK-2-5 containing the heavy chain Y54H mutation showed slightly higher affinity than NK-177 without the mutation at pH 7.4 (Table 2), proving that the heavy chain Y54H mutation contributes less to the pH dependence of the antibody, but has a certain positive effect on the affinity of the antibody to IgE in the body fluid environment.

[0326] There were a large number of uniform histidine substitution mutations in LCDR1 of the 12 antibodies ( Figure 1 , Table 4). The original amino acid sequence of LCDR1 of omalizumab is 30-DYDGDSY-36. Except for D34, which almost never has histidine substitution mutation, the histidine substitution mutations of the other 6 amino acid residues (D30H, Y31H, D32H, G33H, S35H and Y36H on the basis of the light chain of omalizumab) in 12 pH-dependent antibodies all appear at a high frequency of more than two-thirds. Among the 12 antibodies, 5 antibodies contain all the above 6 histidine substitution mutations, and 4 antibodies (including NK-2-12) contain 5 of the above 6 histidine substitution mutations. Among the remaining 3 antibodies, 2 antibodies contain 4 of the above 6 histidine substitution mutations, and 1 antibody contains 3 of the above 6 histidine substitution mutations.

[0327] In the LCDR2 region, 12 antibodies also showed highly uniform histidine substitution mutations ( Figure 1 , Table 4), mainly manifested as S56H and Y57H mutations of the original amino acid sequence 53-YAASY-57 of LCDR2 of omalizumab, and A55H mutations also appeared in some antibodies. Among the 12 pH-dependent antibodies, as many as 11 contained both S56H and Y57H mutations, and all 12 antibodies contained at least two of the A55H, S56H and Y57H mutations, proving that the combination of these three mutations plays an important role in achieving pH-dependent binding with IgE. In addition, in addition to the two point mutations of S56H and Y57H mentioned above, LCDR2 of NK-2-12 also accidentally inserted a histidine residue between the Ala55 and S56H mutant amino acids (the inserted amino acid residue was numbered 55a, and the insertion mutation was named H55a insertion mutation). Comparison of the sequences of the two antibodies NK-2-12 and NK-100 showed that the two differed only in the LCDR2 region. NK-100 contains three consecutive histidine substitution mutations in this region: A55H, S56H, and Y57H, while NK-2-12 retains Ala55, but has three consecutive histidine mutations consisting of H55a insertion mutation and S56H and Y57H ( Figure 1, Table 4). Although both NK-2-12 and NK-100 showed pH dependence on IgE, the affinity of NK-2-12 for IgE was much higher than that of NK-100 at pH 7.4 (Table 2), indicating that the insertion of H55a has an important positive effect. In addition, as mentioned above, the sequence of NK-2-12 is the only sequence that appears frequently 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.

[0328] Table 4. LCDR1 and LCDR2 amino acid sequences of the screened antibodies

[0329]

[0330] Note: “*” indicates H55a insertion mutation.

[0331] In summary, data analysis shows that by introducing D30H, Y31H, D32H, G33H, S35H, Y36H, A55H, H55a insertion mutations, S56H, Y57H mutations or a combination of these mutations on the basis of the omalizumab light chain sequence, antibodies can be prepared that can bind to IgE at human physiological pH (~pH 7.4) but not at pH 6.0.

[0332] 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 to be endocytosed by cells. When the complex of antibody and IgE is endocytosed into the weakly acidic environment of the endosome, the antibody and IgE are separated, and thereafter IgE is transported to the lysosome for degradation, while the antibody is recirculated outside the cell with FcRn, mediating the endocytic degradation of IgE again. Therefore, the pH-dependent antibodies of the present application are conducive to reducing the dosage of medication and have the potential to become drugs.

[0333] Example 3. Correlation between mutation sites and pH dependence

[0334] To further verify the relationship between the above-mentioned LCDR1 and LCDR2 region-related mutations and the pH dependence of the antibody, the inventors, based on NK-2-12, reversed some of the mutation sites contained in the LCDR1 and LCDR2 regions to the corresponding sequences of omalizumab, and tested the binding dissociation constants of the related reverse mutation antibodies with IgE under pH 7.4 and pH 6.0. The antibody expression and purification method and the binding dissociation constant determination method are as described in Example 1. Specifically, based on the NK-2-12 antibody in Table 4, the antibody NK-2-12-L1 was modified and constructed. The specific modified CDR sequence is shown in Table 5, and the remaining CDR sequences are the same as those of the NK-2-12 antibody.

[0335] The results showed that compared with NK-2-12, reverting the LCDR2 sequence of NK-2-12 to the LCDR2 sequence of omalizumab (NK-2-12-L1) hardly changed the affinity of the antibody to IgE at pH 7.4, but at pH 6.0, the affinity of the antibody to IgE was improved, and the binding dissociation constant changed from undetectable (ND) to 10 -5 The order of magnitude (Table 5). This shows that the mutations contained in the LCDR2 region contribute to the pH-dependent binding of the antibody to IgE. But at the same time, it can be seen that even without the mutations in the LCDR2 region, the antibody can still maintain a fairly high level of pH dependence, and the dissociation constants of the antibody binding to IgE at pH 7.4 and pH 6.0 still differ by more than 350 times, proving that the mutations contained in LCDR1 can independently achieve pH-dependent binding of the antibody to IgE to a considerable extent.

[0336] Therefore, the above experiments show that based on the CDR sequence contained in omalizumab, only some amino acid sites of LCDR1 are mutated by histidine, and the obtained antibody is still pH-dependent, that is, the binding dissociation constants of the antibody with IgE at pH 7.4 and pH 6.0 are quite different.

[0337] Table 5. LCDR1 and LCDR2 amino acid sequences of reverse mutant antibodies and their binding dissociation constants with IgE

[0338]

[0339] *H55a indicates insertion mutation, ND indicates not detected.

[0340] Example 4. Screening and obtaining of high affinity FcγRIIb

[0341] 1. Screening of high affinity Fc for FcγRIIb

[0342] The inventors designed a new library construction strategy, randomly constructed a library for the region in IgG Fc that contacts FcγRIIb, and on this basis used the mammalian display screening system previously developed and published by the inventor's team (Chen et al., 2021, doi: 10.7150 / thno.51299) to screen out an IgG Fc mutant that can enhance affinity with the receptor FcγRIIb.

[0343] According to the complex structure of wild-type IgG Fc (wtFc, SEQ ID NO: 6) and FcγRIIa (PDB: 3RY6) and FcγRIIIa (PDB: 1E4K), the amino acid 327-331 in the IgG Fc amino acid sequence (amino acid sequence ALPAP) is one of the key sites for the interaction between Fc and the receptor. The inventors of the present application proposed to use the Overlap PCR method to build a library for the amino acids 327-331 corresponding to the amino acid sequence in the IgG Fc nucleic acid sequence, and change the original five consecutive codons in the region into four consecutive NNK (N represents any base, K represents G or T base) codons, so that the translation product of the gene in the region sequence changes from ALPAP 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 IgG Fc mutants with specific high affinity for FcγRIIb.

[0344] The screening results showed that after three rounds of screening, cells with higher affinity for FcγRIIb were enriched ( Figure 3 A). We randomly selected 20 single cell clones for sequencing and found that the screened sequences were highly uniform, with about 70% of the sequences replacing the ALPAP at amino acids 327-331 of the wild-type IgG Fc amino acid sequence with EWPS (this mutation was named EWPS mutation, Figure 3 B). The amino acid sequence of IgG Fc with EWPS mutation is shown in SEQ ID NO: 7 (the EWPS mutation is marked by a box, and the hinge region of IgG Fc is underlined).

[0345] 2. Identification of the affinity of IgG Fc-EWPS mutants to receptor FcγRIIb

[0346] The inventors used the above method to purify the IgG Fc-EWPS mutant, and then used SPR technology to detect the binding dissociation constants of the IgG Fc-EWPS mutant with the human FcγRIIb extracellular region with a 6×His tag (ACROBiosystems, Catalog No.: CDB-H5228) and the mouse FcγRIIb extracellular region with a 6×His tag (ACROBiosystems, Catalog No.: CDB-M52H7). The results showed that the KD of the IgG Fc-EWPS mutant for human FcγRIIb was 2.47×10 -7 mol / L, which is lower than the affinity dissociation constant (KD = 2.41 × 10-6 ), which proves that the EWPS mutation has indeed significantly increased the affinity of human IgG Fc to human FcγRIIb. At the same time, the KD of IgG Fc-EWPS mutant to mouse FcγRIIb is 2.94×10 -7 mol / L, which is close to the dissociation constant for human FcγRIIb.

[0347] The inventors further compared the IgG Fc-EWPS mutant with human FcγRIIIa F158 (ACROBiosystems, Catalog No.: CDB-H5220), FcγRIIIa V158 (ACROBiosystems, Catalog No.: CD8-H52H4), and FcγRIIa H131 The results showed that compared with wild-type IgG Fc, IgG Fc-EWPS had a significantly higher affinity for FcγRIIb and FcγRIIIa. F158 and FcγRIIa H131 There was no significant change in affinity ( Figure 3 C), and FcγRIIIa V158 These further prove that the EWPS mutation can specifically enhance the affinity of IgG Fc and FcγRIIb.

[0348] In summary, the EWPS mutant of IgG Fc has a high affinity specifically for the receptor FcγRIIb. At the same time, the EWPS mutant of IgG Fc has similar affinity for human FcγRIIb and mouse FcγRIIb, so the EWPS mutation has the advantage of being able to be used in the FcγRIIb non-transgenic mouse model.

[0349] Example 5. Clearance effect of NK-2-12-EWPS on human IgE in mice

[0350] Based on the aforementioned antibody NK-2-12, the inventors introduced the aforementioned EWPS mutation that can enhance the affinity of the antibody to the receptor FcγRIIb into the Fc end of the antibody to form 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 box marks the introduced histidine mutation and EWPS mutation), and the amino acid sequence of the light chain of NK-2-12-EWPS is shown in SEQ ID NO: 10.

[0351] The expressed and purified NK-2-12-EWPS antibody was used to detect the clearance effect of human IgE in wild-type C57 mice (purchased from Vitallife) or C57-derived FcγRIIb humanized mice. During the experiment, the control group and the experimental group were randomly divided into groups, with 5 mice in each group. The experimental method is as follows Figure 4 As shown in A, the IgE injected into the mouse peritoneum is the human anti-HEL IgE described in Example 1. 30 minutes before the intraperitoneal injection of IgE into the mouse, the test antibody (NK-2-12 or NK-2-12-EWPS) was first injected into the mouse peritoneum at a dose of 100 μg / mouse as each experimental group, or Omalizumab was injected as a positive control group, or no injection was made as a negative control group. After the intraperitoneal injection of IgE into the mouse at a dose of 50 μg / mouse, the tail vein blood of the mouse was collected at five time points of 1, 2, 3, 8, and 22 hours (50 μl of blood was collected from each mouse each time), and the serum was obtained by centrifugation after standing at room temperature until it coagulated.

[0352] The method for detecting the total human IgE content in serum is as follows Figure 4 As shown in B, it is a standard sandwich ELISA. The antibody used to coat the 96-well ELISA plate is the mouse IgM anti-human IgE antibody HP6061 (Southern Biotech, catalog number: 9240-01), and the concentration used is the recommended concentration in the antibody manual. As described in the manual, the antibody does not conflict with either omalizumab or FcεRI when binding to human IgE, that is, the antibody can bind to both free IgE and IgE bound to omalizumab or FcεRI. Antibody HP6029 (Southern Biotech, catalog number: 9250-05) is a mouse IgG anti-human IgE HRP enzyme-linked antibody. As described in the manual, the antibody does not conflict with either HP6061 or omalizumab when binding to human IgE. Therefore, the enzyme-linked antibody can be used in combination with the substrate ABTS (Thermo Scientific, catalog number: 002024) according to the recommended concentration in the instructions to detect the total amount of IgE captured by HP6061. When detecting the total IgE content in serum, first use the above two antibodies and IgE of known concentration to establish a standard curve for measuring the IgE concentration in the serum sample to be tested.

[0353] The method for detecting the free human IgE content in serum is an improved sandwich ELISA, such as Figure 4As shown in C. The protein used to coat the 96-well ELISA plate is the IgE receptor FcεRI (Sino Biological, Cat. No.: 13193-HNAH), and the concentration is 1μg / 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 to the IgE captured by FcεRI, and then the HRP enzyme-linked antibody of goat anti-mouse IgM is used with the substrate ABTS to detect the content of HP6061 to reflect the content of free IgE. When detecting the free IgE content in serum, it is also necessary to first use a known concentration of IgE to establish a standard curve through the above method to measure the free IgE concentration in the serum sample to be tested.

[0354] The results of the IgE clearance experiment in wild-type C57 mice are as follows Figure 4 D and 4E. Injection of omalizumab into mice will lead to an extension of the half-life of IgE, as shown by the fact that the IgE content in the serum of this group of mice is always higher than that of the negative control group mice injected with only IgE ( Figure 4 D). This is completely consistent with the clinical observation that injection of omalizumab will further increase the total IgE content in the patient's blood. On the contrary, as a marketed drug, omalizumab has a significant effect on reducing the free IgE concentration in mouse serum ( Figure 4 E). The NK-2-12-EWPS antibody of the present application showed excellent effects in reducing total IgE in mice. In mice injected with NK-2-12-EWPS, IgE in the serum was basically completely eliminated after 2 to 3 hours ( Figure 4 D), which performed much better than omalizumab. Since NK-2-12-EWPS cleared almost all IgE in the mouse serum in a short period of time, the free IgE content in the mouse serum was also extremely low ( Figure 4 E), which was lower than that of mice injected with omalizumab. However, the NK-2-12 antibody without Fc modification showed no effect in reducing total IgE and free IgE in mice ( Figure 4 D, E).

[0355] To be closer to the human body environment, the inventors further verified the clearance effect of NK-2-12-EWPS on human IgE in C57-derived FcγRIIb humanized transgenic mice (Shanghai Model Organisms, catalog number: NM-HU-2000010). The experimental results were similar to those of human IgE clearance in wild-type C57 mice. The injection of omalizumab effectively reduced the free IgE in the mice, but prolonged the half-life of IgE, 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, and both the total IgE content and the free IgE content in the mice were lower than those in the group injected with omalizumab ( Figure 4 F, G).

[0356] These in vivo experiments in mice all showed that NK-2-12-EWPS can reduce the amount of IgE in the body through a mechanism different from that of omalizumab. In particular, NK-2-12-EWPS has the ability to reduce total IgE in the body, which omalizumab does not have. Even in terms of reducing free IgE, NK-2-12-EWPS is much better than omalizumab, so it has great potential for drug development.

[0357] Example 6. Study on the dosage of NK-2-12-EWPS in mice

[0358] In principle, omalizumab reduces the free IgE content by neutralizing IgE. Therefore, in order to obtain sufficient neutralization effect, the dosage of omalizumab cannot be lower than the total amount of IgE in the patient's body, otherwise some IgE will not be neutralized. In fact, the clinical dosage of omalizumab usually needs to be up to 100 times the total amount of IgE in the patient's body (calculated according to the disclosed "Chinese Dosage Table of Omalizumab"). The NK-2-12-EWPS antibody proposed by the inventor reduces the free IgE content in the body by removing all IgE. Therefore, in theory, the dosage of NK-2-12-EWPS does not need to be higher than the total amount of IgE in the patient's body, and the free IgE content in the patient's body can be fully reduced.

[0359] The inventors used the method described in Example 5 in C57-derived FcγRIIb humanized mice, fixed the intraperitoneal injection amount of human IgE for each mouse at 50 μg, and detected the changes in total IgE and free IgE levels in the mice over time at dosages of 50 μg, 20 μg and 10 μg (corresponding to 100%, 40% and 20% of the IgE injection amount).

[0360] The results showed that the use of omalizumab would lead to an increase in the total IgE content in the serum regardless of the dose. The higher the dose of omalizumab, the higher the total IgE content in the serum ( Figure 5 AC). In terms of free IgE, when the dosage of omalizumab was not less than the IgE injection dosage, it showed a good neutralization effect, and the free IgE content was reduced to a lower level from the beginning ( Figure 5D). However, when the dose of omalizumab is lower than the amount of IgE injected, IgE cannot be fully neutralized, and the free IgE content only decreases slowly over time like the negative control group without the injection of therapeutic antibodies, rather than being reduced rapidly ( Figure 5 DF). By longitudinally comparing the free IgE curves when using different doses of omalizumab, we can more clearly see the effect of changing the dosage of omalizumab on the neutralization effect ( Figure 5 G). Whether the dosage of omalizumab is higher than the IgE injection dosage is the watershed for omalizumab to fully reduce serum free IgE, which is also consistent with common sense. Comparing the free IgE content change curves when the dosage of omalizumab is 50μg and the dosage described in Example 5 is 100μg (corresponding to 100% and 200% of the IgE injection dosage, respectively), it is found that the higher dosage of omalizumab does not further reduce the IgE content in mice. Considering the measurement error factor, the curves of the two experiments are basically the same ( Figure 5 G). This proves that the increase in the amount of omalizumab has limited further gains in the neutralization effect, which is consistent with the theory of chemical thermodynamics. According to the binding and dissociation formula, in order to further reduce the free IgE content, it is necessary to increase the amount of omalizumab by an order of magnitude. In addition, since antibodies have binding and dissociation constants, some antigens will dissociate from antibodies and be free. Therefore, although the amount of omalizumab used in the above two experiments was not lower than the total amount of IgE, the free IgE level in mice was always slightly higher than the baseline level ( Figure 5 G, I).

[0361] Unlike the case of omalizumab, when NK-2-12-EWPS was used, even if the antibody dosage was lower than the IgE injection dosage, it still had a good IgE clearance effect. Both the total IgE and free IgE levels in mouse serum decreased rapidly within the first few hours ( Figure 5 AE). When the dosage of NK-2-12-EWPS (20 μg) was only 40% of the IgE injection, the serum IgE content of mice could be reduced to the baseline level within 3 hours ( Figure 5 When the dosage of NK-2-12-EWPS was further reduced to 10 μg (corresponding to only 20% of the IgE injection amount), the serum IgE content of mice failed to decrease to the baseline level within 3 hours ( Figure 5 AE). By longitudinally comparing the free IgE curves at different NK-2-12-EWPS doses, it can be observed that when NK-2-12-EWPS is present, the reduction in its dose mainly affects the IgE content at the first hour time point, without changing the trend of a 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), will the IgE clearance rate be slightly delayed ( Figure 5 H), which is different from the situation with omalizumab.

[0362] Further comparison of the serum free IgE curves when using 20μg (corresponding to 40% of the IgE injection amount) NK-2-12-EWPS and 100μg (corresponding to 200% of the IgE injection amount) omalizumab showed that from the second hour, 20μg NK-2-12-EWPS showed a better free IgE control effect than 100μg omalizumab. Only at the first hour, the free IgE content corresponding to 100μg omalizumab was lower than the free IgE content corresponding to 20μg NK-2-12-EWPS ( Figure 5 I). This indicates that NK-2-12-EWPS, as a human IgE-clearing antibody, takes about an hour to accumulate efficacy after injection 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.

[0363] In summary, NK-2-12-EWPS, as a new concept of human IgE-clearing antibody, has the characteristics of low dosage and good effect compared with omalizumab, and has great drug potential. In addition, considering the actual clinical occasions, after injecting a sufficient amount of omalizumab into the patient, the IgE in the patient's body is almost completely neutralized, so the allergic symptoms are alleviated or disappear. However, since the allergen has not disappeared, IgE is still being produced in the patient's body under its continuous stimulation. 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. The NK-2-12-EWPS proposed by the inventor plays a role in continuously clearing IgE, so even if IgE is still being produced in the patient's body, NK-2-12-EWPS can also maintain the free IgE in the patient's body at an extremely low level for a long time, thereby controlling allergic symptoms for a long time. From this perspective, NK-2-12-EWPS is particularly suitable for chronic allergic patients who need long-term medication.

[0364] Example 7. Clearance effect of NK-2-12-SELF on human IgE in mice

[0365] In a paper published in 2008 by Chu et al. (Chu et al., 2008, doi: 10.1016 / j.molimm.2008.06.027), another mutation scheme that can enhance the affinity of antibodies to receptor FcγRIIb is described, namely the S267E / L328F double point mutation (SELF mutation) scheme. The inventors also introduced the SELF mutation into the Fc end of the antibody NK-2-12 described in Example 1 to form the NK2-12-SELF antibody. The amino acid sequence of IgG Fc with SELF mutation is shown in SEQ ID NO: 8, the amino acid sequence of NK-2-12-SELF heavy chain is shown in SEQ ID NO: 11 (the box marks the introduced histidine mutation and SELF mutation), and the amino acid sequence of NK-2-12-SELF light chain is shown in SEQ ID NO: 5 (the box marks the introduced histidine mutation, and the sequence is the same as the aforementioned NK-2-12 antibody light chain sequence).

[0366] The expressed and purified NK-2-12-SELF antibody was also used to perform a human IgE clearance experiment in wild-type C57 mice or C57-derived FcγRIIb humanized mice 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 is far less than NK-2-12-EWPS ( Figure 4 D, E). In particular, the free IgE concentration in mice was higher than that in the omalizumab group over a longer period of time. However, this does not mean that NK-2-12-SELF does not have drug potential. Because the SELF mutation is different from the EWPS mutation, the latter has a higher affinity for both human and mouse FcγRIIb, while the SELF mutation only has a higher affinity for human FcγRIIb. The inventors did not detect the affinity between the SELF mutation and mouse FcγRIIb, so the clearance ability of human IgE in wild-type C57 mice was poor. In contrast, in C57-derived FcγRIIb humanized mice, 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 IgE in mice to baseline levels more quickly ( Figure 6 C, D).

[0367] Example 8. NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF have the ability to bind to human IgE clearance and longer serum half-life

[0368] Based on the IgG Fc with SELF mutation and IgG Fc with EWPS mutation synthesized in the previous examples, this example further introduced mutations at three sites (i.e., M252Y / S254T / T256E), and the mutated fragments were named: IgG Fc with YTE-EWPS mutation (whose amino acid sequence is shown in SEQ ID NO: 118) and IgG Fc with YTE-SELF mutation (whose amino acid sequence is shown in SEQ ID NO: 119).

[0369] Furthermore, two antibodies, NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF, were constructed using the two Fc fragments and the antibody NK-2-12 obtained in the above example. 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 antibody is shown in SEQ ID NO: 5.

[0370] Four antibodies, NK-2-12-YTE-EWPS, NK-2-12-YTE-SELF, NK-2-12-EWPS and NK-2-12-SELF, were expressed and purified, and injected into the peritoneal cavity of FcRn humanized mice (Shanghai Model Organisms, Cat. No.: NM-HU-00109) for the detection of serum half-life. In the experiment, each antibody was injected into the peritoneal cavity of three randomly grouped FcRn humanized mice at a dose of 10 mg / kg. After that, the tail vein blood of the mice was collected at 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, a total of 14 time points, and the blood was centrifuged after standing at room temperature for coagulation. The human antibody content in the serum was detected by standard sandwich ELISA. The antibody used to coat the 96-well ELISA plate was goat anti-human IgG Fc antibody (Jackson, catalog number: 109-006-098), and HRP enzyme-linked goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109-036-088) combined with substrate ABTS (ThermoScientific, catalog number: 002024) was used to detect the captured human IgG content. The concentrations of both antibodies were the recommended concentrations in the antibody instructions. When detecting the content of human IgG in mouse serum samples, a standard curve was first established using the above two antibodies and a known concentration of human IgG, and then the standard curve was used to calculate the concentration of human IgG in the serum sample.

[0371] The experimental results are as follows Figure 7 As shown, Figure 7 A shows that the serum half-life of NK-2-12-YTE-EWPS is significantly longer than that of NK-2-12-EWPS. The calculated serum half-life of NK-2-12-EWPS is 39.6 hours, while the serum half-life of NK-2-12-YTE-EWPS is 66.9 hours. Similarly, Figure 7 B shows that the calculated serum half-life of NK-2-12-SELF is 62.7 hours, while the serum half-life of NK-2-12-YTE-SELF is increased to 103.8 hours. The serum half-lives of the two antibodies with YTE mutations were significantly prolonged.

[0372] It should be noted that although the test results show that the half-life of antibodies with SELF mutations in mice is relatively long, the real reason is that antibodies with SELF mutations do not bind to mouse FcγRIIb (as described in Example 7), and are therefore not easily endocytosed by mouse cells, thus creating the illusion that antibodies with SELF mutations have a longer half-life in mice. However, since antibodies with EWPS mutations or SELF mutations have similar high affinity to human FcγRIIb and are easily endocytosed by human cells in the human body, antibodies with EWPS mutations or SELF mutations should have similar serum half-lives in the human body.

[0373] 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 not appropriate to make a horizontal comparison of the serum half-lives of NK-2-12-EWPS and NK-2-12-SELF in mice.

[0374] The inventors tested the clearance effects of NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF on humanized IgE at different dosages in C57-derived FcγRIIb humanized mice according to the method described in Example 5. Figure 7 As shown in C-7F, both NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF can reduce the total human IgE and free human IgE concentrations in FcγRIIb humanized mice to baseline levels in a short period of time. Moreover, the experimental results also showed that even if the dosage of the two antibodies was reduced to one-fifth of the original, that is, the dosage was reduced from 100μg to 20μg, the clearance effect of the two antibodies on human IgE was still not significantly reduced. Compared with omalizumab, NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF, even if the dosage was only 20μg, could make the total IgE content and free IgE content in the test mice almost lower than that of the 100μg omalizumab group. Therefore, both NK-2-12-YTE-EWPS and NK-2-12-YTE-SELF antibodies have efficacy far exceeding that of omalizumab in reducing both total IgE and free IgE levels in the body.

[0375] In summary, IgG Fc with YTE-EWPS mutation and IgG Fc with YTE-SELF mutation not only prolong the serum half-life of the antibody, but also enhance the efficacy of the antibody.

[0376] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to IgE, comprising: (a) a heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VHCDR1 of SEQ ID NO: 24, VH CDR2 of SEQ ID NO: 25, and VH CDR3 of SEQ ID NO: 26; and a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 of SEQ ID NO: 27, VL CDR2 of SEQ ID NO: 28, and VL CDR3 of SEQ ID NO: 29; (b) a heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VHCDR1 of SEQ ID NO: 72, VH CDR2 of SEQ ID NO: 73, and VH CDR3 of SEQ ID NO: 74; and a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 of SEQ ID NO: 75, VL CDR2 of SEQ ID NO: 76, and VL CDR3 of SEQ ID NO: 77; or (c) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 80, VH CDR2 of SEQ ID NO: 81, and VH CDR3 of SEQ ID NO: 82; and a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 83, VL CDR2 of SEQ ID NO: 84, and VL CDR3 of SEQ ID NO:

85.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) a heavy chain variable region (VH) as shown in SEQ ID NO: 22, and a light chain variable region (VL) as shown in SEQ ID NO: 23; (b) a heavy chain variable region (VH) as shown in SEQ ID NO: 70, and a light chain variable region (VL) as shown in SEQ ID NO: 71; or (c) a heavy chain variable region (VH) as shown in SEQ ID NO: 78, and a light chain variable region (VL) as shown in SEQ ID NO:

79.

3. The antibody or antigen-binding fragment thereof of claim 1, further comprising a constant region.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises an IgG heavy chain constant region. 5 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the light chain constant region comprised in the light chain of the antibody or antigen-binding fragment thereof is a kappa light chain constant region or a lambda light chain constant region.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO:

12.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region as shown in SEQ ID NO:

13. The antibody or antigen-binding fragment thereof according to claim 1 , comprising an Fc region.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the Fc region is Fc or Fc variant of IgG; wherein The Fc variant has an increased affinity for Fcγ receptor (FcγR) compared to Fc of IgG.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-bonded Fv, scFv and diabody; and / or the antibody is a murinized antibody, a chimeric antibody, a fully human antibody, a bispecific antibody or a multispecific antibody.

11. The antibody or antigen-binding fragment thereof of claim 9, wherein the Fc of IgG has the sequence shown in SEQ ID NO:

6.

12. The antibody or antigen-binding fragment thereof according to claim 9, wherein The Fc variant has the sequence shown in SEQ ID NO:

7.

13. The antibody or antigen-binding fragment thereof according to claim 9, wherein The Fc variant has the sequence shown in SEQ ID NO:

118.

14. The antibody or antigen-binding fragment thereof according to claim 9, wherein The Fc variant has the sequence shown in SEQ ID NO:

8.

15. The antibody or antigen-binding fragment thereof according to claim 9, wherein The Fc variant has the sequence shown in SEQ ID NO:

119.

16. The antibody or antigen-binding fragment thereof according to claim 9, wherein The FcγR is selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, or any combination thereof.

17. The antibody or antigen-binding fragment thereof according to claim 9, wherein The FcγR is derived from a mammal.

18. The antibody or antigen-binding fragment thereof according to claim 9, wherein The FcγR is human FcγRIIb.

19. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

20. A vector comprising the isolated nucleic acid molecule of claim 19. The vector of claim 20 , which is a cloning vector or an expression vector.

22. A host cell comprising the isolated nucleic acid molecule of claim 19 or the vector of claim 20.

23. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, comprising culturing the host cell according to claim 22 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

24. A multispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1-18.

25. The multispecific molecule of claim 24, further comprising at least one second specific binding molecule for a second target.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 or the multispecific molecule according to claim 24 or 25, and a pharmaceutically acceptable carrier and / or excipient.

27. The pharmaceutical composition of claim 26, further comprising an additional pharmaceutically active agent.

28. The pharmaceutical composition of claim 27, wherein the additional pharmaceutically active agent is a drug having anti-allergic effects.

29. The pharmaceutical composition of claim 27, wherein the additional pharmaceutically active agent is an anti-asthmatic drug.

30. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

31. The kit of claim 30, wherein the antibody or antigen-binding fragment thereof carries a detectable label, and the detectable label is selected from an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

32. The kit of claim 31, wherein the antibody or antigen-binding fragment thereof is conjugated with horseradish peroxidase or a chemiluminescent substance.

33. The kit of claim 30, further comprising a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

34. The kit of claim 33, wherein the second antibody further comprises a detectable label, wherein the detectable label is selected from an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

35. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-18, the isolated nucleic acid molecule of claim 19, the vector of claim 20 or 21, the host cell of claim 22, the multispecific molecule of any one of claims 24-26, or the pharmaceutical composition of any one of claims 27-29 for preparing a medicament for treating a type 1 hypersensitivity disease in a subject.

36. The use of claim 35, wherein the medicament is for treating an allergic reaction in a subject.

37. The use of claim 35, wherein the type 1 hypersensitivity disease is selected from allergic rhinitis, allergic cough, anaphylactic shock, food allergy, asthma, chronic urticaria, acute urticaria, acute bronchospasm, atopic dermatitis, inflammatory skin disease, eczema, laryngeal edema, angioedema, nasal polyps, sinusitis, or any combination thereof.

38. The use according to claim 35, wherein the subject is a mammal.

39. The use according to claim 35, wherein the subject is a human, a monkey or a mouse.

40. The use of claim 35, wherein the antibody or antigen-binding fragment thereof is used alone or in combination with another pharmaceutically active agent.

41. The use of claim 35, wherein the antibody or antigen-binding fragment thereof specifically binds to IgE under neutral or alkaline conditions.

42. The use of claim 35, wherein the antibody or antigen-binding fragment thereof does not specifically bind to IgE at pH 6.

43. A method for detecting the presence or amount of IgE in a sample for non-diagnostic purposes, comprising the following steps: (1) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18; (2) Detecting the formation of a complex between the antibody or antigen-binding fragment thereof and IgE or detecting the amount of the complex.

44. The method of claim 43, wherein the antibody or antigen-binding fragment thereof is detectably labeled.

45. The method of claim 43, wherein the IgE is human IgE.

46. ​​The method of claim 43, wherein the antibody or antigen-binding fragment thereof specifically binds to IgE under neutral or alkaline conditions.

47. The method of claim 43, wherein the antibody or antigen-binding fragment thereof does not specifically bind to IgE at pH 6.

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