Modified anti-IgE antibodies and their application

Abstract

The present invention provides antigen-binding proteins and pharmaceutical compositions containing antigen-binding proteins. [Solution] An antigen-binding protein is provided. Compared to the light chain and heavy chain variable region sequences of omalizumab, the antigen-binding protein contains amino acid mutations. The antigen-binding protein is an anti-IgE modified antibody. Further comprising pharmaceutical compositions comprising the antigen-binding protein, and methods thereof for alleviating or treating diseases associated with abnormal IgE levels.

Description

[Technical Field]

[0001] This application relates to the field of biomedicine, particularly to anti-IgE modified antibodies and their use in diseases in which IgE levels are abnormal. [Background technology]

[0002] Immunoglobulin E (IgE) is a protein with a molecular weight of 180 kD. Although IgE accounts for only 0.002% of total serum Ig, it is closely related to the pathological processes of allergic diseases. In various tissues and organs, elevated levels of IgE can trigger an inflammatory immune response, the most common of which are allergic asthma, allergic rhinitis and sinusitis, allergic conjunctivitis, systemic allergies, cutaneous allergies, and urticaria.

[0003] In recent years, biologics, such as anti-IgE monoclonal antibodies, have become a hot spot in research for treating diseases associated with abnormal IgE levels. In 2003, the anti-IgE antibody omalizumab was the first to be approved by the FDA for the treatment of asthma. In 2014, the U.S. Food and Drug Administration (FDA) approved omalizumab for the treatment of chronic roseola. The application of omalizumab has the following problems: (1) Omalizumab targets free IgE but does not target (or cannot effectively target) pathogenic species of IgE / FcεRI (IgE Fc receptor) complexes at pharmaceutically appropriate doses; (2) The dose concentration and interval of omalizumab are directly related to the patient's body weight and free IgE levels, and if the patient's body weight and free IgE levels exceed a certain range, the dose concentration and frequency increase, increasing the complexity and inconvenience of adjusting and managing the patient's disease; (3) The affinity of omalizumab for IgE is not particularly good. Therefore, there is an urgent need for new, modified anti-IgE antibodies with improved efficacy and compliance. [Overview of the Initiative]

[0004] This application provides an antigen-binding protein in which the antibody light chain and heavy chain sequences of omalizumab have been modified and optimized, and which may include at least one of the following features: (1) improved degree of humanization, reduced probability of anti-drug antibody production, and reduced loss of potential efficacy compared to omalizumab; (2) mutation of post-translational modification sites, improving chemical stability (e.g., stability under high temperature or pH environments); (3) improved affinity to human IgE by yeast display method, improved drug efficacy, and the antigen-binding protein is expected to be applicable to patients with high body weight or high concentrations of free IgE; (4) high affinity binding to monkey IgE, useful for pharmacodynamic studies in preclinical animal models; (5) effective blocking of IgE binding to IgE Fc receptor (FcεRI); (6) extended serum half-life, which can reduce the frequency of injections and facilitate adjustment of the patient's condition.

[0005] In one embodiment, the present application provides an antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL contains amino acid mutations at one or more positions selected from the group consisting of E55 and V104, compared to the amino acid sequence shown in SEQ ID NO: 22.

[0006] In some embodiments, VL contains amino acid mutations at one or more positions selected from the group consisting of D30b, V29, D30, Y30a, and G30c.

[0007] In some embodiments, VL contains amino acid mutations at the following groups: D30b, E55, and V104.

[0008] In some embodiments, VL contains amino acid mutations at positions selected from the group consisting of (1) D30b, E55, V104, V29, D30, and Y30a; (2) D30b, E55, V104, D30, Y30a, and G30c; (3) D30b, E55, V104, and Y30a; (4) D30b, E55, V104, Y30a, and G30c; (5) D30b, E55, V104, D30, Y30a, and G30c; and (6) D30b, E55, V104, D30, and Y30a.

[0009] In some embodiments, the amino acid mutation at position E55 includes E55Q.

[0010] In some embodiments, the amino acid mutation at position V104 includes V104L.

[0011] In some embodiments, the amino acid mutation at position D30b includes D30bE.

[0012] In some embodiments, the amino acid mutation at position V29 includes V29L.

[0013] In some embodiments, the amino acid mutation at position Y30a includes any one selected from the group consisting of Y30A, Y30aS, and Y30aD.

[0014] In some embodiments, the amino acid mutation at position D30 includes any one selected from the group consisting of D30Y, D30A, D30E, D30F, D30G, and D30N.

[0015] In some embodiments, the amino acid mutation at position G30c includes any one selected from the group consisting of G30cA, G30cY, and G30cW.

[0016] In some embodiments, VL includes the following amino acid mutations in the group: D30bE, E55Q, and V104L.

[0017] In some embodiments, VL includes the amino acid sequence shown in SEQ ID NO: 57.

[0018] In some embodiments, VL includes any one of the amino acid sequences shown in SEQ ID NOs: 23-34.

[0019] In some embodiments, the antigen-binding protein includes an antibody heavy chain variable region (VH), and VH includes amino acid mutations at one or more positions selected from the group consisting of I37, A49, S50, and D54 as compared to the amino acid sequence shown in SEQ ID NO: 50.

[0020] In some embodiments, VH contains amino acid mutations at one or more positions selected from the group consisting of N60, P61, I67, T30, S31, S96, and H97.

[0021] In some embodiments, VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, and D54.

[0022] In some embodiments, the amino acid mutation at position I37 includes I37V.

[0023] In some embodiments, the amino acid mutation at position A49 includes A49S.

[0024] In some embodiments, the amino acid mutation at position S50 includes S50V.

[0025] In some embodiments, the amino acid mutation at position D54 includes D54A.

[0026] In some embodiments, the amino acid mutation at position N60 includes N60A.

[0027] In some embodiments, the amino acid mutation at position P61 includes P61D.

[0028] In some embodiments, the amino acid mutation at position I67 includes I67F.

[0029] In some embodiments, the amino acid mutation at position T30 includes T30R.

[0030] In some embodiments, the amino acid mutation at position S31 includes S31Q.

[0031] In some embodiments, the amino acid mutation at position S96 includes S96T.

[0032] In some embodiments, the amino acid mutation at position H97 includes H97N.

[0033] In some embodiments, VH includes amino acid mutations from the following groups: N60A, P61D, I67F, I37V, A49S, S50V, and D54A.

[0034] In some embodiments, VH includes the amino acid sequence shown in SEQ ID NO: 58.

[0035] In some embodiments, VH comprises one of the amino acid sequences shown in SEQ ID NOs. 51-56.

[0036] In some embodiments, VL contains amino acid mutations at the following positions: D30b, E55 and V104; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50 and D54.

[0037] In some embodiments, VL and VH contain amino acid mutations at positions selected from the following groups: (1) VL contains amino acid mutations at the following positions: D30b, E55, V104, V29, D30 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54, S96 and H97; (2) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and T30 (3) VL contains amino acid mutations at the following positions: D30b, E55, V104 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and S96; (4) VL contains amino acid mutations at the following positions: D30b, E55, V104 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50 and D54; (5) VL contains amino acid mutations at the following positions: D30b, E55, V104, Y30a and G30c; VH contains amino acid mutations at the following positions (6) VL contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50 and D54; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and H97; (7) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50 and D54; (8) VL contains amino acid mutations at the following positions (9) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and H97; (10) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and S96; and (10) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c;VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54, and S31.

[0038] In some embodiments, the antigen-binding protein includes a constant region of the antibody heavy chain.

[0039] In some embodiments, the constant region of the antibody heavy chain of the antigen-binding protein is derived from the human IgG constant region.

[0040] In some embodiments, the human IgG constant region includes the human IgG1 constant region.

[0041] In some embodiments, the human IgG1 constant region includes one or more amino acid mutations selected from the group consisting of M252Y, S254T, and T256E.

[0042] In some embodiments, the human IgG1 constant region includes one of the amino acid sequences shown in SEQ ID NOs. 60-61.

[0043] In some embodiments, the antigen-binding protein includes a constant region of the antibody light chain.

[0044] In some embodiments, the antibody light chain constant region includes the amino acid sequence shown in SEQ ID NO: 59.

[0045] In another embodiment, this application provides one or more isolated nucleic acid molecules encoding antigen-binding proteins described herein.

[0046] In another embodiment, this application provides a vector comprising the nucleic acid molecule described herein.

[0047] In another aspect, this application provides cells comprising nucleic acid molecules or vectors as described herein.

[0048] In another aspect, the present application provides a method for preparing an antigen-binding protein as described herein, comprising the step of culturing cells under conditions that enable the expression of the antigen-binding protein.

[0049] In another embodiment, the present application provides compositions comprising antigen-binding proteins, nucleic acid molecules, vectors and / or cells, and optionally pharmaceutically acceptable vectors.

[0050] On the other hand, this application provides the use of antigen-binding proteins, nucleic acid molecules, vectors, cells and / or compositions in the preparation of pharmaceuticals for alleviating or treating diseases associated with abnormal levels of IgE.

[0051] In another embodiment, the present application provides a method for alleviating or treating a disease associated with abnormal levels of IgE, comprising the step of administering an antigen-binding protein, nucleic acid molecule, vector, cell and / or composition to a subject in need thereof.

[0052] Other aspects and advantages of this application can be readily understood by those skilled in the art from the following detailed description. Only exemplary embodiments of this application are shown and described in the following detailed description. As those skilled in the art will recognize, those skilled in the art can modify specific embodiments disclosed in this application without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in this application are illustrative and not limiting.

[0053] The special features of the invention relating to this application are described in the appended claims. The features and advantages of the invention relating to this application will be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows. [Brief explanation of the drawing]

[0054] [Figure 1]Figure 1 shows the inhibition rates of free IgE in cynomolgus monkey serum by omalizumab, AB1904AM10, and AB1904AM15 over time. [Figure 2] Figure 2 shows the blocking of human full-length IgE binding to the FcεRIα receptor protein by an anti-IgE antibody. [Figure 3] Figure 3 shows the blocking of human full-length IgE binding to the cell surface CD23 receptor protein by an anti-IgE antibody. [Figure 4] Figures 4A and 4B show the blocking of human full-length IgE-induced histamine release from FcεRI / RBL-2H3 by anti-IgE antibodies. [Figure 5] Figure 5 shows histamine release from human whole blood. [Figure 6] Figure 6 shows the time course of anti-IgE antibody levels in hFcRn mouse serum. [Figure 7] Figure 7 shows the time course of anti-IgE antibodies in cynomolgus monkey serum. [Modes for carrying out the invention]

[0055] Embodiments of the present invention are described below by specific examples, and those skilled in the art will readily understand other advantages and effects of the present invention from the contents disclosed herein.

[0056] Definition of Terms In this application, the term "IgE" typically refers to antibodies within mammals. IgE is a member of the immunoglobulin family that mediates allergic responses to many causes, such as asthma, food allergies, type 1 hypersensitivity, and familial sinusitis. IgE is secreted from B cells and expressed on the surface of B cells. IgE synthesized in B cells is anchored to the B cell membrane by a transmembrane domain linked to a mature IgE sequence via a short membrane-binding region. IgE can also bind to B cells (as well as monocytes, eosinophils, and platelets) by binding its Fc region to the low-affinity IgE receptor (FcεRII). After a mammal is exposed to an allergen, B cells proliferate asexually and synthesize IgE that binds to the allergen. This IgE is released into circulation by B cells, where it is bound by B cells (via FcεRII) and by mast cells and basophils via so-called high-affinity receptors (FcεRI) found on the surface of mast cells and basophils. Thus, such mast cells and basophils are sensitized to allergens. Subsequent exposure to allergens cross-links the FcεRI on these cells, and thus activates their release of histamine and other factors that contribute to clinical hypersensitivity and anaphylaxis.

[0057] In this application, the term “antigen-binding protein” typically refers to a fragment or portion of one or more antibodies that possess the ability to specifically bind to an antigen (e.g., IgE), or a synthesized variant of an antibody fragment that possesses a desired binding ability to an antigen. The antigen-binding function of the expressed antibody may be performed by a fragment or portion of a full-length antibody, or a variant thereof. Examples include bispecific, dual-specific, and multiple-specific formats that can specifically bind to two or more different antigens or to several epitopes or discrete epitope regions of an antigen. Examples of antigen-binding proteins may include single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH) (e.g., described in International Publication No. 2001 / 090190), scFv, bivalent, trivalent, or tetravalent antibodies, bi-scFv, diabody, tribody, triabody, tetrabody, and any of the above epitope-antigen conjugates (e.g., see Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing and creating these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0058] In this application, the term “antibody” generally refers to any immunoglobulin (Ig) molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, variant, variant, or derivative thereof that retains at least some of the epitope-binding properties of an Ig molecule and enables its specific binding to IgE. In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH). The heavy chain constant region consists of four domains—CH1, hinge, CH2, and CH3 (heavy chain gamma, alpha, and delta), or CH1, CH2, CH3, and CH4 (heavy chain mu and epsilon). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved 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. Immunoglobulin molecules can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0059] In this application, the term “monoclonal antibody” generally refers to a preparation of an antibody molecule that shares a common heavy-chain amino acid sequence and a common light-chain amino acid sequence, or any functional fragment, variant, variant, or derivative thereof that retains at least the light-chain epitope binding characteristics of the Ig molecule, as opposed to a “polyclonal” antibody preparation that generally contains a mixture of different antibodies. Monoclonal antibodies can be produced by several known techniques, e.g., phage, bacterial, yeast, or ribosome display, as well as by classical methods, e.g., hybridoma-derived antibodies, e.g., hybridoma techniques, e.g., antibodies secreted by hybridomas by the standard Kohler and Milstein hybridoma method ((1975) Nature 256:495-497).

[0060] In this application, the term "omalizumab" generally refers to the recombinant DNA-derived humanized IgG1κ monoclonal antibody, traded under the registered trademark Xolair, which selectively binds to human immunoglobulin E (IgE). Omalizumab has a molecular weight of approximately 149 kD. It is produced from a suspension culture of Chinese hamster ovary cells in a medium containing the antibiotic gentamicin (see European Patent No. 602126 (and thereunder SPC / GB06 / 005); International Publication No. 93 / 04173; U.S. Patent No. 6267958 (and thereunder PTE); International Publication No. 97 / 04807; International Publication No. 97 / 04801; Presta et al. (1993) J. Immunol. 151:2623-2632). Where omalizumab is described, it refers to the full-length IgG antibody containing the light chain variable region shown in SEQ ID NO: 22 and the heavy chain variable region shown in SEQ ID NO: 50. Omalizumab is currently indicated for the treatment of moderate to severe persistent asthma in patients who have positive skin tests or in vitro reactions to chronic airborne allergens and whose symptoms are not adequately controlled by inhaled corticosteroids.

[0061] In this application, when describing common immunoglobulin regions (CDR - complementarity-determining regions, or FR - framework regions) of VH or VL sequences, they are concatenated in a standard order (VH = HFR1.HCDR1.HFR2.HCDR2.HFR3.HCDR3.HFR4; VL = LFR1.LCDR1.LFR2.LCDR2.LFR3.LCR3.LFR4).

[0062] In the amino acid numbering of antibodies or antigen-binding proteins of this application, a continuous amino acid sequence derived from an antibody or antigen-binding protein may be numbered according to the Chothia numbering rules. In this application, the terms “Chothia scheme,” “Chothia numbering,” or “Chothia definition” are used interchangeably and generally refer to an amino acid residue numbering system (Chothia et al., (1987) J. Mol. Biol., 196, 901-917 (1987)). According to the Chothia numbering rules, for omalizumab, the VL (Sequence ID 22) is defined as follows: LFR1 (L1-L23), LCDR1 (L24-L34), LFR2 (L35-L49), LCDR2 (L50-L56), LFR3 (L57-L88), LCDR3 (L89-L97), and LFR4 (L98-L107), and the VH (Sequence ID 50) is defined as follows: HFR1 (H1-H25), HCDR1 (H2 The terms HFR2 (H33-H51), HFR2 (H52-H56), HFR5 (H57-H94), HFR3 (H95-H102), and HFR4 (H103-H113) are defined as follows: "Lx" or "Hx" (where x is an Arabic numeral) represents an amino acid numbered as x in the light chain (L) variable region or heavy chain (H) variable region. For VL, L24 represents the amino acid at position 24 of VL by Chothia numbering. In the case of VL or VH, the Lx or Hx position may also be represented by the letter of the amino acid at position x plus a number. For example, for the VL of omalizumab, position L24 is arginine (R), and the amino acid at position 24 of VL may be represented by R24. In this application, the two above-mentioned naming conventions for positions (e.g., L24 and R24) may be used interchangeably. According to the Chothia numbering rules, the amino acid positions of the complementarity-determining region of omalizumab are shown in Tables 1-6 below.

[0063] [Table 1]

[0064] [Table 2]

[0065] [Table 3]

[0066] [Table 4]

[0067] [Table 5]

[0068] [Table 6]

[0069] The amino acid mutations described in this application are expressed as "amino acid before mutation + position + amino acid after mutation". For example, the VL amino acid mutation "E55Q" means that the amino acid at position 55 of VL is mutated from glutamic acid (E) to glutamamide (Q).

[0070] In this application, the terms "Kd", "KD", or "K DThe terms "Kd" and "Koff" are interchangeable and generally refer to the dissociation constant of a particular antibody-antigen interaction, as is known in the art. The strength or affinity of an immunobinding interaction can be expressed by the dissociation constant (kD or kd) of the interaction, where a smaller KD indicates a greater or higher affinity. The immunobinding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, the "association rate constant" ("Kon") and the "dissociation rate constant" ("Koff") can be determined by calculating the concentration and the actual rates of association and dissociation. The Koff / Kon ratio cancels out all parameters not related to affinity and is equal to the dissociation constant Kd.

[0071] The affinity of the antibodies or antigen-binding proteins of this application and the degree to which they inhibit binding can be determined by those skilled in the art using prior art, e.g., as described by Scatchard et al. (Ann. KY. Acad. Sci. 51:660-672 (1949)), biofilm layer optical interferometry (BLI) techniques using ForteBio Octet®, or surface plasmon resonance (SPR) using systems such as Biacore. In biofilm layer optical interferometry, the lower end of the internal biosensor is covered by a biofilm layer, allowing for the binding and immobilization of biomolecules. When visible light with a specific bandwidth is projected perpendicularly onto the biofilm layer, the light is reflected at two interfaces of the biofilm layer, forming an interference wave of a specific wavelength. When the immobilized molecules interact with molecules in solution, the thickness of the biofilm layer increases, the interference spectrum curve shifts in the direction of increasing wavelength, and the phase change of the light wave is detected in real time by a workstation. This allows for the detection of changes in the number of molecules, associated concentrations, and dynamic data. In surface plasmon resonance (SPR), a target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase flowing through a flow cell. When ligand binding to the immobilized target occurs, the local reflectivity changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. By analyzing the rate of change of the SPR signal, apparent rate constants for the association and dissociation phases of the binding reaction can be constructed. The ratio of these values ​​gives an apparent equilibrium constant (affinity).

[0072] It will be understood that the affinity of the antigen-binding protein provided herein can be modified using any suitable method known in the art. Accordingly, this application also designs variants of the antigen-binding protein of this application that have improved affinity for IgE. Such variants have been identified through CDR mutations (Yang et al., J. Mol. Biol., 254, 392-403, 1995), strand shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), mutagenesis using Escherichia coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al, J. Mol. Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al, Nature, 391, 288-291, This can be achieved by many affinity maturation protocols, including the one described in 1998.

[0073] In this application, the term “isolated nucleic acid molecule” generally includes DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, for example, double-stranded DNA. These nucleic acids may exist in whole cells, in cell lysates, or in partially purified or substantially pure forms. When nucleic acids are isolated and purified from other cellular components or other impurities (e.g., other cellular nucleic acids or proteins) by standard techniques including alkali / SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis, and other methods known in the art, they are “isolated” or “substantially pure.”

[0074] In this application, the term “vector” generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. The term “vector” includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which other DNA segments can be ligated. Another type of vector is a viral vector, to which other DNA segments can be ligated into a viral genome. Generally, modified vectors may contain an origin of replication, multiple cloning sites, and selection markers. The vector itself usually contains a nucleotide sequence, typically a DNA sequence, an insert (transgene) sequence, and a larger sequence that functions as the “backbone” or “backbone” of the vector. Current vectors may contain further features in addition to the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags.

[0075] In this application, the term “cell” generally refers to a cell into which nucleic acids encoding heterologous polypeptides or constituting shRNAs can or may be introduced. Host cells may include prokaryotic cells that can be used for vector / plasmid proliferation and eukaryotic cells that can be used for nucleic acid expression. For example, eukaryotic cells may be mammalian cells. In another example, mammalian host cells may be selected from the following mammalian cells: CHO cells (e.g., CHO K1 or CHO DG44), BHK cells, NS0 cells, SP2 / 0 cells, HEK 293 cells, HEK 293EBNA cells, PER.C6 cells and COS cells. In a particular example, mammalian cells may be selected from hybridomas, myelomas, and rodent cells. Myeloma cells may include rat myeloma cells (e.g., YB2) and mouse myeloma cells (e.g., NS0, SP2 / 0). Polypeptides or proteins for pharmaceutical applications can be produced in mammalian cells, such as CHO cells, NSO cells, SP2 / 0 cells, COS cells, HEK cells, BHK cells, etc.

[0076] In this application, the term “pharmaceutically acceptable carrier” generally refers to one or more non-toxic substances that do not interfere with the efficacy of the biological activity of the active ingredient. Such formulations may traditionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable carriers may also contain compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. Other intended carriers, excipients, and / or additives that may be used in the formulations described herein include, for example, fragrances, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (e.g., serum albumin, gelatin, casein), salt-forming counterions (e.g., sodium), etc. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences and Practice (Remington: The Science and Practice of Pharmacy, 22nd ed., Pharmaceutical Press (2012) and Physician's Desk Reference)", 66th ed., Medical Economics (2012). Appropriate pharmaceutically acceptable carriers for the desired or required mode of administration, solubility, and / or stability may be routinely selected.

[0077] In this application, the term “disease associated with abnormal levels of IgE” generally refers to any condition or disorder associated with abnormal (e.g., elevated) levels of serum IgE. Exemplary diseases associated with abnormal levels of IgE include, but are not limited to, bronchial asthma, allergic rhinitis, atopic dermatitis, urticaria, allergic response, or atopic dermatitis.

[0078] In this application, the terms “polypeptide,” “peptide,” “protein,” and “proteins” are used interchangeably and generally refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. These terms also encompass modified amino acid polymers. These modifications may include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation (e.g., binding to a labeling component). The term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and its D and L optical isomers, as well as amino acid analogs and peptide mimetic compounds.

[0079] In this application, the terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “nucleotide” are used interchangeably and generally refer to polymeric forms of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications of the nucleotide structure may be carried out before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation to labeled components.

[0080] In addition to the specific proteins and nucleotides described herein, this application may also include their functional variants, derivatives, analogs, homologs, and fragments.

[0081] The term “functional variant” refers to a polypeptide having substantially the same amino acid sequence as the natural sequence or encoded by substantially the same nucleotide sequence, and capable of retaining one or more activities of the natural sequence. In the context of this application, a variant of any given sequence refers to a sequence in which a specific sequence of residues (amino acid or nucleotide residues) is modified so that the polypeptide or polynucleotide substantially retains at least one intrinsic function. Variant sequences can be obtained by the addition, deletion, substitution, modification, exchange, and / or variation of at least one amino acid residue and / or nucleotide residue present in the natural protein and / or polynucleotide, as long as the original functional activity is retained.

[0082] In this application, the term “derivative” generally refers to a polypeptide or polynucleotide comprising any substitution, variation, modification, exchange, deletion and / or addition of one (or more) amino acid residues of / for the polypeptide or polynucleotide of this application, insofar as the resulting polypeptide or polynucleotide substantially retains at least one of its intrinsic functions.

[0083] In this application, the term “analog” generally refers to any mimic of a polypeptide or polynucleotide, i.e., a polypeptide or polynucleotide comprising a chemical compound that retains at least one intrinsic function of the polypeptide or polynucleotide that the mimic imitates.

[0084] In general, amino acid substitutions, such as at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, may be made, provided that the modified sequence substantially retains the desired activity or capability. Amino acid substitutions may include the use of unnatural analogs.

[0085] The proteins or polypeptides used in this application may also have deletions, insertions, or substitutions of amino acid residues that result in silent changes and produce functionally equivalent proteins. Intentional amino acid substitutions may be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the intrinsic function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar heads having similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0086] In this application, the term “homologous” generally refers to an amino acid sequence or nucleotide sequence that has a certain degree of homology to wild-type amino acid sequences and wild-type nucleotide sequences. The term “homologous” may be equivalent to “sequence identity.” A homologous sequence may include an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the target sequence. Typically, a homologous sequence will contain the same active site, etc., as the target amino acid sequence. Homologousity may be conceived from similarity (i.e., amino acid residues with similar chemical properties / functions) or expressed from sequence identity. In this application, a sequence having a certain percentage identity with any one of the SEQ ID NO sequences of an amino acid sequence or nucleotide sequence means a sequence that has said percentage identity over the entire length of the referenced SEQ ID NO.

[0087] To determine sequence identity, sequence alignment can be performed using various means known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate alignment parameters, including any algorithm necessary to achieve optimal alignment between the full-length sequences being compared.

[0088] In this application, the terms "and / or" should be understood to mean either or both of the alternatives.

[0089] In this application, the term "includes" generally means including clearly defined features but not excluding other elements.

[0090] In this application, the term "about" generally refers to a change within a range of 0.5% to 10% higher or lower than a specific value, for example, a change within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% higher or lower than a specific value.

[0091] Embodiment In one embodiment, the present application provides an antigen-binding protein that may contain an antibody light chain variable region (VL), wherein the VL of the antigen-binding protein described herein may contain amino acid mutations at one or two positions selected from the group consisting of E55 and V104, compared to the light chain variable region of omalizumab (i.e., the amino acid sequence shown in SEQ ID NO: 22).

[0092] In this application, the VL of the antigen-binding protein may contain amino acid mutations at one or more positions (e.g., 1, 2, 3, 4, or 5) selected from the group consisting of D30b, V29, D30, Y30a, and G30c.

[0093] For example, the VL of an antigen-binding protein may contain amino acid mutations at the following positions: D30b, E55, and V104.

[0094] For example, VL may contain amino acid mutations at positions D30b, E55, V104, V29, D30, and Y30a.

[0095] For example, VL may contain amino acid mutations at positions D30b, E55, V104, D30, Y30a, and G30c.

[0096] For example, VL may contain amino acid mutations at positions D30b, E55, V104, and Y30a.

[0097] For example, VL may contain amino acid mutations at positions D30b, E55, V104, Y30a, and G30c.

[0098] For example, VL may contain amino acid mutations at positions D30b, E55, V104, D30, Y30a, and G30c.

[0099] For example, VL may contain amino acid mutations at positions D30b, E55, V104, D30, and Y30a.

[0100] In this application, the amino acid mutation at position E55 may include E55Q.

[0101] In this application, the amino acid mutation at position V104 may include V104L.

[0102] In this application, the amino acid mutation at position D30b may include D30bE.

[0103] In this application, the amino acid mutation at position V29 may include V29L.

[0104] In this application, the amino acid mutation at position Y30a may include any one selected from the group consisting of Y30A, Y30aS, and Y30aD.

[0105] In this application, the amino acid mutation at position D30 includes one selected from the group consisting of D30Y, D30A, D30E, D30F, D30G, and D30N.

[0106] In this application, the amino acid mutation at position G30c may include any one selected from the group consisting of G30cA, G30cY, and G30cW.

[0107] In this application, VL may include amino acid mutations from the following groups: D30bE, E55Q, and V104L.

[0108] In this application, VL may include the amino acid sequence shown in Sequence ID No. 57.

[0109] In this application, the VL of the antigen-binding protein may include the amino acid sequences shown in SEQ ID NOs. 23-34.

[0110] For example, the VL of the antigen-binding protein may include amino acid mutations D30bE, E55Q, V104L, V29L, D30Y, and Y30aD.

[0111] For example, VL may include amino acid mutations D30bE, E55Q, V104L, D30A, Y30aD, and G30cW.

[0112] For example, VL may contain amino acid mutations 30bE, E55Q, V104L, and Y30aA.

[0113] For example, VL may include amino acid mutations D30bE, E55Q, V104L, and Y30aD.

[0114] For example, VL may include amino acid mutations D30bE, E55Q, V104L, and Y30aS.

[0115] For example, VL may include amino acid mutations D30bE, E55Q, V104L, Y30aD, and G30cY.

[0116] For example, VL may include amino acid mutations D30bE, E55Q, V104L, D30E, Y30aD, and G30cW.

[0117] For example, VL may include amino acid mutations D30bE, E55Q, V104L, D30G, Y30aD, and G30cA.

[0118] For example, VL may include amino acid mutations D30bE, E55Q, V104L, D30N, Y30aD, and G30cA.

[0119] For example, VL may include amino acid mutations D30bE, E55Q, V104L, D30N, Y30aD, and G30cW.

[0120] For example, VL may include amino acid mutations D30bE, E55Q, V104L, D30F, and Y30aD.

[0121] In this application, the antigen-binding protein may further include a light chain constant region. For example, the light chain constant region may be derived from a human kappa light chain. For example, the light chain constant region may include the amino acid sequence shown in SEQ ID NO: 59.

[0122] In this application, the antigen-binding protein may also include an antibody light chain. The antibody light chain may include an antibody light chain variable region and an antibody light chain constant region. For example, the antibody light chain of the antigen-binding protein described herein may include any one of the amino acid sequences shown in SEQ ID NOs. 23 to 34.

[0123] The antigen-binding protein described in this application may include an antibody heavy chain variable region (VH), and compared to the heavy chain variable region of omalizumab (the amino acid sequence shown in SEQ ID NO: 50), the VH of the antigen-binding protein may contain amino acid mutations at one or more positions (e.g., 1, 2, 3, or 4) selected from the group consisting of I37, A49, S50, and D54.

[0124] In this application, the VH of the antigen-binding protein may further include one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) amino acid mutations selected from the group consisting of N60, P61, I67, T30, S31, S96, and H97.

[0125] For example, the VH of an antigen-binding protein may contain amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, and D54.

[0126] For example, VH may contain amino acid mutations at positions N60, P61, I67, I37, A49, S50, D54, S96, and H97.

[0127] For example, VH may contain amino acid mutations at positions N60, P61, I67, I37, A49, S50, D54, and T30.

[0128] For example, VH may contain amino acid mutations at positions N60, P61, I67, I37, A49, S50, D54, and S96.

[0129] For example, VH may contain amino acid mutations at positions N60, P61, I67, I37, A49, S50, D54, and S31.

[0130] For example, VH may contain amino acid mutations at positions N60, P61, I67, I37, A49, S50, D54, and H97.

[0131] In this application, the amino acid mutation at position I37 may include I37V.

[0132] In this application, the amino acid mutation at position A49 may include A49S.

[0133] In this application, the amino acid mutation at position S50 may include S50V.

[0134] In this application, the amino acid mutation at position D54 may include D54A.

[0135] In this application, the amino acid mutation at position N60 may include N60A.

[0136] In this application, the amino acid mutation at position P61 may include P61D.

[0137] In this application, the amino acid mutation at position I67 may include I67F.

[0138] In this application, the amino acid mutation at position T30 may include T30R.

[0139] In this application, the amino acid mutation at position S31 may include S31Q.

[0140] In this application, the amino acid mutation at position S96 may include S96T.

[0141] In this application, the amino acid mutation at position H97 may include H97N.

[0142] In this application, VH may include amino acid mutations from the following groups: N60A, P61D, I67F, I37V, A49S, S50V, and D54A.

[0143] In this application, VH may include the amino acid sequence shown in SEQ ID NO: 58.

[0144] In this application, the antigen-binding protein VH may contain any one of the amino acid sequences shown in SEQ ID NOs. 51 to 56.

[0145] For example, the antigen-binding protein VH may include amino acid mutations N60A, P61D, I67F, I37V, A49S, S50V, D54A, S96T, and H97N.

[0146] For example, VH may include amino acid mutations N60A, P61D, I67F, I37V, A49S, S50V, D54A, and T30R.

[0147] For example, VH may include amino acid mutations N60A, P61D, I67F, I37V, A49S, S50V, D54A, and S96T.

[0148] For example, VH may include amino acid mutations N60A, P61D, I67F, I37V, A49S, S50V, D54A, and S31Q.

[0149] For example, VH may include amino acid mutations N60A, P61D, I67F, I37V, A49S, S50V, D54A, and H97N.

[0150] In this application, the antigen-binding protein may include HCDR1, HCDR2, and HCDR3, where HCDR1 may include the amino acid sequence shown in SEQ ID NO: 62, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 42, and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 63.

[0151] For example, the antigen-binding protein may include HCDR1, HCDR2, and HCDR3, where HCDR1 may include any one of the amino acid sequences shown in SEQ ID NOs. 36-37, HCDR2 may include the amino acid sequence shown in SEQ ID NOs. 42, and HCDR3 may include any one of the amino acid sequences shown in SEQ ID NOs. 45-48.

[0152] In this application, the antigen-binding protein comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 may contain the amino acid sequence shown in SEQ ID NO: 64, LCDR2 may contain the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 may contain the amino acid sequence shown in SEQ ID NO: 19.

[0153] For example, the antigen-binding protein may include LCDR1, LCDR2, and LCDR3, where LCDR1 may include any one of the amino acid sequences shown in SEQ ID NOs: 3 to 14, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 19.

[0154] In this application, the antigen-binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 may contain the amino acid sequences shown in SEQ ID NOs. 62, 42, 63, 64, 17, and 19, respectively.

[0155] For example, the antigen-binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1 may include any one of the amino acid sequences shown in SEQ ID NOs. 36-37, HCDR2 may include the amino acid sequence shown in SEQ ID NOs. 42, HCDR3 may include any one of the amino acid sequences shown in SEQ ID NOs. 45-48, LCDR1 may include any one of the amino acid sequences shown in SEQ ID NOs. 3-14, LCDR2 may include the amino acid sequence shown in SEQ ID NOs. 17, and LCDR3 may include the amino acid sequence shown in SEQ ID NOs. 19.

[0156] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 45, 3, 17, and 19, respectively.

[0157] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 46, 4, 17, and 19, respectively.

[0158] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 37, 42, 45, 5, 17, and 19, respectively.

[0159] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 47, 6, 17, and 19, respectively.

[0160] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 45, 7, 17, and 19, respectively.

[0161] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 45, 8, 17, and 19, respectively.

[0162] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 48, 9, 17, and 19, respectively.

[0163] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 45, 10, 17, and 19, respectively.

[0164] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 48, 11, 17, and 19, respectively.

[0165] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 45, 12, 17, and 19, respectively.

[0166] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 47, 12, 17, and 19, respectively.

[0167] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 36, 42, 47, 13, 17, and 19, respectively.

[0168] For example, the antigen-binding proteins may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which may contain the amino acid sequences shown in SEQ ID NOs. 38, 42, 45, 14, 17, and 19, respectively.

[0169] In this application, the antigen-binding protein may include framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, and may include the amino acid sequences shown in SEQ ID NOs. 35, 40, 44, 49, 1, 15, 18, and 21, respectively.

[0170] In this application, the antigen-binding protein may further comprise an antibody heavy chain constant region. In certain cases, the antibody heavy chain constant region may be derived from a human IgG constant region. For example, the human IgG constant region may comprise a human IgG1 constant region. In certain cases, the human IgG1 constant region may comprise one or more amino acid mutations selected from the group consisting of M252Y, S254T, and T256E (Chothia numbering rule). For example, the human IgG1 constant region may comprise amino acid mutations from the following group: M252Y, S254T, and T256E (Chothia numbering rule). In certain cases, the antibody heavy chain constant region of the antigen-binding protein described herein may comprise one of the amino acid sequences shown in SEQ ID NOs. 60-61.

[0171] In this application, the antigen-binding protein may also include an antibody heavy chain. The antibody heavy chain may include a variable region and a constant region.

[0172] In this application, the antigen-binding protein may include an antibody light chain variable region (VL) and an antibody heavy chain constant region (VH). Compared to the amino acid sequence shown in SEQ ID NO: 22, the VL may contain amino acid mutations at one or two positions selected from the group consisting of E55 and V104. Compared to the amino acid sequence shown in SEQ ID NO: 50, the VH may contain amino acid mutations at one or more positions (e.g., 1, 2, 3, 4, or 5) selected from the group consisting of I37, A49, S50, and D54.

[0173] In certain cases, the VL antigen-binding protein may contain amino acid mutations at the following positions: D30b, E55, and V104, and the VH antigen-binding protein may contain amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, and D54.

[0174] For example, the antigen-binding protein VL may contain amino acid mutations at positions D30b, E55, V104, V29, D30 and Y30a in the following groups, and VH may contain amino acid mutations at positions N60, P61, I67, I37, A49, S50, D54, S96 and H97 in the following groups.

[0175] For example, the VL antigen-binding protein of this application may contain amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a, and G30c, and the VH may contain amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54, and T30.

[0176] For example, VL may contain amino acid mutations at positions D30b, E55, V104 and Y30a in the following groups, and VH may contain amino acid mutations at positions N60, P61, I67, I37, A49, S50, D54 and S96 in the following groups.

[0177] For example, VL may contain amino acid mutations at the following positions in the group: D30b, E55, V104 and Y30a, and VH may contain amino acid mutations at the following positions in the group: N60, P61, I67, I37, A49, S50 and D54.

[0178] For example, VL may contain amino acid mutations at the following group positions: D30b, E55, V104, Y30a, and G30c, and VH may contain amino acid mutations at the following group positions: N60, P61, I67, I37, A49, S50, and D54.

[0179] For example, VL may contain amino acid mutations at the following positions in the group: D30b, E55, V104 and Y30a, and VH may contain amino acid mutations at the following positions in the group: N60, P61, I67, I37, A49, S50, D54 and H97.

[0180] For example, VL may contain amino acid mutations at the following group positions: D30b, E55, V104, D30, Y30a, and G30c, and VH may contain amino acid mutations at the following group positions: N60, P61, I67, I37, A49, S50, and D54.

[0181] For example, VL may contain amino acid mutations at the following group positions: D30b, E55, V104, D30, and Y30a, and VH may contain amino acid mutations at the following group positions: N60, P61, I67, I37, A49, S50, D54, and H97.

[0182] For example, VL may contain amino acid mutations at the following group positions: D30b, E55, V104, D30, Y30a, and G30c, and VH may contain amino acid mutations at the following group positions: N60, P61, I67, I37, A49, S50, D54, and S96.

[0183] For example, VL may contain amino acid mutations at the following group positions: D30b, E55, V104, D30, Y30a, and G30c, and VH may contain amino acid mutations at the following group positions: N60, P61, I67, I37, A49, S50, D54, and S31.

[0184] In this application, the species of amino acid mutation at each position may be those described above.

[0185] In this application, the antigen-binding protein VL may contain any one of the amino acid sequences shown in SEQ ID NOs. 23 to 34, and VH may contain any one of the amino acid sequences shown in SEQ ID NOs. 51 to 56.

[0186] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 23, and VH may contain the amino acid sequence shown in SEQ ID NO: 51.

[0187] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 24, and VH may contain the amino acid sequence shown in SEQ ID NO: 52.

[0188] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 25, and VH may contain the amino acid sequence shown in SEQ ID NO: 53.

[0189] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 26, and VH may contain the amino acid sequence shown in SEQ ID NO: 54.

[0190] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 27, and VH may contain the amino acid sequence shown in SEQ ID NO: 51.

[0191] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 28, and VH may contain the amino acid sequence shown in SEQ ID NO: 51.

[0192] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 29, and VH may contain the amino acid sequence shown in SEQ ID NO: 55.

[0193] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 30, and VH may contain the amino acid sequence shown in SEQ ID NO: 51.

[0194] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 31, and VH may contain the amino acid sequence shown in SEQ ID NO: 55.

[0195] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 32, and VH may contain the amino acid sequence shown in SEQ ID NO: 51.

[0196] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 32, and VH may contain the amino acid sequence shown in SEQ ID NO: 54.

[0197] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 33, and VH may contain the amino acid sequence shown in SEQ ID NO: 54.

[0198] For example, VL may contain the amino acid sequence shown in SEQ ID NO: 34, and VH may contain the amino acid sequence shown in SEQ ID NO: 56.

[0199] The antigen-binding protein described in the present application has high chemical stability. For example, it is stable at high temperature, low pH, and / or repeated freezing and thawing. For example, at 40°C for 0 to 28 days, or at pH 5.0 and 40°C for 0 to 28 days, the antigen-binding protein of the present application produces at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30% or more) less aggregation compared to omalizumab (e.g., detected by size exclusion high performance liquid chromatography SEC-HPLC). For example, at 40°C for 0 to 28 days, or at pH 5.0 and 40°C for 0 to 28 days, the main peak content of the antigen-binding protein described herein is at least 5% (e.g., at least 10%, 15%, 20% or more) higher than that of omalizumab (e.g., by total column imaging capillary isoelectric focusing (iCIEF detection)).

[0200] The antigen-binding protein described in the present specification, for example, with respect to binding to human IgE CH3-CH4, is less than 5×10 -10 M (e.g., 4×10 -10 M, 3×10 -10 M, 2×10 -10 M, 2.8×10 -10 M, 2.6×10 -10 M, 2.4×10 -10 M, 2.2×10 -10 M, 2.0×10 -10 M, 1.8×10 -10 M, 1.6×10 -10 M, 1.4×10-10 M, 1.2 × 10 -10 M, 1.0×10 -10 (Less than M or smaller) K D Depending on the value; in another example, for binding to human full-length IgE, (detected by Octet Red, for example) 2 × 10 -9 Less than M (for example, 1.5 × 10) -9 M, 1.2 × 10 -9 M, 1×10 -9 (Less than M or smaller) K D Depending on the value; in yet another example, for binding to full-length human IgE, (detected by Biacore, for example) 5 × 10 -9 Less than M (for example, 4.8 × 10) -9 M, 4.6×10 -9 M, 4.4×10 -9 M, 4.2×10 -9 M, 4.0×10 -9 M, 3.8×10 -9 (Less than M or smaller) K D Depending on the value, it has a high binding affinity to human IgE or its fragments.

[0201] The antigen-binding protein described in this application, for example, binds to IgE CH3-CH4 in cynomolgus monkeys, with a detection rate of 3.0 × 10⁻⁶ (detectable by, for example, Octet Red). -10 Less than M (for example, 2.5 × 10) -10 M, 2.4×10 -10 M, 2.3×10 -10 M, 2.2 × 10 -10 M, 2.1 × 10 -10 M, 2.0×10 -10 M, 1.9×10 -10 M, 1.8×10 -10 (Less than M or smaller) K D Depending on the value; in another example, for monkey IgE binding to CH3-CH4, (detected by Biacore, for example) 4.0 × 10 -9 Less than M (for example, 3.9 × 10) -9 M, 3.8×10 -9 M, 3.7×10 -9 M, 3.6×10 -9 M, 3.5×10-9 (Less than M or smaller) K D Depending on the value, it can bind to monkey IgE with high affinity, facilitating pharmacodynamic studies in preclinical animal models.

[0202] The antigen-binding proteins described in this application, for example, block the binding of full-length human IgE to FcεRI, have an EC level of less than 1.5 nM (e.g., less than 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1.0 nM, 0.9 nM or less) (detectable by ELISA, for example) 50 Depending on the value; in another example, for blocking IgE-stimulated histamine release from FcεRI-expressing cells, less than 4.0 nM (e.g., less than 3.9 nM, 3.8 nM, 3.7 nM, 3.6 nM, 3.5 nM, 3.4 nM, 3.3 nM, 3.2 nM, 3.1 nM, or less than 3.0 nM) EC 50 Depending on the value, it is possible to effectively block the binding of IgE to the IgE Fc receptor (FcεRI).

[0203] The antigen-binding proteins of this application have a long half-life in the blood, which can reduce the frequency of injections and facilitate the regulation of the patient's disease. For example, the half-lives of the antigen-binding proteins described herein are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more higher than those of omalizumab, when calculated by free IgE concentration using non-compartment model analysis or ELISA assay.

[0204] nucleic acid molecules, vectors, and cells This application also provides isolated nucleic acid molecules encoding the heavy and / or light chains of the antigen-binding protein of this application. Preferably, the nucleic acid molecules encode the heavy or light chains of the antigen-binding protein of this application. The nucleic acid molecules of this application may include, for example, synthetic DNA, cDNA, genomic DNA, or any combination thereof, produced by chemical processing.

[0205] The nucleic acid molecules encoding the antigen-binding proteins of this application can be obtained by methods well known to those skilled in the art. For example, nucleic acid molecules encoding some or all of the antibody heavy and light chains can be synthesized, if desired, from assayed nucleic acid molecules or based on corresponding amino acid sequences. The nucleic acid molecules encoding the antigen-binding proteins of this application can be prepared using standard techniques of molecular biology. The desired nucleic acid molecules can be synthesized completely or partially using oligonucleotide synthesis techniques. Where appropriate, site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used.

[0206] This application also relates to a cloning or expression vector comprising one or more nucleic acid molecules of this application. Accordingly, this application provides a cloning or expression vector comprising one or more nucleic acid molecules encoding the antigen-binding protein of this application. Alternatively, the cloning or expression vector may comprise two nucleic acid molecules, each encoding the light chain and heavy chain of the antigen-binding protein of this application, and a preferred signal sequence.

[0207] This application also provides host cells comprising one or more cloning or expression vectors comprising one or more nucleic acid molecules encoding the antigen-binding proteins of this application. Any suitable host cell / vector system may be used to express the nucleic acid molecules encoding the antigen-binding proteins of this application. Bacteria, e.g., Escherichia coli (E. coli) and other microbial systems may be used, or eukaryotic, e.g., mammalian host cell expression systems may also be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells.

[0208] Preparation method This application also provides a method for producing the antigen-binding protein described herein, comprising the step of culturing cells under conditions that enable the expression of the antigen-binding protein. The antigen-binding protein may comprise only heavy-chain or light-chain polypeptides, in which case only the heavy-chain or light-chain coding sequence may be transfected into host cells. To produce a product comprising both heavy-chain and light-chain polypeptides, the cell line may be transfected with two vectors, the first vector encoding a light-chain polypeptide and the second vector encoding a heavy-chain polypeptide. Alternatively, one vector may be used, and the vector may comprise sequences encoding both light-chain and heavy-chain polypeptides.

[0209] This application provides a method for culturing host cells, and for expressing, isolating, and purifying antigen-binding proteins or fragments thereof.

[0210] One embodiment provides a method for purifying antigen-binding proteins, comprising the step of performing anion exchange chromatography in unbound mode so that impurities are retained on the column and antibodies are eluted.

[0211] In one example, purification can be performed by affinity capture on a protein A column followed by titration. In another example, purification can be performed by affinity capture on a protein G column followed by HPLC titration. In yet another example, purification can be performed by affinity capture on an IgE column followed by titration. The purification method may also include one or more filtration steps, such as a diafiltration step or an HPLC filtration step.

[0212] Pharmaceutical compositions, methods and uses Since the antigen-binding protein of this application is suitable for the treatment and / or prevention of pathological conditions, this application also provides pharmaceutical or diagnostic compositions comprising the antigen-binding protein of this application in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers. Accordingly, this application also provides the use of the antigen-binding protein of this application in the preparation of pharmaceutical compositions.

[0213] This application also provides a method for preparing a pharmaceutical or diagnostic composition, comprising the step of adding and mixing the antibody or antigen-binding agent of this application together with one or more pharmaceutically acceptable excipients, diluents, or carriers. The antigen-binding protein may contribute as the sole active ingredient in the pharmaceutical or diagnostic composition, or it may be accompanied by other active ingredients, including other antibody or non-antibody components, such as steroids or other drug molecules, in particular drug molecules whose half-live is not associated with IgE binding.

[0214] The compositions may be administered to the patient individually or in combination with other drugs, medications, or hormones (for example, simultaneously, sequentially, or separately).

[0215] On the other hand, this application provides the use of antigen-binding proteins, nucleic acid molecules, vectors, cells and / or compositions in the preparation of pharmaceuticals for alleviating or treating diseases associated with abnormal IgE levels.

[0216] In another embodiment, the present application provides a method for alleviating or treating a disease associated with abnormal levels of IgE, comprising the step of administering an antigen-binding protein, nucleic acid molecule, vector, cell and / or composition to a subject in need thereof.

[0217] A pharmaceutical composition may contain a therapeutically effective dose of the antibody or antigen-binding protein of this application. As used herein, the term “therapeutic dose” refers to the amount of therapeutic agent required to treat, alleviate or prevent a target disease or condition, or to exhibit a detectable therapeutic or prophylactic effect. For any disclosed antibody or antigen-binding protein, the therapeutic dose may first be evaluated in a cell culture assay or an animal model, typically rodents, rabbits, dogs, pigs, or primates. Animal models may also be used to determine appropriate concentration ranges and routes of administration. This information may then be used to determine suitable doses and routes for administration to humans. The exact therapeutic dose for human subjects will depend on the severity of the disease condition, the subject’s overall health, the subject’s age, weight and sex, diet, timing and frequency of administration, combination, sensitivity of response, and tolerance / response to treatment.

[0218] The pharmaceutical compositions of this application may be administered by various routes, including, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, vaginal, or rectal routes. The pharmaceutical compositions of the present invention may also be administered using needle-free injection devices. Typically, the therapeutic compositions are prepared for injection as solutions or suspensions. Solid forms suitable for solution or suspension in a liquid vehicle before injection may also be prepared.

[0219] The therapeutic doses of the pharmaceutical composition containing the antigen-binding protein of this application do not exhibit significant toxicological effects in vivo. Furthermore, continuous doses of the antigen-binding protein of this application can maintain the level of IgE activity in the subject at an appropriate normal (e.g., reduced) level.

[0220] Without being limited by any theory, the following examples are intended solely to illustrate the fusion protein, preparation method, and use of the present invention, and are not intended to limit the scope of the invention. [Examples]

[0221] [Example 1] Design and expression of an optimized modified omalizumab antibody. Based on the light and heavy chain variable regions of omalizumab (the light chain variable region sequence is shown in SEQ ID NO: 22, and the heavy chain variable region sequence is shown in SEQ ID NO: 50), optimization design, humanization mutations in the light and heavy chains, and removal of post-translational modification (PTM) sites are performed to obtain new light and heavy chain variable regions.

[0222] Next, the light and heavy chain protein sequences of the full-length recombinant antibody were codon-optimized, and a codon-optimized DNA fragment (Genscript) was genetically synthesized. After cloning, the synthesized gene fragment having the morphology of IgG1 was inserted into the expression vector pcDNA3.4 (Life Technologies). After amplifying and extracting the expression plasmid, the two plasmids were cotransfected into ExpiCHO cells (ThermoFisher Scientific, A29133). Then, according to the manufacturer's ExpiCHO expression system method, the ExpiCHO cells were expressed in 25 ml of culture medium at 8% carbon dioxide concentration and 36.5°C for 6 × 10⁶ cells. 6The cells were incubated to a density of 100 cells / mL, and the antibodies were transiently expressed by transforming the cells with 10 μg each of antibody light chain and heavy chain expression plasmids using ExpiFectamine transfection reagent; one day after transfection, 150 μL and 4 mL of ExpiCHO enhancer and ExpiCHO auxiliary material were added to the cultured cells, and the cells were cultured for 9 days. The supernatant was then obtained by centrifugation at 3500 rpm at 4°C. After mixing AmMag™ Protein A magnetic beads (Genscript, L00695) and antibody expression supernatant, the mixture was incubated at room temperature for 2 hours. The supernatant was washed twice with PBS before discarding, and then an appropriate amount of elution buffer Protein G or A Sefinose™ Elution Buffer (Sangon, C600481) was added. The mixture was thoroughly mixed before placing it in a tube rack for 5 minutes of incubation. The magnetic beads were resuspended 2-3 times during incubation, and then eluted twice immediately before adding an appropriate amount of neutralization solution 1M Tris-HCl, pH 7.5 (Sangon, B548124) for neutralization and preservation. The purified antibodies obtained are shown in Table 7 (AB1904HzL0H0 indicates omalizumab).

[0223] [Table 7]

[0224] [Example 2] Human IgE affinity assay The antibodies were first tested for affinity with biotinylated human IgE CH3-CH4 (Biotin-hIgE CH3-CH4, Sino Biological, catalog number 29702-H08H, biotin-labeled in-house), and then sorted by the off-rate using the following experimental process. (1) Dilution of antigen and antibody: Both the antigen and antibody were diluted with 1× Kinetic Buffer (Kinetic Buffer, Fortebio, 18-1105) to a concentration of 1 μg / ml for the antigen and 100 nM for the antibody for use. (2) Detection of samples using an instrument (Octet Data Acquisition 11.1.0.11): First, samples were added to a 96-well plate (Greiner bio-one, 655209) at 200 μL / well according to the table below. Next, the software parameters were set, the plate temperature was set to 30°C, and the standard dynamic signal frequency was collected at 5.0 Hz. Then, the CH1 sensor (Fortebio, 18-0039) was pre-wet with 1×PBST for 10 minutes and then tested with the instrument. Each cycle includes the following steps: 1) Immerse in buffer for 60 seconds; 2) Detect whether the antigen binds nonspecifically to the sensor; 3) Regenerate with glycine solution at 10 mM pH 1.7; 4) Immerse in buffer for 60 seconds; 5) Immobilize antibody on the sensor for 30 seconds; 6) Immerse the sensor in buffer for 180 seconds; 7) Antigen-antibody association for 180 seconds; 8) Antigen-antibody dissociation for 10 minutes; 9) Regenerate the sensor. (3) Data analysis: Using Fortebio's Data Analysis 11.1 software, the aggregation rate (K) of 1:1 bound antigen-antibody was analyzed. a ) and dissociation rate (K d ) is measured, and the equilibrium dissociation constant (K) of the antibody is measured. D The result is shown in Table 2.

[0225] [Example 3] Detection of the physical and chemical properties of antibodies 3.1 SEC-HPLC purity analysis (1) The sample was diluted to 1 mg / mL, thoroughly mixed, and centrifuged at 12000 rpm for 5 minutes to obtain the supernatant, which was then transferred to a sample bottle and placed on an HPLC sample tray. The chromatographic conditions were set as follows:

[0226] [Table 8] (2) After equilibrating the column with the mobile phase (200 mM phosphate buffer, pH 6.8), the sample was injected and analyzed. The data was analyzed using chromatography software, and the peak area percentage of each peak was calculated using the peak area normalization method.

[0227] 3.2 HIC-HPLC analysis (1) The sample was diluted to 1 mg / mL, thoroughly mixed, and centrifuged at 12000 rpm for 5 minutes to obtain the supernatant, which was then transferred to a sample bottle and placed on an HPLC sample tray. The chromatographic conditions were set as follows:

[0228] [Table 9] (3) Gradient elution analysis was performed using mobile phase A (50 mM phosphate buffer / 1 M ammonium sulfate, pH 7.0) and mobile phase B (50 mM phosphate buffer, pH 7.0), and data analysis was performed using chromatography software. The hydrophilicity coefficient of each sample was calculated based on the retention time.

[0229] 3.3 Analysis of Melting Temperature (Tm) Values Following the instructions for the Protein Thermal Shift™ Starter Kit (catalog number 4461146), the sample to be tested was diluted to 1 mg / mL with sample buffer. Then, 13 μL of the test solution was added to a PCR tube, followed by the addition of 5 μL of Protein Thermal Shift™ Buffer and 2 μL of 10× Staining solution to bring the reaction volume to 20 μL. Before mixing, the mixture was centrifuged at 12000 rpm for 5 minutes to remove air bubbles. The sample to be tested was placed in a PCR instrument, analyzed, and the Tm value of the sample was recorded.

[0230] 3.4 ICIEF analysis The sample solution was added to the following well-mixed system: 70 μL of 1% methylcellulose (catalog no. 101876), 4M urea (catalog no. U6504), 8 μL of the amphoteric electrolyte Pharmalyte pH 3-10 (catalog no. 17-0456-01), and 2 μL each of pI markers 5.5 (catalog no. A58325) and 9.5 (catalog no. A358357). An appropriate volume of ultrapure water was added to make 200 μL, and the mixture was thoroughly mixed. All samples were centrifuged at 6000 rpm for 3 minutes to remove air bubbles, and the supernatant was then transferred to a sample bottle, placed on a sample tray, and the sample position was recorded. After the start of the procedure, the result files were imported into Chrom Perfect software for spectral integration and calculation of the isoelectric point and percentage of each peak. The physical and chemical test results of the antibodies are shown in Table 8.

[0231] [Table 10]

[0232] The results show that AB1904L1p1H3p2 (light chain variable region: SEQ ID NO: 23, heavy chain variable region: SEQ ID NO: 51) has higher affinity, simultaneously removes potential post-translational modification sites, and further improves chemical stability.

[0233] [Example 4] Yeast affinity maturation for novel antibodies AB1904L1P1H3P2 was selected for yeast affinity maturation, and AB1904Am15 was obtained from the antibody AB1904Am1 shown in Table 9. The YTE(M252Y / S254T / T256E) modification mutation was performed on the Fc fragment, and these were named AB1904Am13, AB1904Am14, and AB1904Am15, respectively.

[0234] [Table 11] The physical and chemical properties of the antibodies in Table 9 were tested using the method of Example 3, and the results are shown in Table 10.

[0235]

Table 12

[0236] [Example 5] The antibody binds to human IgE CH3-CH4, human full-length IgE, and cynomolgus monkey IgE CH3-CH4 (detected by Octet Red). 5.1 The antibodies described herein can bind to human IgE CH3-CH4. The affinity of antibodies AB1904Am1 to AB1904Am12 for human IgE CH3-CH4 (hIgE CH3-CH4, Sino Biological, Catalog No.: 29702-H08H) was determined by Octet RED96e (Fortebio). Both the antigen and the antibodies were diluted using 1×PBST (1×PBS: Sango, B548117-0500; 0.02% Tween 20: sigma-alorich, P1379). The antigen was used at a concentration of 50 nM, and the antibodies were used at a concentration of 33.3 nM. On-machine detection was performed according to the method of Example 2. The results are shown in Table 11, indicating that the antibodies have high affinity for human IgE CH3-CH4 and that the K D values are similar to or lower than those of omalizumab.

[0237]

Table 13

[0238] 5.2 The antibodies described herein can bind to human full-length IgE. The affinity of the antibodies for human full-length IgE (FL hIgE, ABBIOTEC, Catalog No. 250205) was determined according to the method of Example 2. The results are shown in Table 12. All the antibodies can bind to human full-length IgE with high affinity.

[0239]

Table 14

[0240] 5.3 The antibodies described herein can bind to cynomolgus monkey IgE CH3-CH4. The affinity of the antibody with biotinylated cynomolgus IgE CH3-CH4 (Biotin - cyno IgE CH3-CH4, Kactus Biosystems) was tested according to the method of Example 2, and the results are shown in Table 13. All antibodies were able to bind to biotinylated cynomolgus IgE CH3-CH4 with high affinity.

[0241]

Table 15

[0242] [Example 6] The antibody binds to human full-length IgE and cynomolgus monkey IgE CH3-CH4 (detected by Biacore). First, the Biotin CAPTure reagent was captured on a CAP chip (GE Healthcare, catalog number 28-9202-34) at a flow rate of 2 μL / min for 300 seconds. HBS-EP+ was used as the experimental buffer. The binding of a specific concentration of antibody to the captured antigen was determined for each injection cycle, and each cycle included antigen capture, injection of different concentrations of antibody, and regeneration. 2 μg / ml of biotinylated human full-length IgE (Biotin-FL hIgE, Abbiotec, catalog number 250205, biotinylated by the inventors themselves) or 0.25 μg / ml of biotinylated cynomolgus IgE CH3-CH4 (Biotin-cyno IgE, customized by Kactus Biosystems) was captured in two channels at a flow rate of 10 μL / min for 60 seconds, and one channel was used as a blank reference channel. Serial diluted antibodies (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0 nM) were injected into channels 1 and 2 in sequence at a flow rate of 30 μL / min, and association and dissociation were determined. Chip regeneration was performed with a regeneration buffer (6 M guanidine hydrochloride, 0.25 M sodium hydroxide) at a flow rate of 10 μL / min to remove the captured antigen and antibody. The data was analyzed using Biacore 8K analysis software. The model used for software analysis was for 1:1 binding.

[0243] The results shown in Table 14 indicate that all antibodies described in this application are not significantly different from or less different from omalizumab. D The values ​​indicate that it was able to bind to both biotinylated human full-length IgE and cynomolgus monkey IgE CH3-CH4.

[0244] [Table 16]

[0245] [Example 7] The antibody binds to human and cynomolgus monkey FcRn (detected by Octet Red). The affinity of antibodies to human FcRn (Acro, FCN-H52W7) and cynomolgus monkey FcRn (Acro, FCM-C52W9) was determined using Octet RED96e (Fortebio) according to the method of Example 2. Here, human FcRn and cynomolgus monkey FcRn were each diluted to 5 μg / ml with 1×PBS (10 mM Na2HPO4.12H2O, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 6.0), and the biotin-conjugated THE™ His-tagged mouse monoclonal antibody was diluted to 2 μg / ml; the antibody to be tested was diluted with 1×PBST (1xPBS, 0.02% Tween 20, pH 6.0), with antibody dilution concentrations ranging from 1000 nM to 31.3 nM. The results are shown in Table 15. All antibodies were able to bind to cynomolgus monkey and human FcRn.

[0246] [Table 17]

[0247] [Example 8] The antibody blocks the binding of biotinylated human full-length IgE to FcεRI. FcεRIα (R&D, 6678-Fc) was diluted to 0.5 μg / ml in enzyme-labeled plates coated with PBS at 100 μl / well. Sealing film was applied before incubation at 37°C for 1 hour, and the plates were then washed three times with a washing solution (PBS + 0.05% TWEEN-20). A blocking solution (PBS + 0.05% TWEEN-20 + 2% BSA) was then prepared using the washing solution, and 300 μl of the blocking solution was added to each well. The plates were incubated at 37°C for 1 hour. Biotinylated human full-length IgE (Biotin-FL hIgE, Abbiotec, catalog number 250205, biotinylated in-house) at a concentration of 160 ng / ml was prepared in blocking solution; then, anti-IgE antibody was prepared in blocking solution, starting at an initial concentration of 4 μg / ml, by 3-fold serial dilutions (dilution to 7 concentration points + 1 0 concentration point); then, 60 μL of anti-IgE antibody was added to 60 μL of Biotin-FL hIgE and incubated at 37°C for 2 hours. The blocked ELISA plates were then removed, washed three times with washing solution, and the anti-IgE antibody and biotinylated human full-length protein complex solution, pre-incubated at 37°C, was added to 100 μL / well, and then incubated at 37°C for 1 hour. The plate was washed three times with washing solution, SA-HRP (sigma, S2438-250UG) was diluted to 1 / 5000 with blocking solution and added to the ELISA plate at 100 μl / well, and incubated at 37°C for 1 hour; the plate was washed three times with washing solution, then 100 μl / well of TMB chromogenic solution (Biopanda, product number: TMB-S-003) was added, and after reaction at room temperature in a dark room for 10 minutes, 50 μl / well of stop solution (Solarbio, product number: C1058) was added to stop the reaction before determining the absorbance value at a wavelength of 450 nm. The results shown in Figure 2 indicate that the antibody described in this application can block the binding of biotinylated IgE to FcεRI, and EC 50 This indicates that the value is lower than that of omalizumab.

[0248] [Example 9] The antibody blocks the binding of biotinylated human full-length IgE to CD23 on the surface of IM-9 cells. IM-9 cells were cultured at 37°C under 5% carbon dioxide using RPMI1640 + 10% FBS (RPMI1640, FBS, Thermo Fisher). The IM-9 cells were harvested, washed once with FACS buffer (PBS + 2% FBS), and 2.5 × 10⁶ cells were collected. 6 The cells were resuspended in FACS buffer solution at a cell density of 100 cells / ml. The cell suspension (100 μl per well) was added to a U-bottom 96-well cell culture plate and then refrigerated at 4°C for 30 minutes. Biotinylated human full-length IgE (hIgE, ABBIOTEC, catalog no. 250205, biotinylated in-house) was prepared in FACS buffer solution to a concentration of 20 μg / ml; then anti-IgE antibody was prepared using FACS buffer at a starting concentration of 60 μg / ml in 3-fold serial dilutions (diluted to 7 concentration points + 1 0 concentration point); then 60 μL of anti-IgE antibody was added to 60 μL biotin-FL hIgE and incubated at 4°C for 1 hour. Simultaneously, 2.5 μl of Fc Block (BD Pharmingen, catalog number 564220) was added to the cells in each well and incubated at 4°C for 1 hour; the 96-well cell culture plate was removed from the refrigerator at 4°C, centrifuged at 2000 rpm for 5 minutes before discarding the supernatant, then 100 μl / well of the pre-incubated anti-IgE antibody and biotinylated human full-length IgE protein complex solution was added, incubated at 4°C for 1 hour, centrifuged at 2000 rpm for 5 minutes, discarded the supernatant, and washed three times with FACS buffer solution. FITC-conjugated streptavidin (Invitrogen, catalog number SA1001) was diluted 1:500, and 100 μL was added to each well. The cells were incubated at 4°C for 1 hour, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded before washing three times with FACS buffer solution. The cells were then diluted 1:2 with FACS buffer solution and detected using a Guava easyCyte 6-2L Benchtop Flow Cytometer. The results shown in Figure 3 demonstrate that the antibody described in this application was able to block the binding of biotinylated human full-length IgE to CD23 on the surface of IM-9 cells.

[0249] [Example 10] The antibody blocks histamine release induced by biotinylated human full-length IgE stimulation from FcεRI / RBL-2H3. FcεRI / RBL-2H3 cells (constructed by Genechem gene) were cultured in FcεRI / RBL-2H3 medium (MEM + 15% FBS + 1 mM sodium pyruvate + 1 μg / ml Puromycin) at 37°C under 5% carbon dioxide conditions. Human full-length IgE protein (ABBIOTEC, catalog no. 250205) at a concentration of 2.4 μg / ml was prepared using cell culture medium; then anti-IgE antibody was prepared using cell culture medium, starting at a concentration of 15 μg / ml and undergoing 2-fold serial dilutions (9 concentration points + 1 zero concentration). 50 μL of human full-length IgE protein was first added to a 96-well flat-bottom cell culture plate, followed by 50 μL of the prepared anti-IgE antibody. Simultaneously, FcεRI / RBL-2H3 cells were harvested and 2 × 10⁶ cells were added. 6 The cells were resuspended in culture medium at a cell concentration of 10 cells / ml and added to the corresponding 96-well flat-bottom cell culture plate at a rate of 50 μl / well. The cell culture plate was then incubated overnight in a cell incubator at 37°C under 5% carbon dioxide. The following day, after aspirating and discarding the supernatant, the cells were washed twice with assay buffer (Tyrode's buffer (130mM NaCl, 5mM KCl, 10nM HEPES, 1.4mM CaCl2, 1mM MgCl2.6H2O, 5.6mM Glucose, 0.1% BSA), pH 7.4) and 200 μl of 1 μg / ml polyclonal anti-IgE antibody (R&D, catalog number: G-107-C) was added to each well before incubation for 30 minutes in a cell incubator at 37°C and 5% carbon dioxide; the supernatant was aspirated and detected using the Histamine Dynamic kit (CISBIO, product number: 62HTMDPEG) according to the detection method based on the kit instructions. The results are shown in Figures 4A and 4B, and the antibody was able to inhibit FL hIgE-induced histamine release from induced FcεRI / RBL-2H3 and IC 50 The values ​​are lower than those of omalizumab, indicating that the inhibitory effect is superior to that of omalizumab.

[0250] [Example 11] Human whole blood histamine release assay Volunteers should not take any allergenic drugs, antihistamines, oral corticosteroids, or any substances that block H2 receptors 24 hours before blood donation. Whole blood was treated with heparin as an anticoagulant.

[0251] Histamine release was performed according to the operating procedure of the kit (Sigma, catalog number: IB89145) as follows: Before use, the antibodies to be tested were diluted to 6000, 600, 60, and 6 nM with PBS, and the positive control anti-IgE serum in the kit was diluted 1000-fold with the histamine release buffer solution. The loading samples and operations for performing the histamine release process are shown in Table 16 below.

[0252] [Table 18]

[0253] The histamine ELISA was performed according to the procedure of the kit (Sigma, catalog number: IB89128) as follows: 50 μl of supernatant or standard and control material was diluted 1-fold with deionized water and added to the corresponding wells. Then, 25 μl of acetylation buffer and 25 μl of acetylation solution were added to each well, and the mixture was shaken at 600 rpm for 45 minutes at room temperature. Finally, 100 μl of deionized water was added, and the mixture was shaken at 600 rpm for 15 minutes at room temperature. After acetylation, 25 μl of the sample was added to a histamine-coated plate, followed by the addition of 100 μl of antihistamine serum, and then the plate was sealed with sealing film before shaking at 600 rpm for 3 hours at room temperature. After incubation, the liquid in the well plate was discarded, and then 100 μl of enzyme-conjugated secondary antibody was added to each well. Subsequently, 300 μl of washing solution was added to each well for four washes before shaking at 600 rpm for 30 minutes at room temperature. The liquid in the well plate was discarded, and then 100 μl of substrate was added to each well. Subsequently, 300 μl of washing solution was added to each well for four washes before shaking at 600 rpm for 30 minutes at room temperature. Finally, 100 μl of stop solution was added to stop the reaction, and absorbance values ​​at 450 nm and 570 nm were collected within 10 minutes using an Envision microplate reader (PerkinElmer).

[0254] The results are shown in Figure 5, indicating that anti-IgE serum can crosslink or activate IgE bound to FcεRI, inducing histamine release, whereas omalizumab, AB1904A m10, and AB1904A m15 did not.

[0255] [Example 12] Antibody stability test The stability of candidate antibodies is comprehensively evaluated under high temperature, low pH, and repeated freeze-thaw stability experimental conditions. Key stability test parameters primarily focus on critical product purity indicators, including SEC-HPLC, iCIEF, and non-reducing CE-SDS.

[0256] 11.1 High-temperature stability test This study focuses on investigating the stability of candidate antibodies under high-temperature conditions. Samples were placed under experimental conditions of 40±2°C, and SEC-HPLC purity, charge isomer purity, and nrCE-SDS purity were detected on days 0, 3, 7, 14, 21, and 28. The experimental results are as follows:

[0257] [Table 19]

[0258] 11.2 Low pH Stability Test The stability of candidate antibodies under low pH conditions was primarily investigated. The pH of the samples was adjusted to 5.0 using hydrochloric acid, and the samples were placed at 40±2°C. SEC-HPLC purity, charge isomer purity, and nrCE-SDS purity were detected at 0, 6, 24, 48, and 72 hours, respectively. The experimental results are as follows:

[0259] [Table 20]

[0260] 11.3 Repeated freeze-thaw stability test The stability of candidate antibodies under repeated freeze-thaw conditions was primarily investigated. The cycle was defined as the period from freezing the sample at -70±10°C to thawing at room temperature. Samples were tested under 3 and 6 cycles, respectively. The experimental results are as follows:

[0261] [Table 21]

[0262] The high-temperature stability, low-pH stability, and repeated freeze-thaw stability of AB1904Am10, AB1904Am15, and omalizumab were tested. The results of the high-temperature and low-pH stability tests showed that the candidate antibodies AB1904Am10 and AB1904Am15 were more stable than omalizumab under high-temperature and low-pH conditions. In particular, the reduced amount of aggregation in SEC-HPLC detection and the reduction of the major peak in iCIEF detection indicate that candidate antibodies AB1904Am10 and AB1904Am15 are more tolerant and stable to temperature and low pH, thereby being more stable during production, storage, and use. The results of the repeated freeze-thaw stability tests showed that all important quality indicators for purity of AB1904Am10 and AB1904Am15 were sufficiently stable after six repeated freeze-thaw cycles.

[0263] [Example 13] Pharmacokinetic (PK) studies in a humanized FcRn mouse model Humanized FcRn mice were used as test animals to evaluate the pharmacokinetics of three test drugs, omalizumab, AB1904Am10, and AB1904Am15, after a single subcutaneous administration. All animal experiment protocols were reviewed and approved by IACUC. hFcRn mice were purchased from Beijing Biositu, were male, 6-8 weeks old, weighing 23-26 g, and housed in an SPF animal room, fed a standard pellet diet, with free-range feeding, at room temperature of 18-24°C, relative humidity of 40-50%, and a 12-hour day-night cycle. A total of 12 experimental animals were randomly divided into three groups of four, and each group received a single subcutaneous dose of 10 mg / kg, with a total dose volume of 10 mL / kg. Blood samples were collected before administration, and at 2, 6, 24 hours (1 day), 2, 3, 4, 7, 10, 14, 21, 28, 35, and 42 hours after administration. 60 μL of whole blood was collected by cheek puncture into an EP tube, left upright at room temperature for 30 minutes, and then centrifuged (2000 g, 4°C, 5 minutes) to separate the serum. Each sample was divided into two parts (test tube and backup tube), 10 μL / tube, and stored at -80°C.

[0264] The pharmacokinetics of three drugs at different time points were analyzed using the Elisa indirect method. The coating antigen was anti-human IgG1 antibody (Fc-specific) (Abeam, catalog number: ab1927), 2 μg / ml, 100 μl / well, 37°C, 2 hours. After washing the plates, they were incubated overnight at 4°C with 200 μl / well of block solution. Serum samples were added at 50 μl / well, 37°C, 1 hour. The detection antibody was mouse anti-human Fab HRP (1:10000) (Abcam, catalog number: ab87422), 100 μl / well, 37°C, 0.5 hours. Staining was performed using TMB dye solution (KPL, catalog number: 52-00-03), and OD450 values ​​were collected using a microplate reader (Molecular Devices, SpectraMax M3). Drug concentrations were obtained according to a standard curve, and PK parameters were obtained by processing the data using a PK Solver non-compartment model.

[0265] The results are shown in Figure 6. After administration of omalizumab, the half-life (t1 / 2) was 10.9 days, and the peak concentration (Cmax) was 53214.1 ng / ml; the AUC was 900661 ng / ml × d; after administration of AB1904AM10, the half-life (t1 / 2) was 13.5 days, and the peak concentration (Cmax) was 60441.4 ng / ml; the AUC was 1220787 ng / ml × d; after administration of AB1904AM15, the half-life (t1 / 2) was 26.6 days, and the peak concentration (Cmax) was 57355.7 ng / ml; the AUC was 1284785 ng / ml × d.

[0266] [Example 14] Pharmacokinetic-pharmacodynamic (PK / PD) studies in cynomolgus monkey models The pharmacokinetics and pharmacodynamics of three test drugs, omalizumab, AB1904Am10, and AB1904Am15, after single subcutaneous administration were investigated using cynomolgus monkeys as the control animals.

[0267] All animal experiment protocols were reviewed and approved by IACUC. The nine SPF-grade male cynomolgus monkeys used in the experiments were 4 years of age or older, weighing 4.1–5.7 kg, and were housed in an SPF-grade animal room with free-feeding access. The dose was 10 mg / kg; the route of administration was subcutaneous injection; the volume of administration was calculated according to the specific concentration of the drug. Cynomolgus monkeys were divided into three groups of three TAs (Triangulation Areas); for administration: the dose volume was calculated based on body weight, the drug was injected subcutaneously as a single dose, and the administration time was recorded; for sampling times: before administration, 2 hours, 6 hours, 24 hours (1 day), 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, and 56 days after administration, 300 μL of whole blood was collected from the cephalic vein and saphenous vein into EP tubes for each time, and the blood collection time was recorded; for sample processing: the whole blood was left upright at room temperature for 1 hour, then centrifuged at 6000 g, 25°C, for 10 minutes to separate the serum, and each sample was divided into 5 parts (1 test tube and 4 backup tubes), 30 μL / tube, and then stored at -80°C.

[0268] The pharmacokinetics of three drugs at different time points were analyzed using the Elisa indirect method. The coating antigen was anti-human IgG1 antibody (Fc-specific) (Abeam, catalog number: ab1927), 2 μg / ml, 100 μl / well, 37°C, 2 hours. After washing the plates, they were incubated overnight at 4°C with 200 μl / well blocking solution. Serum samples were added at 50 μl / well, 37°C, for 1 hour. The detection reagent was human IgE-Biotin (Abbiotec, catalog number: 250205, biotinylation labeled by the inventors themselves) + SA-HRP (sigma, catalog number: S2438-250UG), added at 100 μl / well, 37°C, for 0.5 hours. The samples were colored using a TMB dye solution (KPL, catalog number: 52-00-03), and values ​​at OD450 were collected using a microplate reader (Molecular Devices, SpectraMax M3). Drug concentrations were obtained according to a standard curve, and PK parameters were obtained by processing the data using a PK Solver non-compartment model.

[0269] The results are shown in Figure 7. After administration of omalizumab, the half-life (t1 / 2) was 6.35 days, the peak concentration (Cmax) was 112493 ng / ml, and the AUC was 1704601 ng / ml × d; after administration of AB1904AM10, the half-life (t1 / 2) was 11.15 days, the peak concentration (Cmax) was 83940 ng / ml, and the AUC was 1324493 ng / ml × d; after administration of AB1904AM15, the half-life (t1 / 2) was 12.2 days, the peak concentration (Cmax) was 142880 ng / ml, and the AUC was 3061056 ng / ml × d.

[0270] Next, free IgE in serum was detected by the Elisa indirect method. The coated antigen was AB1904Am15, 0.5 μg / ml, 100 μl / well, and incubated overnight at 4°C. After washing the plates, they were incubated at 37°C for 1 hour with 200 μl / well blocking solution. Serum samples were added at 50 μl / well at 37°C for 1.5 hours. 500 ng / ml biotin-labeled omalizumab was added at 100 μL per well for 1 hour; then SA-HRP (1:10000, sigma, catalog number: S2438-250UG) was added at 100 μl / well at 37°C for 1 hour. Stain development was performed using TMB dye solution (KPL, catalog number: 52-00-03), and the OD450 was read using a microplate reader (Molecular Devices, SpectraMax M3). Free IgE concentrations were obtained according to a standard curve.

[0271] The results are shown in Figure 1. After administration of omalizumab, the recovery time to the initial free IgE concentration was approximately 21 days; after administration of AB1904Am10, the recovery time to the initial free IgE concentration was approximately 28 days; and after administration of AB1904Am15, the recovery time to the initial free IgE concentration was approximately 42-56 days.

[0272] SEQUENCE LISTING <110> SHANGHAI JEMINCARE PHARM CO., LTD. JIANGXI JEMINCARE GROUP CO., LTD. <120> Anti-IgE engineered antibody and application thereof <130> PA26-057 <150> CN 202010663029.3 <151> 2020-07-10 <150> CN 202110750959.7 <151> 2021-07-01 <160> 64 <170> PatentIn version 3.5 <210> 1 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> LFR1 <400> 1 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 2 Arg Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 3 Arg Ala Ser Gln Ser Val Asp Tyr Glu Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Arg Ala Ser Gln Ser Leu Tyr Asp Glu Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 5 Arg Ala Ser Gln Ser Val Ala Asp Glu Trp Asp Ser Tyr Met Asn 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 6 Arg Ala Ser Gln Ser Val Asp Ala Glu Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 7 Arg Ala Ser Gln Ser Val Asp Asp Glu Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 8 Arg Ala Ser Gln Ser Val Asp Asp Glu Tyr Asp Ser Tyr Met Asn 1 5 10 15 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 9 Arg Ala Ser Gln Ser Val Asp Ser Glu Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 10 Arg Ala Ser Gln Ser Val Glu Asp Glu Trp Asp Ser Tyr Met Asn 1 5 10 15 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 11 Arg Ala Ser Gln Ser Val Phe Asp Glu Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 12 Arg Ala Ser Gln Ser Val Gly Asp Glu Ala Asp Ser Tyr Met Asn 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 13 Arg Ala Ser Gln Ser Val Asn Asp Glu Ala Asp Ser Tyr Met Asn 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 14 Arg Ala Ser Gln Ser Val Asn Asp Glu Trp Asp Ser Tyr Met Asn 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LFR2 <400> 15 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 16 Ala Ala Ser Tyr Leu Glu Ser 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 17 Ala Ala Ser Tyr Leu Gln Ser 1 5 <210> 18 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LFR3 <400> 18 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 19 Gln Gln Ser His Glu Asp Pro Tyr Thr 1 5 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LFR4 <400> 20 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LFR4 <400> 21 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 22 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 22 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 23 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 23 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Glu 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 24 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 24 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Leu Tyr Asp Glu 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 25 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 25 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ala Asp Glu 20 25 30 Trp Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 26 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 26 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Ala Glu 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 27 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 27 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Asp Glu 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 28 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 28 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Asp Glu 20 25 30 Tyr Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 29 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 29 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Ser Glu 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 30 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 30 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Glu Asp Glu 20 25 30 Trp Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 31 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 31 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Phe Asp Glu 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 32 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 32 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Gly Asp Glu 20 25 30 Ala Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 33 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 33 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asn Asp Glu 20 25 30 Ala Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 34 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 34 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asn Asp Glu 20 25 30 Trp Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> HFR1 <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser 20 25 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 36 Gly Tyr Ser Ile Thr Ser Gly Tyr 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 37 Gly Tyr Ser Ile Arg Ser Gly Tyr 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 38 Gly Tyr Ser Ile Thr Gln Gly Tyr 1 5 <210> 39 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> HFR2 <400> 39 Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10 15 Ala Ser Ile <210> 40 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> HFR2 <400> 40 Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 1 5 10 15 Ser Val Ile <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 41 Thr Tyr Asp Gly Ser 1 5 <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 42 Thr Tyr Ala Gly Ser 1 5 <210> 43 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> HFR3 <400> 43 Thr Asn Tyr Asn Pro Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp 1 5 10 15 Asp Ser Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 20 25 30 Asp Thr Ala Val Tyr Tyr Cys Ala Arg 35 40 <210> 44 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> HFR3 <400> 44 Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 1 5 10 15 Asp Ser Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 20 25 30 Asp Thr Ala Val Tyr Tyr Cys Ala Arg 35 40 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 45 Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val 1 5 10 <210> 46 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 46 Gly Thr Asn Tyr Phe Gly His Trp His Phe Ala Val 1 5 10 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 47 Gly Thr His Tyr Phe Gly His Trp His Phe Ala Val 1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 48 Gly Ser Asn Tyr Phe Gly His Trp His Phe Ala Val 1 5 10 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HFR4 <400> 49 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 50 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 50 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro Ser Val 50 55 60 Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 51 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Thr Tyr Ala Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 52 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Thr Tyr Ala Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Asn Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 53 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Arg Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Thr Tyr Ala Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Thr Tyr Ala Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 55 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Thr Tyr Ala Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Gln Gly 20 25 30 Tyr Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Thr Tyr Ala Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 57 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL <220> <221> misc_feature <222> (29)..(29) <223> Xaa = Leu or Val <220> <221> misc_feature <222> (30)..(30) <223> Xaa = Ala, Asp, Glu, Phe, Gly, Asn or Tyr <220> <221> misc_feature <222> (31)..(31) <223> Xaa = Ala, Asp, Ser or Tyr <220> <221> misc_feature <222> (33)..(33) <223> Xaa = Ala, Gly, Trp or Tyr <400> 57 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Xaa Xaa Xaa Glu 20 25 30 Xaa Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 58 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH <220> <221> misc_feature <222> (30)..(30) <223> Xaa = Arg or Thr <220> <221> misc_feature <222> (31)..(31) <223> Xaa = Gln or Ser <220> <221> misc_feature <222> (100)..(100) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (101)..(101) <223> Xaa = His or Asn <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Xaa Xaa Gly 20 25 30 Tyr Ser Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ser Val Ile Thr Tyr Ala Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Xaa Xaa Tyr Phe Gly His Trp His Phe Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 59 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> light chain constant region <400> 59 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 60 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> IgG constant region (none mutation) <400> 60 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 61 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> IgG1 constant region (YTE mutation) <400> 61 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 general formula <220> <221> misc_feature <222> (5)..(5) <223> Xaa = Arg or Thr <220> <221> misc_feature <222> (6)..(6) <223> Xaa = Gln or Ser <400> 62 Gly Tyr Ser Ile Xaa Xaa Gly Tyr 1 5 <210> 63 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 general formula <220> <221> misc_feature <222> (2)..(2) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (3)..(3) <223> Xaa = His or Asn <400> 63 Gly Xaa Xaa Tyr Phe Gly His Trp His Phe Ala Val 1 5 10 <210> 64 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 general formula <220> <221> misc_feature <222> (6)..(6) <223> Xaa = Leu or Val <220> <221> misc_feature <222> (7)..(7) <223> Xaa = Ala, Asp, Glu, Phe, Gly, Asn or Tyr <220> <221> misc_feature <222> (8)..(8) <223> Xaa = Ala, Asp, Ser or Tyr <220> <221> misc_feature <222> (10)..(10) <223> Xaa = Ala, Gly, Trp or Tyr <400> 64 Arg Only Ser Gln Ser No No No Glu No Asp Ser Tyr Met Asn 1 5 10 15

Claims

1. An antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL contains amino acid mutations at one or more positions selected from the group consisting of E55 and V104, compared to the amino acid sequence shown in SEQ ID NO:

22.

2. The antigen-binding protein according to claim 1, wherein VL contains an amino acid mutation at one or more positions selected from the group consisting of D30b, V29, D30, Y30a, and G30c.

3. The antigen-binding protein according to any one of claims 1 to 2, wherein VL contains amino acid mutations at the following positions: D30b, E55, and V104.

4. VL (1) D30b, E55, V104, V29, D30, and Y30a; (2) D30b, E55, V104, D30, Y30a and G30c; (3) D30b, E55, V104 and Y30a; (4) D30b, E55, V104, Y30a and G30c; (5) D30b, E55, V104, D30, Y30a and G30c; and (6) D30b, E55, V104, D30 and Y30a The antigen-binding protein according to any one of claims 1 to 3, comprising an amino acid mutation at a position selected from any group consisting of the following.

5. The antigen-binding protein according to any one of claims 1 to 4, wherein the amino acid mutation at position E55 includes E55Q.

6. The antigen-binding protein according to any one of claims 1 to 5, wherein the amino acid mutation at position V104 includes V104L.

7. The antigen-binding protein according to any one of claims 2 to 6, wherein the amino acid mutation at position D30b includes D30bE.

8. The antigen-binding protein according to any one of claims 2 to 7, wherein the amino acid mutation at position V29 includes V29L.

9. The antigen-binding protein according to any one of claims 2 to 8, wherein the amino acid mutation at position Y30a is selected from the group consisting of Y30A, Y30aS, and Y30aD.

10. The antigen-binding protein according to any one of claims 2 to 9, wherein the amino acid mutation at position D30 is selected from the group consisting of D30Y, D30A, D30E, D30F, D30G, and D30N.

11. The antigen-binding protein according to any one of claims 2 to 10, wherein the amino acid mutation at position G30c is selected from the group consisting of G30cA, G30cY, and G30cW.

12. The antigen-binding protein according to any one of claims 2 to 11, wherein VL comprises amino acid mutations from the following groups: D30bE, E55Q, and V104L.

13. The antigen-binding protein according to any one of claims 1 to 12, wherein VL comprises the amino acid sequence shown in SEQ ID NO:

57.

14. The antigen-binding protein according to any one of claims 1 to 13, wherein VL comprises one of the amino acid sequences shown in SEQ ID NOs. 23 to 34.

15. An antigen-binding protein according to any one of claims 1 to 14, comprising an antibody heavy chain variable region (VH), wherein the VH contains amino acid mutations at one or more positions selected from the group consisting of I37, A49, S50, and D54, compared to the amino acid sequence shown in SEQ ID NO:

50.

16. The antigen-binding protein according to claim 15, wherein VH contains an amino acid mutation at one or more positions selected from the group consisting of N60, P61, I67, T30, S31, S96, and H97.

17. The antigen-binding protein according to any one of claims 15 to 16, wherein VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, and D54.

18. VH (1) N60, P61, I67, I37, A49, S50, D54, S96 and H97; (2) N60, P61, I67, I37, A49, S50, D54 and T30; (3) N60, P61, I67, I37, A49, S50, D54 and S96; (4) N60, P61, I67, I37, A49, S50, D54 and S31; and (5) N60, P61, I67, I37, A49, S50, D54 and H97 The antigen-binding protein according to any one of claims 15 to 17, comprising an amino acid mutation at a position selected from any group consisting of the following.

19. The antigen-binding protein according to any one of claims 15 to 18, wherein the amino acid mutation at position I37 includes I37V.

20. The antigen-binding protein according to any one of claims 15 to 19, wherein the amino acid mutation at position A49 includes A49S.

21. The antigen-binding protein according to any one of claims 15 to 20, wherein the amino acid mutation at position S50 includes S50V.

22. The antigen-binding protein according to any one of claims 15 to 21, wherein the amino acid mutation at position D54 includes D54A.

23. The antigen-binding protein according to any one of claims 16 to 22, wherein the amino acid mutation at position N60 includes N60A.

24. The antigen-binding protein according to any one of claims 16 to 23, wherein the amino acid mutation at position P61 includes P61D.

25. The antigen-binding protein according to any one of claims 16 to 24, wherein the amino acid mutation at position I67 includes I67F.

26. The antigen-binding protein according to any one of claims 16 to 25, wherein the amino acid mutation at position T30 includes T30R.

27. The antigen-binding protein according to any one of claims 16 to 26, wherein the amino acid mutation at position S31 includes S31Q.

28. The antigen-binding protein according to any one of claims 16 to 27, wherein the amino acid mutation at position S96 includes S96T.

29. The antigen-binding protein according to any one of claims 16 to 28, wherein the amino acid mutation at position H97 includes H97N.

30. The antigen-binding protein according to any one of claims 15 to 29, wherein VH comprises amino acid mutations from the following groups: N60A, P61D, I67F, I37V, A49S, S50V, and D54A.

31. The antigen-binding protein according to any one of claims 15 to 20, wherein VH comprises the amino acid sequence shown in SEQ ID NO:

58.

32. The antigen-binding protein according to any one of claims 15 to 31, wherein VH comprises one of the amino acid sequences shown in SEQ ID NOs. 51 to 56.

33. VL and VH are: (1) VL contains amino acid mutations at the following positions: D30b, E55, V104, V29, D30 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54, S96 and H97; (2) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and T30; (3) VL contains amino acid mutations at the following positions: D30b, E55, V104 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and S96; (4) VL contains amino acid mutations at the following positions: D30b, E55, V104 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50 and D54; (5) VL contains amino acid mutations at the following positions: D30b, E55, V104, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50 and D54; (6) VL contains amino acid mutations at the following positions: D30b, E55, V104 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and H97; (7) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50 and D54; (8) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30 and Y30a; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and H97; (9) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and S96; and (10) VL contains amino acid mutations at the following positions: D30b, E55, V104, D30, Y30a and G30c; VH contains amino acid mutations at the following positions: N60, P61, I67, I37, A49, S50, D54 and S31 The antigen-binding protein according to any one of claims 15 to 32, comprising an amino acid mutation at a position selected from any group consisting of the following.

34. An antigen-binding protein according to any one of claims 1 to 33, comprising an antibody heavy chain constant region.

35. The antigen-binding protein according to claim 34, wherein the antibody heavy chain constant region is derived from the human IgG constant region.

36. The antigen-binding protein according to claim 35, wherein the human IgG constant region includes the human IgG1 constant region.

37. The antigen-binding protein according to claim 36, wherein the human IgG1 constant region contains one or more amino acid mutations selected from the group consisting of M252Y, S254T, and T256E.

38. The antigen-binding protein according to claim 37, wherein the human IgG1 constant region comprises one of the amino acid sequences shown in SEQ ID NOs. 60 to 61.

39. An antigen-binding protein according to any one of claims 1 to 38, comprising an antibody light chain constant region.

40. The antigen-binding protein according to claim 39, wherein the constant region of the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

59.

41. One or more isolated nucleic acid molecules encoding an antigen-binding protein according to any one of claims 1 to 40.

42. A vector comprising the nucleic acid molecule described in claim 41.

43. A cell comprising the nucleic acid molecule described in claim 41 or the vector described in claim 42.

44. A method for preparing an antigen-binding protein according to any one of claims 1 to 40, comprising the step of culturing the cells according to claim 43 under conditions that enable the expression of the antigen-binding protein according to any one of claims 1 to 40.

45. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 40, a nucleic acid molecule according to claim 41, a vector according to claim 42 and / or a cell according to claim 43, and optionally a pharmaceutically acceptable carrier.

46. Use of an antigen-binding protein according to any one of claims 1 to 40, a nucleic acid molecule according to claim 41, a vector according to claim 42, a cell according to claim 43, and / or a pharmaceutical composition according to claim 45 in the preparation of a pharmaceutical for alleviating or treating a disease associated with abnormal levels of IgE.

47. A method for alleviating or treating a disease associated with abnormal levels of IgE, comprising the step of administering an antigen-binding protein according to any one of claims 1 to 40, a nucleic acid molecule according to claim 41, a vector according to claim 42, cells according to claim 43, and / or a pharmaceutical composition according to claim 45, to a subject in need thereof.

48. A polypeptide comprising an antigen-binding protein according to any one of claims 1 to 40.

49. An immunoconjugate comprising an antigen-binding protein according to any one of claims 1 to 40.

50. A kit comprising an antigen-binding protein according to any one of claims 1 to 40, a nucleic acid molecule according to claim 41, a vector according to claim 42, a cell according to claim 43, and / or a pharmaceutical composition according to claim 45.

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