Isolated antigen binding proteins and uses thereof

文档序号：182917 发布日期：2021-11-02 浏览：47次中文

阅读说明：本技术 分离的抗原结合蛋白及其用途 (Isolated antigen binding proteins and uses thereof ) 是由孙乃超周若芸马海立高永娟成超刘恒于 2020-04-30 设计创作，主要内容包括：分离的抗原结合蛋白,其包含重链可变区中的至少一个CDR,所述重链可变区包含如SEQ ID NO:72-73中任一项所示的氨基酸序列或其变体；且其包含轻链可变区中的至少一个CDR,所述轻链可变区包含如SEQ ID NO:74-75中任一项所示的氨基酸序列或其变体。(An isolated antigen binding protein comprising at least one CDR in a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 72-73 or a variant thereof; and which comprises at least one CDR in a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 74-75 or a variant thereof.)

1. An isolated antigen binding protein comprising at least one CDR in a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 72-73 or a variant thereof; and which comprises at least one CDR in a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 74-75 or a variant thereof.

2. The isolated antigen binding protein of claim 1, comprising HCDR1, HCDR2 and/or HCDR3 in the heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs 33-36; wherein the HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 1, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 66, and the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 3.

3. The isolated antigen binding protein of any of claims 1-2, comprising LCDR1, LCDR2 and/or LCDR3 in the light chain variable region having an amino acid sequence as set forth in any of SEQ ID NOs 37-40, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID No.5, the LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 67, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID No. 68.

4. The isolated antigen binding protein of any of claims 1-3, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs 37-40.

5. The isolated antigen binding protein of any of claims 1-4, comprising an antibody light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs 43-44.

6. The isolated antigen binding protein of any of claims 1-5, wherein the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs 33-36.

7. The isolated antigen binding protein of any one of claims 1-6, comprising an antibody heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOs 45-46.

8. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-7.

9. A cell comprising the nucleic acid molecule of claim 8.

10. Use of the isolated antigen binding protein of any one of claims 1-7, the nucleic acid molecule of claim 8, in the manufacture of a medicament for the prevention, alleviation and/or treatment of an IgE-related disease or disorder.

Technical Field

The application relates to the field of biomedicine, in particular to an isolated antigen binding protein and application thereof.

Background

Allergic diseases (also called allergic diseases) are the sixth chronic disease worldwide, affect various tissues and organs of the body, cause various uncomfortable symptoms once in onset, seriously reduce the quality of life and even endanger life. For example, allergic rhinitis, sinusitis, nasal polyps can be caused in the nasal cavity; asthma, allergic cough can be caused in other parts of the respiratory tract; allergic conjunctivitis can be caused in the eyes; secretory otitis media can occur in the ear; allergic pharyngitis and allergic tonsillitis can be caused at pharynx; can cause urticaria, angioedema, eczema, atopic dermatitis, contact dermatitis; allergic and eosinophilic gastroenteritis may occur in the digestive tract; even in the brain, hyperactivity, twitching, self-closure, etc. may be caused. According to the statistics of the World Allergy Organization (WAO), the incidence rate of allergic diseases worldwide is increased by at least 3 times in the last 30 years, and the global total disease rate is up to 22 percent at present. Estimated by this growth rate, 50% of the population in industrialized countries will be afflicted with allergic diseases within 20 years. According to the Allergic Child site survey, about 25% and 20% of residents in the united kingdom and new zealand respectively suffer from pollen allergy, and 5 people die of allergy caused by drugs, food or mosquito bites in the united states on average every day. The hospitalization in australia from 1994 to 2004 for systemic anaphylaxis turned over in two, with a 5-fold increase in children under five years of age. In 2013, it was reported that over 1 hundred million allergic patients exist in China, meaning that 1 in 13 people in China has allergic disease. This rate doubled in 2016, with 1 in every 5 Chinese people allergic.

Most allergic diseases are caused by immunoglobulin e (ige) -mediated hypersensitivity reactions. The high affinity receptor for IgE (fceri) is critical for mediating allergic manifestations. In addition to mast cells and basophils, fceri is found in many other cell types (including eosinophils, platelets) and antigen presenting cells (e.g., monocytes and dendritic cells). Since IgE plays an important role in mediating most allergic reactions, it is essential to develop therapeutic strategies against allergic diseases that control IgE levels.

Disclosure of Invention

The present application provides an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 72-73 or a variant thereof; and which comprises at least one CDR in a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 74-75 or a variant thereof.

In certain embodiments, the isolated antigen binding protein has one or more of the following properties:

1) can be at 2.4 × 10^-9(ii) KD of M or lower binds to IgE, wherein the KD value is determined by MSD-SET;

2) can inhibit the binding of IgE to its receptor Fc epsilon RIA.

In certain embodiments, the isolated antigen binding protein comprises HCDR1 in the heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs 33-36.

In certain embodiments, the isolated antigen binding protein comprises HCDR2 in the heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs 33-36.

In certain embodiments, the isolated antigen binding protein comprises HCDR3 in the heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs 33-36.

In certain embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 1.

In certain embodiments, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 66.

In certain embodiments, the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 2 and 4.

In certain embodiments, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 3.

In certain embodiments, the isolated antigen binding protein comprises LCDR1 in the light chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs 37-40.

In certain embodiments, the isolated antigen binding protein comprises LCDR2 in the light chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs 37-40.

In certain embodiments, the isolated antigen binding protein comprises LCDR3 in the light chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs 37-40.

In certain embodiments, the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 5.

In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO. 67.

In certain embodiments, the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 6and 8.

In certain embodiments, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 68.

In certain embodiments, the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 7 and 9.

In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen-binding fragment comprises a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv and/or dAb.

In certain embodiments, the light chain variable region comprises the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

In certain embodiments, the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1 and L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs 10, 14, and 69.

In certain embodiments, the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOS 23-24.

In certain embodiments, the L-FR2 is located between the LCDR1 and the LCDR2 and the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 11, 15, and 25.

In certain embodiments, the L-FR3 is located between the LCDR2 and the LCDR3 and the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 12, 16, and 70.

In certain embodiments, the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOS 26-27.

In certain embodiments, the N-terminus of L-FR4 is linked to the C-terminus of LCDR3 and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO 13.

In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOS 37-40.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Kappa light chain constant region.

In certain embodiments, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 41.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain comprising the amino acid sequence set forth in any one of SEQ ID NOS 43-44.

In certain embodiments, the heavy chain variable region comprises the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In certain embodiments, the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1 and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs 18 and 28.

In certain embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 19, 22, and 71.

In certain embodiments, the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 29-30.

In certain embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 20 and 31.

In certain embodiments, the N-terminus of H-FR4 is linked to the C-terminus of HCDR3 and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 21 and 32.

In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOS 33-36.

In certain embodiments, an antibody heavy chain constant region is included and is derived from a human IgG heavy chain constant region.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 42.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOS 45-46.

In certain embodiments, the antigen is IgE.

In certain embodiments, the IgE is human IgE.

In another aspect, the present application also provides an isolated nucleic acid molecule encoding an isolated antigen binding protein described herein.

A vector comprising a nucleic acid molecule described herein.

In another aspect, the present application also provides a cell comprising a nucleic acid molecule described herein or a vector described herein.

In another aspect, the present application also provides a method of making an isolated antigen binding protein described herein, the method comprising culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In another aspect, the present application also provides an immunoconjugate comprising the isolated antigen binding protein described herein.

In another aspect, the present application also provides a pharmaceutical composition comprising an isolated antigen binding protein, a nucleic acid molecule, a vector, a cell and/or an immunoconjugate as described herein, and optionally a pharmaceutically acceptable adjuvant.

In another aspect, the present application provides the use of an isolated antigen binding protein, a nucleic acid molecule, a vector, a cell, an immunoconjugate and/or a pharmaceutical composition as described herein in the manufacture of a medicament for the prevention, amelioration and/or treatment of an IgE-associated disease or disorder.

In another aspect, the present application provides a method of preventing, ameliorating, or treating an IgE-related disease or disorder, the method comprising administering to a subject in need thereof an isolated antigen binding protein described herein, an immunoconjugate described herein, and/or a pharmaceutical composition described herein.

In another aspect, the present application also provides the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, the immunoconjugate and/or the pharmaceutical composition described herein for use in the prevention, alleviation or treatment of an IgE-associated disease or disorder.

In another aspect, the present application also provides a method of detecting IgE in a sample, the method comprising administering an isolated antigen binding protein described herein.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows the results of the detection of the murine monoclonal antibody of the present application blocking the binding of human IgE-Fc to its receptor Fc epsilon RIA.

FIG. 2 shows the results of the detection of the SE2 and SE5 chimeric antibodies and their corresponding murine antibodies blocking the binding of human IgE-Fc to its receptor FceRIa as described herein.

FIG. 3 shows the results of the detection of SE2 and SE5 humanized antibodies, their corresponding chimeric antibodies and their corresponding murine antibodies blocking the binding of human IgE-Fc to its receptor FceRIa as described herein.

Figure 4 shows the results of the detection of blocking of human IgE binding to its receptor fcepsilon RIa by the humanized SE2 antibody described herein and omalizumab.

Figure 5 shows the results of the detection of blocking of human IgE binding to its receptor fcepsilon RIa by the humanized SE5 antibody described herein and omalizumab.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means or synthesized artificially. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not affect the activity of the substance, mixed with artificial or synthetic substances.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized synthetically.

In the present application, the term "variant" generally refers to a molecule that has undergone amino acid modification (e.g., group substitution, etc.) or insertion, substitution, and/or deletion of one or more amino acids in the original protein sequence, while retaining the function of the original sequence. For example, the variant may have a better biological activity (or function) than the original sequence. For example, the reservation need not be a full reservation. For example, the variant may substantially retain the function of the original sequence, e.g., retain at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the function of the original sequence. For example, the amino acid sequence of the variant may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 99.9% homologous to the original amino acid sequence. For example, the amino acid sequence of the variant may include an amino acid sequence obtained by conservative mutation known in the art of the original amino acid sequence. The amino acid sequence variant shown as SEQ ID NO. 75 may include a molecule in which an amino acid modification (e.g., a group substitution, etc.) or an insertion, substitution, and/or deletion of one or more amino acids is performed on the amino acid sequence shown as SEQ ID NO. 75, while retaining the function of the original sequence. For example, the amino acid sequence as set forth in SEQ ID NO. 75 may be substantially functional, e.g., at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% functional, of the amino acid sequence as set forth in SEQ ID NO. 75. For example, it may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 99.9% identical to the amino acid sequence shown as SEQ ID NO. 75. In the present application, identity can be determined, for example, by the Needle program of the EMBOSS software package (EMBOSS: European molecular biology open software suite, Rice et al, 2000, Trends in Genetics 16: 276-. Optional parameters used are gap penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM 62). The output of the Needle labeled "longest identity" (obtained using the nobrief option) is used as the percent identity and is calculated as follows: (same residue X100)/(length of alignment-total number of gaps in alignment).

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability obtained by artificial means from a natural state or artificially synthesized. The "isolated antigen binding protein" may comprise a portion that binds an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding portion to an antigen. The antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, scaffolds of antibody origin comprising mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein, as well as fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., Korndorfer et al, 2003, Proteins: Structure, Function, andBioinformatics,53(1): 121-; roque et al, Biotechnol.prog.20:639-654 (2004). In addition, peptide antibody mimetics ("PAMs") may also be included as well as scaffolds based on antibody mimetics using fibronectin components may be used as scaffolds.

In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F (ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function (e.g., specifically bind human IgE). Typically, such fragments should include an antigen binding domain. For example, the basic 4 chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. For example, an IgM antibody consists of 5 elementary heterotetrameric units with another polypeptide called the J chain, while an IgA antibody consists of 2-5 elementary 4-chain units that can polymerize in association with the J chain to form multivalent combinations. For IgG, the 4-chain unit can typically be about 150,000 daltons. Each L chain may be linked to an H chain by a covalent disulfide bond, while two H chains may be linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain may also have spaced intrachain disulfide bridges. Each H chain may have a variable domain/variable region (VH) at the N-terminus, followed by three constant domains/constant regions (CH) for the alpha and gamma chains, respectively, followed by four CH domains for the mu and epsilon isotypes. Each L chain may have a variable domain/region (VL) at the N-terminus and a constant domain/region (CL) at its other end. In certain embodiments, VL corresponds to VH and CL corresponds to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The VH and VL may pair together to form an antigen binding site. For the structure and properties of antibodies of different classes see, for example, Basic and Clinical Immunology,8th Edition, Daniel P.Sties, Abba I.Terr and Tristram G.Parsolw (eds), Appleton & Lange, Norwalk, conn.,1994, page 71 and chapter 6. In certain embodiments, L chains from any vertebrate species can be classified into one of two distinctly different classes, termed κ and λ, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of its heavy chain constant domain/constant region (CH), immunoglobulins can also be assigned to different classes or classes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses, e.g., humans comprise the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK 1. An antigen-binding fragment.

In the present application, the term "antigen-binding fragment" generally refers to a portion of a full-length antibody (e.g., a target-binding or variable region) or a functional fragment or analog thereof. Examples of the antibody fragment may include Fab, Fab ', F (ab')2, Fv fragments, and the like. "functional fragment or analog" generally refers to a compound that has substantially the same qualitative biological activity as a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is a compound that can bind to an IgE immunoglobulin so that IgE can be prevented from having or substantially reduced the ability to bind to its high affinity receptor Fc £ RI. An "Fv" fragment is generally the smallest antibody fragment that contains an intact target recognition and binding site. The region may consist of a dimer of one heavy chain variable domain non-covalently linked to one light chain variable domain (VH-VL dimer).

In the present application, the term "framework region" generally refers to the portion of an antibody variable region known in the art that is present between the more diverse (i.e., hypervariable) CDRs. Generally, such framework regions are typically referred to as frameworks 1 to 4(FR1, FR2, FR3 and FR4) and provide a framework for presenting six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space to form an antigen-binding surface. Namely four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR 4). For example, the VL of an isolated antigen binding protein described herein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4. The VH of an isolated antigen binding protein described herein may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In this application, the term "variable" generally refers to the fact that certain segments of an antibody variable region (e.g., heavy chain variable region, light chain variable region) differ in sequence between antibodies. Typically, the variable regions mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. Typically, the variability is not evenly distributed throughout the variable domain, but is concentrated in several segments called Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs) in the light and heavy chain variable domains. The portion of the variable region that is relatively conserved is called the Framework Region (FR). The variable domains of native heavy and light chains may each comprise four FR regions, most in a β -sheet configuration, connected by three CDRs, and in some cases forming part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FRs and can, together with the CDRs from the other chain, contribute to the formation of an antigen binding site for the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). In general, the constant region is not directly involved in binding of an antibody to an antigen, but exhibits various effector functions, such as participation of an antibody in antibody-dependent cellular cytotoxicity.

In this application, the term "light chain variable region" generally refers to the amino-terminal domain of an antibody light chain. The light chain variable region may be referred to as "VL". These domains are typically the most variable parts of the antibody light chain (relative to other antibodies of the same type), and may include Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs) and Framework Regions (FRs).

In this application, the term "heavy chain variable region" generally refers to the amino-terminal domain of an antibody heavy chain. The heavy chain variable region may be referred to as "VH". These domains are typically the most variable parts of an antibody heavy chain (relative to other antibodies of the same type) and may include Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs) and Framework Regions (FRs).

In the present application, the terms "subject" and "patient" are used interchangeably and refer generally to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, and mice, among others. Animals include all vertebrates, e.g., mammals and non-mammals, such as dogs, cats, sheep, cattle, pigs, rabbits, chickens, etc. For example, the subject for performing the present method of treating shenqi is a human. Subjects in need of treatment include patients already suffering from a disease or disorder associated with IgE as well as persons predisposed to developing such a disorder.

In the present application, the term "cell" can generally be or has been nucleic acid molecules or carrier recipients of single cells, cell lines or cell cultures. The cell may comprise a nucleic acid molecule as described herein or a vector as described herein. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may comprise cells transfected in vitro with a vector described herein. The cell may be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293T cell.

In the present application, the term "pharmaceutical composition" generally refers to compositions that are suitable for administration to a patient, e.g., a human patient. For example, a pharmaceutical composition described herein, which can comprise an isolated antigen binding protein described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. For example, the acceptable ingredients of the composition are not toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable adjuvant" refers generally to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like, compatible with pharmaceutical administration. Are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

In the present application, the term "inhibit" generally means capable of completely or partially blocking or reducing the binding of IgE to its receptor fceri.

In the present application, the term "prevention" generally refers to prophylactic administration of a combination to healthy patients to prevent the outbreak of the diseases and conditions described herein. Furthermore, the term "prevention" refers to the prophylactic administration of such a combination to a patient at a pre-stage of the allergic disease to be treated. The term "prevention" does not require 100% elimination of the possibility of an event. More specifically, it means that the likelihood of an event occurring in the presence of the pharmaceutical composition or method is reduced.

In this application, the term "treating" generally refers to applying or administering a therapeutic agent to a patient, or to a tissue or cell line isolated from a patient, who has a disease, disease symptoms, or disease predisposition, for the purpose of treating, curing, alleviating, ameliorating, altering, remediating, ameliorating, increasing, or affecting the disease, disease symptoms, or disease predisposition. May include improving the disease state, eliminating the lesion, or improving prognosis.

In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In this application, the term "directly or indirectly connected" usually refers to the relative "directly connected" or "indirectly connected". "directly connected" generally refers to a direct connection. For example, the direct linkage may be a case where the linked substances (e.g., amino acid sequence segments) are directly linked without a spacer component (e.g., amino acid residue or derivative thereof); for example, the amino acid sequence segment X is directly linked to the further amino acid sequence segment Y via an amide bond formed by the C-terminal amino acid of the amino acid sequence segment X and the N-terminal amino acid of the amino acid sequence segment Y. "indirectly linked" generally refers to the situation where the linked substances (e.g., segments of amino acid sequences) are indirectly linked with a spacer (e.g., amino acid residues or derivatives thereof) therebetween, e.g., the C-terminus of L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly linked in the isolated antigen binding proteins described herein.

In this application, the term "complementarity determining regions" or the term "CDRs" generally refers to amino acid sequences that collectively define the binding affinity and specificity of the variable region of the binding site of an antigen binding protein (e.g., a natural immunoglobulin, chimeric antibody or humanized antibody) (see, e.g., Chothia et al, J.mol.biol.196: 901-917 (1987); Kabat et al, U.S. Deptof Health and Human Services NIH Publication No.913242 (1991)). Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR 3). Naturally occurring camel antibodies consisting of only heavy chains can also remain functionally normal and stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996).

In the present application, the term "fcsria" generally refers to the alpha chain of a high affinity receptor (fcsri, also known as Fc epsilon RI,) that is the Fc region of immunoglobulin e (ige). Fcsri is a tetrameric receptor complex capable of binding to the Fc region of the epsilon heavy chain of IgE, consisting of one alpha chain (i.e., fcsria), one beta chain (fcsria) and two gamma chains (fcsry). In general, the α chain can serve as a binding site for an antibody (e.g., IgE), the γ chain can serve as a site for initiation of downstream signals, and the β chain can serve to amplify downstream signals. Fc epsilon RI is expressed on epidermal langerhans cells, eosinophils, mast cells and basophils. Due to its cellular distribution, this receptor plays a major role in allergic reactions. Fcsri is also expressed on antigen presenting cells and is involved in the production of important immune mediators (e.g., cytokines, interleukins, leukotrienes, prostaglandins, etc.) that promote inflammation.

In the present application, the term "IgE" or "IgE immunoglobulin" or "immunoglobulin E" generally refers to a class of antibodies (or immunoglobulins (igs)) produced by plasma cells found in mammals. The IgE monomer consists of two heavy chains (epsilon chain) and two light chains, where the epsilon chain contains 4 Ig-like constant regions (C epsilon 1-C epsilon 4). The major functions of IgE are known to include immunity to parasites (e.g., helminths such as schistosoma mansoni, plasmodium falciparum, trichina or fasciola hepatica, etc.). IgE may also be involved in defense against venom (e.g., bee, snake bites, etc.). IgE also has an important role in type I hypersensitivity reactions, such as allergic asthma, most types of sinusitis, allergic rhinitis, food allergy and specific types of chronic urticaria and atopic dermatitis. IgE also plays a key role in the response to allergens, for example: allergic reaction to drugs. In humans, IgE belongs to the class of polypeptides of antibodies essentially encoded by the well-known immunoglobulin epsilon genes. IgE may include membrane-anchored (mIgE), or non-membrane-anchored, also known as circulating IgE.

In this application, the term "IgE-associated disease or disorder" refers to any condition or disorder that is mediated by or associated with IgE. Usually including overproduction of immunoglobulin IgE and/or hypersensitivity to IgE. Such as allergic diseases.

In the present application, the term "KD", also called "K_D”、“K_D"," affinity constant "or" equilibrium dissociation constant "generally refers to the dissociation rate constant (k) at equilibrium, or by the dissociation rate constant (k) in a titration measurement_d) Divided by the binding rate constant (k)_a) The obtained value. Using the binding Rate constant (k)_a) Dissociation rate constant (k)_d) And equilibrium dissociation constant (K)_D) Refers to the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) for an antigen (e.g., human IgE). Methods for determining the association and dissociation rate constants are well known in the art. Using fluorescence-based techniquesThe technique provides high sensitivity and the ability to examine the sample at equilibrium in physiological buffer. For example, the K can be determined by Octet_DValues, other experimental pathways and instruments such as BIAcore (biomolecular interaction analysis) assays may also be used. In addition, the KD values were determined by electrochemiluminescence analysis-solution equilibrium titration (MSD-SET). The measurement methods are described in Estep P. et al, MAbs,2013.5(2): p.270-8.

In the present application, the term "comprising" or "comprises" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 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% above or below the stated value.

Detailed Description

In one aspect, the present application provides an isolated antigen binding protein, which may comprise at least one CDR in a heavy chain variable region, which may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 72-73 or a variant thereof; and which may comprise at least one CDR in a light chain variable region which may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 74-75 or a variant thereof.

Antigen binding proteins

The present application provides antigen binding proteins, for example, the isolated antigen binding protein may comprise at least one CDR in a heavy chain variable region that may comprise any amino acid sequence conforming to the general formula shown as SEQ ID No. 72 or a variant thereof.

For example, the isolated antigen binding protein may comprise a CDR in the heavy chain variable region which may comprise any amino acid sequence conforming to the general formula shown in SEQ ID NO:72 or a variant thereof.

For example, the isolated antigen binding protein may comprise two CDRs in a heavy chain variable region that may comprise any amino acid sequence conforming to the general formula shown as SEQ ID NO:72 or a variant thereof.

For example, the isolated antigen binding protein may comprise three CDRs in a heavy chain variable region which may comprise any amino acid sequence conforming to the general formula shown as SEQ ID No. 72 or a variant thereof.

For example, the isolated antigen binding protein may comprise at least one CDR in the heavy chain variable region which may comprise any amino acid sequence conforming to the general formula shown in SEQ ID NO:73 or a variant thereof.

For example, the isolated antigen binding protein may comprise two CDRs in the heavy chain variable region, which may comprise any amino acid sequence conforming to the general formula shown as SEQ ID NO:73 or a variant thereof.

For example, the isolated antigen binding protein may comprise three CDRs in a heavy chain variable region that may comprise any amino acid sequence conforming to the general formula shown as SEQ ID No. 73 or a variant thereof.

For example, the CDRs in the heavy chain variable region may be referred to as HCDRn, n representing the number of CDRs, which may typically be 1, 2 or 3, etc.

For example, the CDRs in the heavy chain variable region may include HCDR1, HCDR2 and HCDR 3.

For example, the variant may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 99.9% identity to any amino acid sequence corresponding to the general formula shown in any of SEQ ID NOS 72-73.

For example, the isolated antigen binding protein may comprise HCDR1 in the heavy chain variable region or a variant thereof having an amino acid sequence as set forth in any one of SEQ ID NOS 33-36.

For example, the isolated antigen binding protein may comprise HCDR2 in the heavy chain variable region or a variant thereof having an amino acid sequence as set forth in any one of SEQ ID NOS 33-36.

For example, the isolated antigen binding protein may comprise HCDR3 in the heavy chain variable region or a variant thereof having an amino acid sequence as set forth in any one of SEQ ID NOS 33-36.

For example, the isolated antigen binding protein may comprise HCDR1 and HCDR2 in the heavy chain variable region or a variant thereof having an amino acid sequence as set forth in any one of SEQ ID NOS: 33-36.

For example, the isolated antigen binding protein may comprise HCDR2 and HCDR3 in the heavy chain variable region or a variant thereof having an amino acid sequence as set forth in any one of SEQ ID NOS: 33-36.

For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3 in the heavy chain variable region or a variant thereof having an amino acid sequence as set forth in any one of SEQ ID NOS: 33-36.

For example, the variant may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 99.9% identity to the amino acid sequence set forth in any one of SEQ ID NOS 33-36.

For example, the isolated antigen binding protein may comprise at least one CDR in the light chain variable region which may comprise any amino acid sequence conforming to the general formula shown in SEQ ID NO:74 or a variant thereof.

For example, the isolated antigen binding protein may comprise a CDR in the light chain variable region which may comprise any amino acid sequence conforming to the general formula shown in SEQ ID NO:74 or a variant thereof.

For example, the isolated antigen binding protein may comprise two CDRs in the light chain variable region, which may comprise any amino acid sequence conforming to the general formula shown as SEQ ID NO:74 or a variant thereof.

For example, the isolated antigen binding protein may comprise three CDRs in the light chain variable region, which may comprise any amino acid sequence conforming to the general formula shown as SEQ ID NO:74 or a variant thereof.

For example, the isolated antigen binding protein may comprise two CDRs in a light chain variable region which may comprise any amino acid sequence conforming to the general formula shown as SEQ ID NO:75 or a variant thereof.

For example, the isolated antigen binding protein may comprise three CDRs in a light chain variable region which may comprise any amino acid sequence conforming to the general formula shown as SEQ ID NO:75 or a variant thereof.

For example, the variant may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 99.9% identity to any amino acid sequence conforming to the general formula shown in any of SEQ ID NOS: 74-75.

For example, the CDRs in the light chain variable region may be referred to as LCDRn, n represents the CDR number, and may typically be 1, 2 or 3, etc.

For example, the CDRs in the light chain variable region may include LCDR1, LCDR2 and LCDR 3.

For example, the isolated antigen binding protein may comprise LCDR1 in the light chain variable region or a variant thereof having the amino acid sequence set forth in any one of SEQ ID NOs 37-40.

For example, the isolated antigen binding protein may comprise LCDR2 in the light chain variable region or a variant thereof having the amino acid sequence set forth in any one of SEQ ID NOs 37-40.

For example, the isolated antigen binding protein may comprise LCDR3 in the light chain variable region or a variant thereof having the amino acid sequence set forth in any one of SEQ ID NOs 37-40.

For example, the isolated antigen binding protein may comprise LCDR1 and LCDR2 in the light chain variable region or variant thereof having the amino acid sequence set forth in any one of SEQ ID NOs: 37-40.

For example, the isolated antigen binding protein may comprise LCDR2 and LCDR3 in the light chain variable region or variant thereof having the amino acid sequence set forth in any one of SEQ ID NOs: 37-40.

For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3 in the light chain variable region or a variant thereof having the amino acid sequence set forth in any one of SEQ ID NOS 37-40.

For example, exemplary conventions that may be used to define the CDRs may include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Typically, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of the structural loop region, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et Al, J.mol.biol.273:927-948 (1997); and Martin et al, Proc.Natl.Acad.Sci.USA 86: 9268-. Public databases known in the art may also be used to identify CDR sequences within antibodies.

Complementarity Determining Region (CDR)

In the present application, the CDR may be an amino acid sequence of the heavy chain variable region and/or light chain variable region of the isolated antigen binding protein that is capable of mediating specific binding of the antigen binding protein to the corresponding antigen.

For example, the CDR can be an amino acid sequence fragment in the heavy chain variable region as set forth in any one of SEQ ID NOs 72-73 and 33-36, which can be defined by the Kabat definition, the Chothia definition, and/or the AbM definition.

For example, the CDR can be an amino acid sequence fragment in the light chain variable region as set forth in any one of SEQ ID NOs 74-75 and 37-40, which can be defined by the Kabat definition, the Chothia definition, and/or the AbM definition.

For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 1 or a variant thereof.

For example, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 66 or a variant thereof.

For example, the HCDR2 can comprise the amino acid sequence set forth in any one of SEQ ID NOs 2 and 4 or a variant thereof.

For example, the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3 or a variant thereof.

For example, the variant may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 99.9% identity to any of the amino acid sequences set forth in any of SEQ ID NOs 1-4 and 66.

For example, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO.5 or a variant thereof.

For example, the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 67 or a variant thereof.

For example, the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 6and 8 or a variant thereof.

For example, the LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 68 or a variant thereof.

For example, the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID nos. 7 and 9 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1 and the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 1 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR2 and the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 66 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR2 and the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 2 and 4 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR3 and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No.5 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 67 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 6and 8, or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 68, or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 7 and 9, or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1 and HCDR2, the HCDR1 may comprise the amino acid sequence shown in SEQ ID No. 1 or a variant thereof, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 66 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1 and HCDR2, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 1 or a variant thereof, and the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID nos. 2 and 4 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR2 and HCDR3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 66 or a variant thereof, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR2 and HCDR3, the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 2 and 4 or a variant thereof, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 3 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1 and HCDR3, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID No. 1 or a variant thereof, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 3 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID No. 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 66 or a variant thereof, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID No. 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID nos. 2 and 4 or a variant thereof, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR1 and LCDR2, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No.5 or a variant thereof, and the LCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 67 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR1 and LCDR3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No.5 or a variant thereof, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 68 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR2 and LCDR3, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 67 or a variant thereof, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 68 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR2 and LCDR3, the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 6and 8 or a variant thereof, and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs 7 and 9 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No.5 or a variant thereof, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID No. 67 or a variant thereof, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID No. 68 or a variant thereof.

For example, the isolated antigen binding protein may comprise LCDR1, LCDR2, and LCDR3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID No.5 or a variant thereof, the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID nos. 6and 8 or a variant thereof, and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID nos. 7 and 9 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 66 or a variant thereof, the HCDR3 may comprise the amino acid sequence of SEQ ID No. 3 or a variant thereof, the LCDR1 may comprise the amino acid sequence of SEQ ID No.5 or a variant thereof, the LCDR2 may comprise the amino acid sequence of SEQ ID No. 67 or a variant thereof, and the LCDR3 may comprise the amino acid sequence of SEQ ID No. 68 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence of any one of SEQ ID NOs 2 and 4 or a variant thereof, the HCDR3 may comprise the amino acid sequence of SEQ ID No. 3 or a variant thereof, the LCDR1 may comprise the amino acid sequence of SEQ ID No.5 or a variant thereof, the LCDR2 may comprise the amino acid sequence of SEQ ID No. 67 or a variant thereof, and the LCDR3 may comprise the amino acid sequence of SEQ ID No. 68 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence of any one of SEQ ID NOs 2 and 4 or a variant thereof, the HCDR3 may comprise the amino acid sequence of SEQ ID No. 3 or a variant thereof, the LCDR1 may comprise the amino acid sequence of SEQ ID No.5 or a variant thereof, the LCDR2 may comprise the amino acid sequence of any one of SEQ ID NOs 6and 8 or a variant thereof, and the LCDR3 may comprise the amino acid sequence of any one of SEQ ID NOs 7 and 9 or a variant thereof.

For example, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 1 or a variant thereof, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 66 or a variant thereof, the HCDR3 may comprise the amino acid sequence of SEQ ID No. 3 or a variant thereof, the LCDR1 may comprise the amino acid sequence of SEQ ID No.5 or a variant thereof, the LCDR2 may comprise the amino acid sequence of any one of SEQ ID nos. 6and 8 or a variant thereof, and the LCDR3 may comprise the amino acid sequence of any one of SEQ ID nos. 7 and 9 or a variant thereof.

Frame Region (FR)

In the present application, the light chain variable region comprises the framework regions L-FR1, L-FR2, L-FR3, and/or L-FR 4.

For example, the light chain variable region may comprise the framework regions L-FR1, L-FR2 and L-FR 3.

For example, the light chain variable region may comprise the framework regions L-FR1, L-FR2 and L-FR 4.

For example, the light chain variable region may comprise the framework regions L-FR2, L-FR3 and L-FR 4.

For example, the light chain variable region may comprise the framework regions L-FR1, L-FR3 and L-FR 4.

For example, the light chain variable region may comprise the framework regions L-FR1, L-FR2, L-FR3 and L-FR 4.

For example, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, e.g., the C-terminus of L-FR1 is directly linked to the N-terminus of LCDR1, e.g., the C-terminus of L-FR1 is indirectly linked to the N-terminus of LCDR1, e.g., said indirect linkage may comprise 1, 2, 3, 4, 5 or more amino acids apart from the C-terminal amino acid residue of L-FR1 and the N-terminal amino acid residue of LCDR1, which may be any amino acid that occurs naturally or is modified.

For example, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOS: 10, 14 and 69 or a variant thereof.

For example, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOS 23-24 or a variant thereof.

For example, the L-FR2 is located between the LCDR1 and the LCDR 2.

For example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR 2.

For example, the indirect linkage may include the linkage of two moieties (e.g., the L-FR2 and the LCDR1, or the L-FR2 and the LCDR2) separated by 1, 2, 3, 4, 5, or more amino acids, which may be any amino acid that occurs naturally or is modified.

For example, the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 11, 15 and 25 or a variant thereof.

For example, the L-FR3 is located between the LCDR2 and the LCDR 3.

For example, the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR 3.

For example, the indirect linkage may include the linkage of two moieties (e.g., the L-FR3 and the LCDR2, or the L-FR3 and the LCDR3) separated by 1, 2, 3, 4, 5, or more amino acids, which may be any amino acid that occurs naturally or is modified.

For example, the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOS 12, 16 and 70 or a variant thereof.

For example, the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOS 26-27 or a variant thereof.

For example, the N-terminus of the L-FR4 is linked directly or indirectly to the C-terminus of the LCDR 3.

For example, the indirect linkage may include 1, 2, 3, 4, 5 or more amino acids between the N-terminal amino acid residue of the L-FR4 and the C-terminal amino acid residue of the LCDR3, which may be any naturally occurring or modified amino acid.

For example, the L-FR4 contains the amino acid sequence shown in SEQ ID NO. 13 or a variant thereof.

In the present application, the heavy chain variable region comprises the framework regions H-FR1, H-FR2, H-FR3, and/or H-FR 4.

For example, the light chain variable region may include the framework regions H-FR1, H-FR2 and H-FR 3.

For example, the light chain variable region may include the framework regions H-FR1, H-FR2 and H-FR 4.

For example, the light chain variable region may include the framework regions H-FR2, H-FR3 and H-FR 4.

For example, the light chain variable region may include the framework regions H-FR1, H-FR3 and H-FR 4.

For example, the light chain variable region may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR 4.

For example, the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR 1. For example, the C-terminus of the H-FR1 is directly linked to the N-terminus of the HCDR1, e.g., the C-terminus of the H-FR1 is indirectly linked to the N-terminus of the HCDR1, e.g., the indirect linkage can include 1, 2, 3, 4, 5, or more amino acids between the C-terminal amino acid residue of the H-FR1 and the N-terminal amino acid residue of the HCDR1, which can be any naturally occurring or modified amino acid.

For example, the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOS 18 and 28 or a variant thereof.

For example, the H-FR2 is located between the HCDR1 and the HCDR 2.

For example, the N-terminus of the H-FR2 is linked directly or indirectly to the C-terminus of the HCDR1, and the C-terminus of the H-FR2 is linked directly or indirectly to the N-terminus of the HCDR 2.

For example, the indirect linkage may include the linkage of two moieties (e.g., the H-FR2 and the HCDR1, or the H-FR2 and the HCDR2) separated by 1, 2, 3, 4, 5, or more amino acids, which may be any naturally occurring or modified amino acid.

For example, the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOS 19, 22 and 71 or a variant thereof.

For example, the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOS: 29-30 or a variant thereof.

For example, the H-FR3 is located between the HCDR2 and the HCDR 3.

For example, the N-terminus of the H-FR3 is linked directly or indirectly to the C-terminus of the HCDR2, and the C-terminus of the H-FR3 is linked directly or indirectly to the N-terminus of the HCDR 3.

For example, the indirect linkage may include the linkage of two moieties (e.g., the H-FR3 and the HCDR2, or the H-FR3 and the HCDR3) separated by 1, 2, 3, 4, 5, or more amino acids, which may be any naturally occurring or modified amino acid.

For example, the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 20 and 31 or a variant thereof.

For example, the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR 3.

For example, the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 21 and 32 or a variant thereof.

Variable, constant, heavy and light chains

In the present application, the isolated antigen binding protein may comprise a heavy chain variable region (VH) and/or a light chain variable region (VL). For example, the heavy chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NOs 72-73 or a variant thereof. For example, the light chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs 74-75 or a variant thereof.

For example, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 33-36 or a variant thereof. For example, the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 37-40 or a variant thereof.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. For example, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. For example, the antibody heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO. 42 or a variant thereof.

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human Ig Kappa (Kappa) constant region. For example, the antibody light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 41 or a variant thereof.

In the present application, the isolated antigen binding protein may comprise an antibody Heavy Chain (HC), and the antibody heavy chain may comprise the amino acid sequence set forth in any one of SEQ ID NOS 45-46 or a variant thereof.

In the present application, the isolated antigen binding protein may comprise an antibody Light Chain (LC), and the antibody light chain may comprise the amino acid sequence set forth in any one of SEQ ID NOS 43-44 or a variant thereof.

For example, the isolated antigen binding protein may comprise the antibody heavy chain and the antibody light chain.

For example, the antibody heavy chain may comprise the amino acid sequence set forth in any one of SEQ ID NOS 45-46 and the antibody light chain may comprise the amino acid sequence set forth in any one of SEQ ID NOS 43-44. The heavy chain may comprise a heavy chain variable region and a heavy chain constant region. The heavy chain variable region may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 72-73. The heavy chain constant region may comprise the amino acid sequence shown as SEQ ID NO 42. The heavy chain variable region may comprise a heavy chain complementarity determining region (HCDR) and a heavy chain framework region (H-FR). The heavy chain complementarity determining region (HCDR) may comprise HCDR1, HCDR2, and HCDR 3. The HCDR1 can comprise the amino acid sequence shown as SEQ ID NO. 1, the HCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 66, and the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 3. The heavy chain framework region (H-FR) may comprise H-FR1, H-FR2, H-FR3, H-FR 4. The H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NOS 18 and 28, H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NOS 19, 22 and 71, H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NOS 20 and 31, and H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NOS 21 and 32.

The light chain may comprise a light chain variable region and a light chain constant region. The light chain variable region may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 74-75. The light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 41. The light chain variable region may comprise a light chain complementarity determining region (LCDR) and a light chain framework region (L-FR). The light chain complementarity determining region (LCDR) may comprise LCDR1, LCDR2, and LCDR 3. The LCDR1 can comprise the amino acid sequence shown as SEQ ID NO.5, the LCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 67, and the LCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 68. The light chain framework region (L-FR) may comprise L-FR1, L-FR2, L-FR3, L-FR 4. The L-FR1 may comprise an amino acid sequence shown in any one of SEQ ID NOS: 10, 14 and 69, L-FR2 may comprise an amino acid sequence shown in any one of SEQ ID NOS: 11, 15 and 25, L-FR3 may comprise an amino acid sequence shown in any one of SEQ ID NOS: 12, 16 and 70, and L-FR4 may comprise an amino acid sequence shown in SEQ ID NO: 13.

For example, the isolated antigen binding protein may be SE2 antibody, SE5 antibody, SE2 chimeric antibody, SE5 chimeric antibody, humanized SE2 antibody, humanized SE5 antibody in the examples of the present application.

Furthermore, the isolated antigen binding proteins described herein may comprise heavy and/or light chain sequences modified with one or more conserved sequences as in SE2, SE5, SE2, SE5, humanized SE2 or humanized SE5 antibodies. By "conservative sequence modification" is meant an amino acid modification that does not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions may be those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in a CDR region of an isolated antigen binding protein described herein can be replaced with other amino acid residues of the ipsilateral chain set. One skilled in the art will recognize that some conservative sequence modifications will not abolish antigen binding, as seen in particular, for example, in Brummell et al, (1993) Biochem 32: 1180-8; de Wildt et al, (1997) prot.Eng.10: 835-41; komissarov et al, (1997) J.biol.chem.272: 26864-26870; hall et al, (1992) J.Immunol.149: 1605-12; kelley and O' Connell (1993) biochem.32: 6862-35; Adib-Conquy et al, (1998) int.Immunol.10:341-6and Beers et al, (2000) Clin.Can.Res.6: 2835-43.

Properties and types of isolated antigen binding proteins

In the present application, the isolated antigen binding protein has one or more of the following properties:

1) can be at 2.4 × 10^-9(ii) KD of M or lower binds to IgE, wherein the KD value is determined by MSD-SET;

2) can inhibit the binding of IgE to its receptor Fc epsilon RIA.

For example, the IgE is human IgE. For example, the isolated antigen binding protein may have a KD value of ≦ 2 × 10 for binding to human-derived IgE antigen as determined by MSD-SET^-9M、≤1.5×10^-9M、≤1×10^-9M M、≤8×10^-10M、≤7×10^-10M、≤6×10^-10M、≤5×10^-10M、≤4.5×10^-10M、≤4×10^-10M、≤3.5×10^-10M、≤3×10^- ¹⁰M、≤2.5×10^-10M、≤2×10^-10M、≤1.8×10^-10M、≤1.5×10^-10M、≤1×10^-10M、≤9×10^-11M、≤8×10^-11M、≤7×10^-11M、≤6×10^-11M、≤5×10^-11M、≤4×10^-11M、≤3×10^-11M、≤2×10^-11M、≤1×10^-11M、≤9×10^-12M。

For example, the isolated antigen binding protein can have an IC50 value for inhibition of binding of IgE to its receptor Fc epsilon RIA as measured by ELISA methods of less than or equal to 2000ng/mL, less than or equal to 1800ng/mL, less than or equal to 1500ng/mL, less than or equal to 1300ng/mL, less than or equal to 1000ng/mL, less than or equal to 950ng/mL, less than or equal to 900ng/mL, less than or equal to 850ng/mL, less than or equal to 800ng/mL, less than or equal to 750ng/mL, less than or equal to 700ng/mL, less than or equal to 650ng/mL, less than or equal to 600ng/mL, less than or equal to 550ng/mL, less than or equal to 500ng/mL, less than or equal to 450ng/mL, less than or equal to 430ng/mL, or less than or equal to 400 ng/mL. SE5 and

for example, the isolated antigen binding protein may have an IC50 value of 4.9X 10 for inhibiting the binding of IgE to its receptor Fc epsilon RIA as measured by ELISA methods^-9M、≤4.5×10^-9M、≤4.0×10^-9M、≤3.5×10^-9M、≤3.0×10^-9M、≤2.5×10^-9M、≤2.0×10^-9M、≤1.5×10^-9M、≤1.0×10^-9M、≤9.0×10^-10M、≤8.0×10^- ¹⁰M、≤7.0×10^-10M、≤6.0×10^-10M、≤5.0×10^-10M、≤4.0×10^-10M、≤3.0×10^-10M、≤2.5×10^-10M。

In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof. For example, an isolated antigen binding protein described herein can include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, murine antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, triabodies, tetrabodies, Fv fragments, scFv fragments, Fab 'fragments, F (ab')2 fragments, and camelized single domain antibodies.

For example, the antibody can be a humanized antibody. For example, an isolated antigen binding protein described herein can be an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest (e.g., a human IgE antigen and comprises Framework Regions (FRs) having substantially the amino acid sequence of a human antibody and Complementarity Determining Regions (CDRs) having substantially the amino acid sequence of a non-human antibody For example, a humanized antibody may further comprise at least a portion of an immunoglobulin constant region (e.g., Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domains of the light chain or and the heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. For example, a humanized antibody may contain only a humanized light chain. For example, a humanized antibody may contain only humanized heavy chains. For example, a humanized antibody may contain only the humanized variable domains of the light chain and/or the humanized heavy chain.

For example, the antigen binding fragment may comprise a Fab, Fab ', F (ab)2, Fv fragment, F (ab')2, scFv, di-scFv and/or dAb.

Immunoconjugates

In the present application, the isolated antigen binding protein may be crosslinked with other compounds (e.g., therapeutic agents) to form the immunoconjugate. Such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents, such as SN-38. In an ADC, the antibody and therapeutic agent may be cross-linked by a linker that is cleavable, for example, a peptide linker, a disulfide linker, or a hydrazone linker. For example, the linker may be a peptide linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Cit, Ser or Glu. ADCs may be as in us patent 7,087,600; 6,989,452, respectively; and 7,129,261; PCT publications WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; U.S. patent publication 20060024317; 20060004081, respectively; and 20060247295.

Nucleic acid molecule, vector, cell, preparation method, pharmaceutical composition and application

In another aspect, the present application also provides isolated nucleic acid molecules that can encode the isolated antigen binding proteins described herein. An isolated nucleic acid molecule described herein can be an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized that can encode an isolated antigen binding protein described herein.

For example, the nucleic acid molecule may comprise the nucleotide sequence set forth in any one of SEQ ID nos. 50-61.

For example, the nucleic acid molecule may comprise a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.2%, 99.4%, 99.6%, 99.8% or 99.9% identical to the nucleotide sequence set forth in any one of SEQ ID No. 50-61.

In another aspect, the present application also provides a vector, which may comprise a nucleic acid molecule as described herein. The vector may be used to express the genetic material element carried by the vector in a host cell by transformation, transduction or transfection of the host cell. For example, the carrier may include: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). As another example, the vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. In addition, the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In another aspect, the present application also provides a cell, which may comprise a nucleic acid molecule described herein or a vector described herein. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation.

For example, the cell may comprise a prokaryotic cell, a yeast cell, or a higher eukaryotic cell. Prokaryotes suitable for this purpose include gram-negative and gram-positive bacteria, for example, enterobacteria, such as E.coli, Enterobacter, Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella), Serratia marcescens (Serratia), and Shigella (Shigella), as well as bacilli, Pseudomonas and Streptomyces.

Eukaryotic microorganisms such as filamentous fungi or yeast may also be suitable cloning or expression hosts for the vectors described herein. The most commonly used among lower eukaryotic host microorganisms is Saccharomyces cerevisiae. A variety of other genera, species and strains are also commonly available and can be used in the present application, for example schizosaccharomyces pombe (schizosaccharomyces pombe), kluyveromyces, candida, trichoderma, streptomyces crassa (Neurospora crassa), and filamentous fungi, such as Neurospora, Penicillium, cervicitis (To1 ypocladium) and aspergillus (aspergilli 11us) host cells, such as aspergillus nidulans (a. nidulans) and aspergillus niger (a. niger).

For example, vertebrate cells and vertebrate cells propagated in culture (tissue culture) suitable cloning or expression hosts for the vectors shown herein.

For example, the cell may comprise a mammalian host cell line. For example, monkey kidney cells, human embryonic kidney cell lines, baby hamster kidney cells, chinese hamster ovary cells, mouse sertoli (sertoli) cells, human cervical cancer cells (HELA), canine kidney cells, human lung cells, human liver cells, mouse breast cancer cells, or NSO cells.

For example, the cell may also include a cell transfected in vitro with a vector of the invention. For example, the cell can be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell may be a mammalian cell. For example, the mammalian cell can be a HEK293 cell.

In another aspect, the present application also provides methods of making an isolated antigen binding protein described herein, which methods can include culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

In another aspect, the present application also provides the use of the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of an IgE-associated disease or disorder.

In another aspect, the present application also provides a method of preventing, ameliorating, or treating an IgE-related disease or disorder, which may comprise administering to a subject in need thereof an isolated antigen binding protein, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition described herein.

In another aspect, the present application also provides the isolated antigen binding protein, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition, which can be used for preventing, alleviating or treating an IgE-related disease or disorder.

For example, an IgE-related disease or disorder can include a therapeutically applicable disease or disorder for which Omalizumab (trade name Xolair) has been approved worldwide (e.g., U.S., china, europe, etc.). For example, the disease or condition may include an allergic disease.

For example, the subject may include human and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In another aspect, the present application also provides a pharmaceutical composition comprising an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.

For example, the pharmaceutical composition may additionally contain one or more other therapeutic agents suitable for treating or preventing an IgE-related disease or disorder.

For example, the pharmaceutically acceptable adjuvant enhances or stabilizes the composition, or facilitates the preparation of the composition. For example, the pharmaceutically acceptable adjuvants may include physiologically compatible solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

For example, the administration can be performed in different ways, e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

For example, the pharmaceutical composition may be administered by a variety of methods known in the art, varying depending on the desired result, route of administration, and/or mode of administration. For example, administration may be intravenous, intramuscular, intraperitoneal or subcutaneous, or proximal to the target site. For example, the pharmaceutical compositions are formulated so that they can be administered intravitreally into the eye. For example, depending on the route of administration, the isolated antigen binding proteins (e.g., antibodies, bispecific and multispecific molecules) may be coated with a material to protect the compound from the action of acids or other natural conditions that can inactivate the compound.

For example, the pharmaceutical composition may be sterile and fluid. Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, or by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

For example, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition. For example, prolonged absorption of the injectable pharmaceutical composition can be brought about by including in the pharmaceutical composition an agent that delays absorption (e.g., aluminum monostearate or gelatin).

For example, the isolated antigen binding protein, the nucleic acid molecule, the vector and/or the cell are prepared for storage in admixture with an optional pharmaceutically acceptable carrier, excipient or stabilizer. For example, the pharmaceutical composition may be in the form of a lyophilized formulation or an aqueous solution.

For example, the pharmaceutically acceptable adjuvant may include a pharmaceutically acceptable carrier, excipient, or stabilizer.

For example, the acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate citrate, acetate, and other organic acids.

For example, a pharmaceutical composition comprising an isolated antigen binding protein described herein can be in a water soluble form.

For example, a pharmaceutical composition for in vivo administration may be sterile. This can be easily achieved by filtration through sterile filtration membranes or other methods. For example, administration of a pharmaceutical composition comprising an isolated antigen binding protein described herein in a sterile aqueous solution can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, otically, transdermally, topically (e.g., gel, ointment, lotion, cream, etc.), intraperitoneally, intramuscularly, intrapulmonary, parenterally, rectally, or intraocularly. In some cases, e.g., treatment of wounds, inflammation, etc., the isolated antigen binding protein may be applied directly as a solution or spray.

For example, the pharmaceutical compositions may be prepared according to methods well known and conventionally practiced in the art. For example, the pharmaceutical composition may be prepared under GMP conditions. Typically, a therapeutically effective dose or effective dose of an IgE binding protein is used in the pharmaceutical compositions of the present application. For example, IgE binding proteins can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). The actual dosage level of the active ingredient in the pharmaceutical compositions of the present application can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response, composition, and mode of administration for a particular patient without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors, e.g., the activity of the particular composition or ester, salt or amide thereof used herein, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and/or substances used in combination with the particular composition used, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and other factors.

For example, subcutaneous administration may be used in situations where a patient may self-administer a pharmaceutical composition. Many protein therapeutics are not sufficiently effective to allow formulation of a therapeutically effective dose for subcutaneous administration in the maximum acceptable volume. The antigen binding proteins disclosed herein may be suitable for subcutaneous administration, e.g., may have increased potency, increased plasma half-life, and increased solubility.

It is known in the art that protein therapeutics can be delivered by IV infusion or bolus injection. Antigen binding proteins disclosed herein can also be delivered using such methods.

In another aspect, the present application provides a method of detecting IgE in a sample, the method comprising administering an isolated antigen binding protein described herein.

For example, a sample obtained from a subject is contacted with an isolated antigen binding protein (e.g., an IgE binding protein) described herein. For example, where an IgE binding protein is labeled with a detectable label or reporter molecule or an anti-IgE binding protein is used as a capture ligand to selectively isolate IgE from a patient sample. Alternatively, unlabeled anti-IgE binding proteins may be bound to a second antibody, which itself is detectably labeled, for use in detection applications. The detectable label or reporter molecule may be a radioisotope, e.g., such as³H、¹⁴C、³²P、³⁵S or¹²⁵I; but also fluorescent or chemiluminescent moieties, such as fluorescein isothiocyanate, rhodamine; or an enzyme, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase; specific exemplary assays that can be used to detect or measure IgE in a sample include enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA) and Fluorescence Activated Cell Sorting (FACS).

Samples that may be used in the IgE detection of the present application may include any tissue or fluid sample obtained from a subject under normal or pathological conditions that contains a detectable amount of IgE protein or fragments thereof. For example, the level of IgE protein in a particular sample obtained from a healthy subject (e.g., a subject not suffering from an IgE-related disease) can be measured to initially establish a baseline or standard level of IgE. This baseline level of IgE is then compared to the level of IgE measured in a sample obtained from an individual suspected of having an IgE-associated disease or disorder or a symptom associated with the disorder.

The IgE binding proteins may be free of additional labels, or may contain an N-terminal or C-terminal label. For example, the label is biotin. In a binding assay, the position of the label (if present) can determine the orientation of the peptide relative to the surface on which the peptide is bound. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will keep the C-terminal portion of the peptide away from the surface.

The present application also includes the following embodiments:

1. an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 72-73 or a variant thereof; and which comprises at least one CDR in a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs 74-75 or a variant thereof.

2. The isolated antigen binding protein of embodiment 1, having one or more of the following properties:

1) can be at 2.4 × 10^-9(ii) KD of M or lower binds to IgE, wherein the KD value is determined by MSD-SET;

2) can inhibit the binding of IgE to its receptor Fc epsilon RIA.

3. The isolated antigen binding protein of any of embodiments 1-2, comprising HCDR1 in the heavy chain variable region as set forth in any one of amino acid sequences SEQ ID NOs 33-36.

4. The isolated antigen binding protein of any of embodiments 1-3, comprising HCDR2 in the heavy chain variable region as set forth in any one of amino acid sequences SEQ ID NOs 33-36.

5. The isolated antigen binding protein of any of embodiments 1-4, comprising HCDR3 in the heavy chain variable region as set forth in any one of amino acid sequences SEQ ID NOs 33-36.

6. The isolated antigen binding protein of embodiment 3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO. 1.

7. The isolated antigen binding protein of embodiment 4, wherein the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 66.

8. The isolated antigen binding protein of embodiment 7, wherein the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 2 and 4.

9. The isolated antigen binding protein of embodiment 5, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO. 3.

10. The isolated antigen binding protein of any one of embodiments 1-9, comprising LCDR1 in the variable region of the light chain as set forth in any one of amino acid sequences of SEQ ID NOs 37-40.

11. The isolated antigen binding protein of any of embodiments 1-10, comprising LCDR2 in the variable region of the light chain as set forth in any one of amino acid sequences of SEQ ID NOs 37-40.

12. The isolated antigen binding protein of any of embodiments 1-11, comprising LCDR3 in the variable region of the light chain as set forth in any one of amino acid sequences of SEQ ID NOs 37-40.

13. The isolated antigen binding protein of embodiment 10, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 5.

14. The isolated antigen binding protein of embodiment 11, wherein the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO 67.

15. The isolated antigen binding protein of embodiment 14, wherein the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 6and 8.

16. The isolated antigen binding protein of embodiment 12, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68.

17. The isolated antigen binding protein of embodiment 16, wherein the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 7 and 9.

18. The isolated antigen binding protein of any one of embodiments 1-17, comprising an antibody or antigen binding fragment thereof.

19. The isolated antigen binding protein of embodiment 18, wherein the antigen binding fragment comprises Fab, Fab', F (ab)₂Fv fragment, F (ab')₂scFv, di-scFv and/or dAb.

20. The isolated antigen binding protein of any one of embodiments 1-19, wherein the light chain variable region comprises the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

21. The isolated antigen binding protein of embodiment 20, wherein the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1 and the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOS 10, 14 and 69.

22. The isolated antigen binding protein of embodiment 21, wherein said L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOS 23-24.

23. The isolated antigen binding protein of any one of embodiments 20-22, wherein the L-FR2 is located between the LCDR1 and the LCDR2 and the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 11, 15, and 25.

24. The isolated antigen binding protein of any one of embodiments 20-23, wherein the L-FR3 is located between the LCDR2 and the LCDR3 and the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs 12, 16, and 70.

25. The isolated antigen binding protein of any one of embodiments 20-24, wherein said L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOS 26-27.

26. The isolated antigen binding protein of any of embodiments 20-25, wherein the N-terminus of L-FR4 is linked to the C-terminus of the LCDR3 and the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOS 13 and 17.

27. The isolated antigen binding protein of any of embodiments 1-26, wherein the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 37-40.

28. The isolated antigen binding protein of any one of embodiments 1-27, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human Kappa light chain constant region.

29. The isolated antigen binding protein of embodiment 28, wherein the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 41.

30. The isolated antigen binding protein of any one of embodiments 1-29, comprising an antibody light chain comprising the amino acid sequence set forth in any one of SEQ ID NOs 43-44.

31. The isolated antigen binding protein of any one of embodiments 1-30, wherein the heavy chain variable region comprises the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

32. The isolated antigen binding protein of embodiment 31, wherein the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1 and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOS 18 and 28.

33. The isolated antigen binding protein of any one of embodiments 31-32, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs 19, 22, and 71.

34. The isolated antigen binding protein of any one of embodiments 31-33, wherein said H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOS: 29-30.

35. The isolated antigen binding protein of any one of embodiments 31-34, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NOs 20 and 31.

36. The isolated antigen binding protein of any one of embodiments 31-35, wherein the N-terminus of H-FR4 is linked to the C-terminus of the HCDR3 and the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs 21 and 32.

37. The isolated antigen binding protein of any of embodiments 1-36, wherein the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs 33-36.

38. The isolated antigen binding protein of any one of embodiments 1-37, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

39. The isolated antigen binding protein of any one of embodiments 1-38, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region.

40. The isolated antigen binding protein of any one of embodiments 1-39, wherein the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 42.

41. The isolated antigen binding protein of any one of embodiments 1-40, comprising an antibody heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOS 45-46.

42. The isolated antigen binding protein of any one of embodiments 1-41, wherein the antigen is IgE.

43. The isolated antigen binding protein of embodiment 42, wherein the IgE is human IgE.

44. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of embodiments 1-43.

45. A vector comprising the nucleic acid molecule of embodiment 44.

46. A cell comprising the nucleic acid molecule of embodiment 44 or the vector of embodiment 45.

47. A method of making the isolated antigen binding protein of any one of embodiments 1-43, the method comprising culturing the cell of embodiment 46 under conditions such that the isolated antigen binding protein of any one of embodiments 1-43 is expressed.

An immunoconjugate comprising the isolated antigen binding protein of any one of embodiments 1-43.

49. A pharmaceutical composition comprising an isolated antigen binding protein of any one of embodiments 1-43, a nucleic acid molecule of embodiment 44, a vector of embodiment 45, a cell of embodiment 46, and/or an immunoconjugate of embodiment 48, and optionally a pharmaceutically acceptable adjuvant.

50. Use of the isolated antigen binding protein of any one of embodiments 1-43, the nucleic acid molecule of embodiment 44, the vector of embodiment 45, the cell of embodiment 46, the immunoconjugate of embodiment 48, and/or the pharmaceutical composition of embodiment 49 in the manufacture of a medicament for preventing, ameliorating, and/or treating an IgE-associated disease or disorder.

51. A method of preventing, ameliorating, or treating an IgE-associated disease or disorder, the method comprising administering to a subject in need thereof an isolated antigen binding protein of any one of embodiments 1-43, an immunoconjugate of embodiment 48, and/or a pharmaceutical composition of embodiment 49.

52. An isolated antigen binding protein of any one of embodiments 1-43, a nucleic acid molecule of embodiment 44, a vector of embodiment 45, a cell of embodiment 46, an immunoconjugate of embodiment 48, and/or a pharmaceutical composition of embodiment 49 for use in the prevention, amelioration, or treatment of an IgE-associated disease or disorder.

53. A method of detecting IgE in a sample, the method comprising administering the isolated antigen binding protein of any one of embodiments 1-43.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

EXAMPLE 1 preparation of anti-human IgE murine monoclonal antibody

(1) Immunization of mice

Four-week-old BALB/c mice were injected subcutaneously with 20. mu.g of the Fc region (CH2-4) protein (IgE-Fc) of recombinantly expressed human IgE in Freund's complete adjuvant. Injections were given every three to four weeks for a total of five times. Finally, a single injection of 20. mu.g of recombinant human IgE-Fc protein was given intraperitoneally.

(2) ELISA method for determining binding of mouse serum and human IgE-Fc antigen

The microplate (Costar) was coated with recombinant human IgE-Fc protein overnight at room temperature. The coating solution was discarded, each well was blocked with 2.5% skim milk dissolved in Phosphate Buffered Saline (PBS) for 0.5 hour, and the wells were washed with PBS containing 0.05% Tween-20. Diluted serum and irrelevant antibody (Irrevalent mAb) as a negative control were then added separately, incubated at room temperature for 1 hour, the wells were washed with PBS containing 0.05% Tween-20, and then 50. mu.l of HRP-labeled goat anti-mouse IgG polyclonal antibody (Jackson Laboratory) was added per well as a detection antibody. After serum testing, mice were identified that contained high levels of serum anti-recombinant human IgE-Fc protein antibodies.

(3) Cell fusion, screening and murine monoclonal antibody preparation

After identifying mice with high levels of anti-human IgE-Fc antibody serum by serum testing, the spleens of the mice were removed and fused with a mouse myeloma Sp2/0 cell line. Mixing 5x 10⁸Sp2/0 cell and 5x 10⁸The splenocytes were fused in a 50% polyethylene glycol (PEG, molecular weight 1450) and 5% Dimethyl Maple (DMSO) solution. Iscove's medium (containing 10% fetal bovine serum, 100 units/mL penicillin, 100. mu.g/mL streptomycin, 10mM hypoxanthine, 0.4. mu.M aminopterin and 1.6mM thymidine) was used to adjust spleen cell counts to 7.5X 10⁵0.2mL of the suspension was added to the wells of a 96-well plate. The cells were incubated at 37 ℃ in a 5% CO2 incubator. After 10 days, the medium in each well was removed to test the binding ability of the murine antibody to human IgE-Fc, and positive wells that bound human IgE-Fc were selected. And subcloning the fused cells in the hole containing the antibody capable of combining with human IgE-Fc, and screening by an ELISA method to obtain a hybridoma cell strain expressing the high-affinity mouse monoclonal antibody.

Purified murine monoclonal antibodies were prepared to obtain antibody samples to be tested. Specific antibody-producing cell clones were cultured in RPMI1640 medium supplemented with 10% FCS. When the cell density reaches about 5x 10⁵At individual cells/ml, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged to collect a culture supernatant. Use of eggsWhite G column was used for purification of the antibody. The monoclonal antibody eluate was dialyzed against 150mM NaCl. The dialyzed solution was filter sterilized through a 0.2 μm filter to obtain an antibody sample to be tested. Monoclonal antibodies SE3(11/5), SE5(4/23), SE2 and SE5 were prepared.

Example 2 detection of blocking Activity by murine monoclonal antibody

The blocking activity of the purified murine monoclonal antibody to block the binding of human IgE-Fc to its receptor Fc epsilon RIA was examined using ELISA method. Specifically, the recombinant expressed human Fc epsilon RIA-Fc protein (amino acid sequence shown in SEQ ID NO: 48) was diluted with PBS to a final concentration of 2.5. mu.g/mL, 100. mu.L/well coated plate (purchased from Corning, cat. No.: 42592), and incubated overnight at 4 ℃. The following day the coating solution was discarded, blocked with 2.5% skim milk in PBS for 1 hour, and the wells were washed with PBS containing 0.05% Tween (Tween) 20. mu.g/mL of the murine or control antibody omabead prepared in example 1 (from Novartis, cat # Xolair) was diluted 2-fold in gradient from 5. mu.g/mL (i.e., 5. mu.g/mL, 2.5. mu.g/mL, 1.25. mu.g/mL, 0.625. mu.g/mL up to 0.002441. mu.g/mL) by adding a premix containing 1. mu.g/mL of biotin-labeled human IgE-Fc (amino acid sequence shown in SEQ ID NO: 49) and the gradient diluted murine or control antibody, which was premixed for 10 minutes, and incubated at room temperature for 1 hour. The supernatant was discarded, and the wells were washed with PBS containing 0.05% Tween (Tween)20, diluted 1:1000 with HRP-labeled streptavidin (purchased from Biochemie, cat # D111054-0001), and then detected. As shown in FIG. 1, each of the purified murine monoclonal antibodies was able to block the binding of human IgE-Fc to its receptor FceRIa to different degrees, and SE2 and SE5 showed stronger blocking activities than those of the control antibody omalizumab.

EXAMPLE 3 preparation of anti-human IgE chimeric antibodies

1) Cloning of heavy chain variable region of murine antibody

To obtain DNA sequences encoding the heavy and light chain variable regions of the murine antibody, cDNA was prepared by isolating mRNA from mouse hybridoma cells using mRNA purification kit (NEB) (SMARTer RACE kit, Clontech). The heavy chain variable region DNA fragment was cloned from cDNA by Polymerase Chain Reaction (PCR). A mixture of 0.4. mu.M of primer 1(5'-ctaatacgactcactatagggcAAGCAGTGGTATCAACGCAGAGT-3', shown as SEQ ID NO: 62) and 2. mu.M of primer 2(5'-ctaatacgactcactatagggc-3', shown as SEQ ID NO: 63) was used as the 5' -primer in PCR. The 3 '-primer of PCR was primer 3(5' -catcccagggtcaccatggagtta-1, shown in SEQ ID NO: 64). Primer 3 is homologous antisense to the mouse IgG1 heavy chain constant region. The DNA fragment of the heavy chain variable region obtained after gel purification was cloned into TOPO-TA vector (Invitrogen) and sequenced to obtain the nucleic acid sequence (shown as SEQ ID NO: 50) and amino acid sequence (shown as SEQ ID NO: 33) encoding the variable region of the SE2 mouse hybridoma heavy chain, the amino acid sequences of the complementarity determining regions CDR-H1 (shown in SEQ ID NO: 1), CDR-H2 (shown in SEQ ID NO: 2) and CDR-H3 (shown in SEQ ID NO: 3) were determined by the Kabat definition, and the variable region nucleic acid sequence (shown as SEQ ID NO: 51) and the amino acid sequence (shown as SEQ ID NO: 34) encoding the heavy chain of the SE5 mouse hybridoma, as well as the amino acid sequences CDR-H1 (shown as SEQ ID NO: 1), CDR-H2 (shown as SEQ ID NO: 4) and CDR-H3 (shown as SEQ ID NO: 3) of the complementarity determining regions thereof. The complementarity determining regions are defined in Kabat E.et al Sequences of Proteins of Immunological Interest 5 th edition U.S. department of Health and Human Services, NIH Publication No. 91-3242.

2) Cloning of light chain variable region of murine antibody

In a manner similar to that of step 1), a light chain variable region DNA fragment was cloned from cDNA using the above 5 '-primer mixture (primer 1 and primer 2) and another 3' -primer (primer 4, 5'-gactgaggcacctccagatgttaa-3', shown in SEQ ID NO: 65) homologous to and antisense to the mouse immunoglobulin light chain constant region. These obtained DNA fragments were cloned into TOPO-TA vector and sequenced to obtain the variable region nucleic acid sequence (as shown in SEQ ID NO: 54) and amino acid sequence (as shown in SEQ ID NO: 38) encoding the light chain of SE2 mouse hybridoma, and the amino acid sequences CDR-L1 (as shown in SEQ ID NO: 5), CDR-L2 (as shown in SEQ ID NO: 6) and CDR-L3 (as shown in SEQ ID NO: 7) of the complementarity determining region thereof, and the variable region nucleic acid sequence (shown as SEQ ID NO: 55) and the amino acid sequence (shown as SEQ ID NO: 39) encoding the SE5 mouse hybridoma light chain, and the amino acid sequences CDR-L1 (shown as SEQ ID NO: 5), CDR-L2 (shown as SEQ ID NO: 8) and CDR-L3 (shown as SEQ ID NO: 9) of the complementarity determining regions thereof.

3) Chimeric antibody preparation

The heavy chain and light chain variable region sequences of the murine monoclonal antibody cloned in step 2) were inserted into a eukaryotic expression vector of pCDNA3.1 (purchased from vast Lingzi) containing a signal peptide (shown in SEQ ID NO: 47) and a human IgG1 heavy chain constant region (shown in SEQ ID NO: 42), or containing a signal peptide (shown in SEQ ID NO: 47) and a human Kappa light chain constant region (shown in SEQ ID NO: 41), respectively, by a seamless cloning method, and after confirming the correctness by DNA sequencing, the plasmid was prepared by extracting a kit from a plasmid of NucleoBond Xtra Midi Plus (MACHEREY-NAGEL) after amplification in a DH5 alpha strain. The prepared chimeric monoclonal antibody heavy and light chain plasmids were co-transferred to Expi293 cells (purchased from Thermo fisher) using pei (polytcience) to transiently express the monoclonal antibody. Transiently expressed antibodies were purified using a protein a column and the monoclonal antibody eluate was dialyzed against PBS. The dialyzed solution was filter sterilized through a 0.2 μm filter to obtain an antibody sample to be tested.

Example 4 detection of blocking Activity of chimeric antibody

To further verify the activity of the cloned and recombinantly expressed chimeric monoclonal antibody, the blocking activity of the purified chimeric antibody to block the binding of human IgE-Fc to its receptor fceria was examined using an ELISA method similar to example 2. Specifically, the recombinant expressed human Fc epsilon RIA-Fc protein (amino acid sequence shown in SEQ ID NO: 48) was diluted with PBS to a final concentration of 1. mu.g/mL, 100. mu.L/well was coated on an enzyme-labeled plate (purchased from Corning, cat. No.: 42592), and the plate was incubated overnight at 4 ℃. The following day the coating solution was discarded, blocked with 2.5% skim milk in PBS for 1 hour, and the wells were washed with PBS containing 0.05% Tween (Tween) 20. A premix of pre-mixed 10 min containing 100ng/mL biotin-labeled human IgE-Fc (amino acid sequence as shown in SEQ ID NO: 49) and a gradient dilution of chimeric or murine antibody was added, the chimeric or murine antibody was diluted 3-fold in a gradient starting at 2. mu.g/mL (i.e., 2. mu.g/mL, 666.67ng/mL, 222.22ng/mL, 74.07ng/mL … … up to 2.74ng/mL and finally 0ng/mL) and incubated at room temperature for 1 hour. The supernatant was discarded, and the wells were washed with PBS containing 0.05% Tween (Tween)20, diluted 1:1000 with HRP-labeled streptavidin (purchased from Biochemie, cat # D111054-0001), and then detected. According to the following formula: the inhibition rate (%) at the antibody concentration X (OD 450 reading at the antibody concentration of 0-OD 450 reading at the antibody concentration of X)/OD 450 reading X100 at the antibody concentration of 0, and the inhibition rate was calculated for each antibody and each concentration. The detection result is shown in figure 2, the chimeric recombinant monoclonal antibody constructed by the mouse-derived monoclonal variable region sequence obtained by cloning has similar blocking activity with the mouse-derived mother antibody purified by the hybridoma.

Example 5 preparation of humanized antibody against human IgE

1) Humanization of anti-human IgE murine monoclonal antibodies

Based on the antibody variable region structure of homology modeling, key amino acid sites which may influence the antigen-antibody interaction are determined, and the key amino acid sites are subjected to back mutation, and a CDR grafting method is used to obtain humanized amino acid sequences of the variable regions of the SE2 and SE5 antibodies, wherein the amino acid sequence of the humanized SE2 heavy chain variable region is shown as SEQ ID NO. 35, the amino acid sequence of the humanized SE2 light chain variable region is shown as SEQ ID NO. 39, the amino acid sequence of the humanized SE5 heavy chain variable region is shown as SEQ ID NO. 36, and the amino acid sequence of the humanized SE5 light chain variable region is shown as SEQ ID NO. 40. The DNA sequence obtained by reverse translation of the amino acid sequence is synthesized, the SE2 heavy chain humanized nucleotide sequence is shown as SEQ ID NO. 52, the SE2 light chain humanized nucleotide sequence is shown as SEQ ID NO. 56, the SE5 heavy chain humanized nucleotide sequence is shown as SEQ ID NO. 53, the SE5 light chain humanized nucleotide sequence is shown as SEQ ID NO. 57, and the obtained DNA is used for constructing a recombinant antibody expression vector.

2) Preparation of humanized antibody against human IgE

The heavy chain and light chain variable regions of the humanized monoclonal antibody obtained in step 1) were inserted into a eukaryotic expression vector of pCDNA3.1 (purchased from vast Lingzi) containing a signal peptide (shown as SEQ ID NO: 47) and a human IgG1 heavy chain constant region (shown as SEQ ID NO: 42), or a signal peptide (shown as SEQ ID NO: 47) and a human Kappa light chain constant region (shown as SEQ ID NO: 41), respectively, by a seamless cloning method, and after confirming the correctness by DNA sequencing, plasmids were prepared using a reagent kit extracted from a NucleoBond Xtra Midi Plus (MACHEREY-NAGEL) plasmid. The prepared humanized monoclonal antibody heavy and light chain plasmids were co-transformed with Expi293 cells (purchased from Thermo fisher) using pei (polytcience) to transiently express the monoclonal antibody. Transiently expressed antibodies were purified using a protein a column and the monoclonal antibody eluate was dialyzed against PBS. The dialyzed solution was filter sterilized through a 0.2 μm filter to obtain an antibody sample to be tested.

Example 6 detection of blocking Activity of anti-human IgE humanized antibody

1) The blocking activity of purified murine, chimeric and humanized antibodies to block the binding of human IgE-Fc to its receptor fceria was examined using ELISA methodology similar to that of example 2. Specifically, the recombinant expressed human Fc epsilon RIA-Fc protein (amino acid sequence shown in SEQ ID NO: 48) was diluted with PBS to a final concentration of 1. mu.g/mL, 100. mu.L/well was coated on an enzyme-labeled plate (purchased from Corning, cat. No.: 42592), and the plate was incubated overnight at 4 ℃. The following day the coating solution was discarded, blocked with 2.5% skim milk in PBS for 1 hour, and the wells were washed with PBS containing 0.05% Tween (Tween) 20. A premix of pre-mixed 10 min containing 100ng/mL biotin-labeled human IgE-Fc (amino acid sequence as shown in SEQ ID NO: 49) and gradient dilutions of each antibody, each antibody was diluted 3-fold from 1. mu.g/mL (i.e., 1. mu.g/mL, 333.33ng/mL, 111.11ng/mL, 37.04ng/mL … … up to 1.37ng/mL, final spot of 0ng/mL), and incubated at room temperature for 1 hour. After discarding the supernatant, the wells were washed with PBS containing 0.05% Tween (Tween)20 and detected with HRP-labeled streptavidin (purchased from Biochemie, cat # D111054-0001). As shown in FIGS. 3 to 5, the detection results show that the recombinant expressed humanized monoclonal antibodies SE2 and SE5 have blocking activities similar to those of the corresponding chimeric antibody and murine parent antibody, respectively (FIG. 3).

2) The blocking activity of SE2 humanized antibody, SE5 humanized antibody and omalizumab blocking the binding of human IgE-Fc to its receptor FceRIa was tested according to the method of step 1) of this example. In a comparative experiment between SE2 humanized antibody and omalizumab, antibody concentrations were diluted 3-fold in a gradient from 3. mu.g/mL (i.e., 3. mu.g/mL, 1. mu.g/mL, 333.33ng/mL, 111.11ng/mL, 37.04ng/mL … … up to 4.12ng/mL and finally 0 ng/mL); in a comparative experiment of SE5 humanized antibody and omalizumab, antibody concentrations were diluted from 10nM with a 3-fold gradient (i.e., 10nM, 3.33nM, 1.11nM, 370.37pM … … up to 13.72pM, and finally 0 pM). Control antibodies (i.e., Control in FIG. 5, which is the her2 antibody pertuzumab isotype, sequences from the IMGT antibody library) were included in the SE5 comparison.

According to the following formula: the inhibition rate (%) at the antibody concentration X (OD 450 reading at the antibody concentration of 0-OD 450 reading at the antibody concentration of X)/OD 450 reading X100 at the antibody concentration of 0, and the inhibition rate was calculated for each antibody and each concentration. The results are shown in FIGS. 4-5, in which the blocking IC50 values of SE2 and omalizumab are about 416.7ng/mL and 2110ng/mL, respectively, and the blocking IC50 values of SE5 and omalizumab are about 266.2pM and 4996pM, respectively, for both the SE2 humanized antibody and the SE5 humanized antibody, compared to the control antibody omalizumab.

Example 7 affinity assay for anti-human IgE humanized antibodies

Equilibrium affinity of humanized antibodies and human IgE was determined using the hypersensitivity electrochemiluminescence assay-solution equilibrium titration method (MSD-SET) as described by Estep P. et al, MAbs,2013.5(2): p.270-8. Solution Equilibrium Titration (SET) was performed in PBS + 0.1% IgG-free BSA buffer (PBSF), where the antigen human IgE (purchased from: abcam, cat # ab65866) was kept constant at 10-100pM and incubated with 3-fold serial dilutions of humanized antibody starting from 0.1-100 nM. Specifically, the antigen human IgE remained constant at 13.33pM when assayed against SE2 and SE5, and the humanized antibodies SE2 and SE5 were serially three-fold diluted from 166.67pM to 0.0085 pM; the antigen human IgE remained constant at 133.3pM when assayed against omalizumab, which was serially diluted 3-fold from 66.67nM to 1.13 pM. 20nM antibody diluted in PBS was coated onto standard binding MSD-ECL plates (purchased from MSD, cat # L15XA-3) overnight at 4 ℃. MSD-ECL plates were blocked with 1% Casein for 30 min while shaking at 700 rpm. The MSD-ECL plates were then washed 3 times with wash buffer (PBS + 0.05% Tween 20). Samples of antigen human IgE incubated with 2-fold serial dilutions of humanized antibody were added to MSD-ECL plates, incubated for 150s with shaking at 700rpm, and then washed once. PBSF containing 250ng/mL sulfotag-labeled streptavidin was added to the MSD-ECL plates, and after 5 minutes, the MSD-ECL plates were washed 3 times with wash buffer (PBS + 0.05% Tween 20). Reading was performed on an MSD Sector Imager instrument (model: MESO Quickplex SQ 120) by adding 1 Xreading buffer T (purchased from MSD, cat # R92 TC-1). The percentage of free antigen was plotted as a function of titrated antibody in Prism-GraphPad6 software and fitted to a quadratic equation to extract KD. The results of the measurements are shown in Table 1 below.

TABLE 1MSD-SET method for testing the equilibrium affinity of humanized and control antibodies to human IgE

	KD.(pM)
		Humanized antibody SE2	10.880
Humanized antibody SE5	9.995
		Omalizumab	2489

From the results shown in table 1, it can be seen that the equilibrium affinities of the binding of the humanized recombinant monoclonal antibodies SE2 and SE5 to human IgE are about 10.880pM and 9.995pM, respectively, and the affinity of the control antibody omalizumab is about 2489pM, which is significantly improved.

Sequence listing

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<223> SE5 heavy chain humanized H-FR2

<400> 29

Asn Trp Ile Arg Gln Pro Pro Gly Asn Gly Leu Glu Trp Leu Gly

1 5 10 15

<210> 30

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 heavy chain humanized H-FR2

<400> 30

Asn Trp Ile Arg Gln Pro Pro Gly Asn Gly Leu Glu Trp Met Gly

1 5 10 15

<210> 31

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5/SE2 heavy chain humanized H-FR3

<400> 31

Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys

1 5 10 15

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Val Tyr

20 25 30

<210> 32

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5/SE2 heavy chain humanized H-FR4

<400> 32

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 33

<211> 136

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 heavy chain variable region amino acid sequence

<400> 33

Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Phe

1 5 10 15

Leu Ser Asp Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr

35 40 45

Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Arg Tyr Asn Pro

65 70 75 80

Ser Leu Lys Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln

85 90 95

Phe Phe Leu Gln Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr

100 105 110

Phe Cys Val Tyr Gly Ser Ser Gln Arg Phe Phe Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 34

<211> 136

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 heavy chain variable region amino acid sequence

<400> 34

Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Phe

1 5 10 15

Leu Ser Asp Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr

35 40 45

Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60

Glu Trp Leu Gly Tyr Ile Asn Tyr Ser Gly Thr Thr Arg Tyr Asn Pro

65 70 75 80

Ser Leu Lys Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln

85 90 95

Phe Phe Leu Gln Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr

100 105 110

Phe Cys Val Tyr Gly Ser Ser Gln Arg Phe Phe Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 35

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 heavy chain variable region humanized amino acid sequence

<400> 35

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Gly Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Arg Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Val Tyr Gly Ser Ser Gln Arg Phe Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 36

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 heavy chain variable region humanized amino acid sequence

<400> 36

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Gly Leu Glu Trp

35 40 45

Leu Gly Tyr Ile Asn Tyr Ser Gly Thr Thr Arg Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Val Tyr Gly Ser Ser Gln Arg Phe Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 37

<211> 135

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 light chain variable region amino acid sequence

<400> 37

Met His His Thr Ser Met Gly Ile Lys Met Glu Ser Gln Ile Gln Val

1 5 10 15

Phe Val Phe Val Phe Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Leu

20 25 30

Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Ile Gly Asp Arg Val

35 40 45

Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Asp Val Ala Trp

50 55 60

Tyr His Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala

65 70 75 80

Ser Phe Arg Tyr Ala Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

85 90 95

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu

100 105 110

Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Leu Phe Thr Phe Gly Ser

115 120 125

Gly Thr Lys Leu Glu Ile Lys

130 135

<210> 38

<211> 138

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 light chain variable region amino acid sequence

<400> 38

Met Gly Glu Met His His Thr Ser Met Gly Ile Lys Met Glu Ser Gln

1 5 10 15

Ile Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser Gly Val Asp Gly

20 25 30

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

35 40 45

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Asp

50 55 60

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

65 70 75 80

Tyr Ser Ala Ser Trp Arg Tyr Ala Gly Val Pro Asp Arg Phe Thr Gly

85 90 95

Ser Arg Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala

100 105 110

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Asn Leu Phe Thr

115 120 125

Phe Gly Ser Gly Thr Lys Leu Glu Ile Gln

130 135

<210> 39

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 light chain variable region humanized amino acid sequence

<400> 39

Asp Ile Leu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Ser Ile Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Arg Tyr Ala Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Leu Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 40

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 light chain variable region humanized amino acid sequence

<400> 40

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Ser Ile Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Trp Arg Tyr Ala Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Asn Leu Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized SE2/SE5 light chain constant region

<400> 41

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> 42

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> humanized SE5/SE2 heavy chain constant region

<400> 42

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 Asp Glu

225 230 235 240

Leu 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> 43

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> full Length sequence of humanized SE2 light chain

<400> 43

Asp Ile Leu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Ser Ile Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Arg Tyr Ala Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Leu Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 44

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> full Length sequence of humanized SE5 light chain

<400> 44

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Ser Ile Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Trp Arg Tyr Ala Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Asn Leu Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 45

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> full-length sequence of humanized SE2 heavy chain

<400> 45

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Gly Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Arg Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Val Tyr Gly Ser Ser Gln Arg Phe Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 46

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> full-length sequence of humanized SE5 heavy chain

<400> 46

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Gly Leu Glu Trp

35 40 45

Leu Gly Tyr Ile Asn Tyr Ser Gly Thr Thr Arg Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys

85 90 95

Val Tyr Gly Ser Ser Gln Arg Phe Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 47

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Signal peptide amino acid sequence in vector

<400> 47

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 48

<211> 405

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fc epsilon RIA-Fc protein amino acid sequence

<400> 48

Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile

1 5 10 15

Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe

20 25 30

Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu

35 40 45

Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly

50 55 60

Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr

65 70 75 80

Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val

85 90 95

Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn

100 105 110

Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys

115 120 125

Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu

130 135 140

Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr

145 150 155 160

Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Glu Pro Lys

165 170 175

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

180 185 190

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

195 200 205

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

210 215 220

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

225 230 235 240

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

245 250 255

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

260 265 270

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

275 280 285

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

290 295 300

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

305 310 315 320

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

325 330 335

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

340 345 350

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

355 360 365

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

370 375 380

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

385 390 395 400

Leu Ser Pro Gly Lys

405

<210> 49

<211> 350

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgE-Fc amino acid sequence

<400> 49

Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser

1 5 10 15

Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu

20 25 30

Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp

35 40 45

Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu

50 55 60

Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His

65 70 75 80

Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His

85 90 95

Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly

100 105 110

Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg

115 120 125

Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp Leu Ala Pro Ser Lys

130 135 140

Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asn

145 150 155 160

His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val

165 170 175

Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr

180 185 190

Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg

195 200 205

Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala

210 215 220

Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Ala

225 230 235 240

Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu

245 250 255

His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro

260 265 270

Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val

275 280 285

Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val

290 295 300

His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val

305 310 315 320

Asn Pro Gly Lys Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu

325 330 335

Trp His Glu Gly His His His His His His His His His His

340 345 350

<210> 50

<211> 408

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 heavy chain variable region nucleic acid sequence

<400> 50

atgagagtgc tgattctttt gtggctgttc acagcctttc ctggtttcct gtctgatgtg 60

cagcttctgg agtcgggacc tggcctggtg aaaccttctc agtctctgtc cctcacctgc 120

actgtcactg gctactcaat caccagtgat tatgcctgga actggatccg gcagtttcca 180

ggaaacaaac tggagtggat gggctacata agctacagtg gtagtactag gtacaatcca 240

tctctcaaaa gtcgaatttc tatcagtcga gacacatcca agaaccagtt cttcctgcag 300

ttgaattctg tgacttctga ggacacagcc acatatttct gtgtttacgg aagtagccag 360

agattctttg actactgggg ccaaggcacc actctcacag tctcctca 408

<210> 51

<211> 408

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 heavy chain variable region nucleic acid sequence

<400> 51

atgagagtgc tgattctttt gtggctgttc acagcctttc ctggtttcct gtctgatgtg 60

cagcttctgg agtcgggacc tggcctggtg aaaccttctc agtctctgtc cctcacctgc 120

actgtcactg gctactcaat caccagtgat tatgcctgga actggatccg gcagtttcca 180

ggaaacaaat tggagtggct gggctacata aactacagtg gtaccactcg ctacaatcca 240

tctctcaaaa gtcgaatttc tatcagtcgc gacacatcca agaaccagtt cttcctgcag 300

ttgaattctg tgacttctga ggacacagcc acatatttct gtgtttacgg aagtagccag 360

agattctttg actactgggg ccaaggcacc actctcacag tctcctca 408

<210> 52

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 heavy chain variable region humanized nucleotide sequence

<400> 52

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60

acctgtaccg tgtccggcta cagcatcacc agcgactacg cctggaactg gatcagacag 120

cctcctggca atggactgga atggatgggc tacatcagct acagcggcag caccagatac 180

aaccccagcc tgaagtcccg gatcaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat accgccgtgt acttttgtgt gtacggcagc 300

agccagcggt tcttcgatta ttggggccag ggcaccctgg tcaccgtttc ttct 354

<210> 53

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 heavy chain variable region humanized nucleotide sequence

<400> 53

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60

acctgtaccg tgtccggcta cagcatcacc agcgactacg cctggaactg gatcagacag 120

cctcctggca atggactgga atggctgggc tacatcaact acagcggcac caccagatac 180

aaccccagcc tgaagtcccg gatcaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat accgccgtgt acttttgtgt gtacggcagc 300

agccagcggt tcttcgatta ttggggccag ggcaccctgg tcaccgtttc ttct 354

<210> 54

<211> 405

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 light chain variable region nucleic acid sequence

<400> 54

atgcatcaca ccagcatggg catcaaaatg gagtcacaga ttcaggtctt tgtattcgtg 60

tttctctggt tgtctggtgt tgacggagac attctgatga cccagtctca caaattcatg 120

tccacatcaa ttggagacag ggtcagcatc acctgcaagg ccagccagga tgtgagtatt 180

gatgtagcct ggtatcacca gaaaccagga caatctccta aattactgat ttactcggct 240

tcgtttcggt acgctggagt ccctgatcgc ttcactggca gtggatctgg gacggatttc 300

actctcacca tcagcagtgt gcaggctgaa gacctggcag tttattactg tcagcaacat 360

tatagtttat tcacgttcgg ctcggggaca aagttggaaa ttaaa 405

<210> 55

<211> 414

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 light chain variable region nucleic acid sequence

<400> 55

atgggggaaa tgcatcacac cagcatgggc atcaaaatgg agtcacagat tcaggtcttt 60

gtattcgtgt ttctctggtt gtctggtgtt gacggagaca ttgtgatgac ccagtctcac 120

aaattcatgt ccacatcagt aggagacagg gtcagcatca cctgcaaggc cagtcaggat 180

gtgagtattg atgtagcctg gtatcaacag aaaccaggac aatctcctaa actactgatt 240

tactcggctt cgtggcggta cgctggagtc cctgatcgct tcactggcag tcgatctggg 300

acggattata ctctcaccat cagcagtgtg caggctgaag acctggcagt ttattactgt 360

cagcaacatt ataatttatt cacgttcggc tcggggacaa agttggaaat acaa 414

<210> 56

<211> 318

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 light chain variable region humanized nucleotide sequence

<400> 56

gacatcctga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60

atcaattgca aggccagcca ggacgtgtcc atcgacgtgg catggtatca gcagaagcct 120

ggccagcctc ctaagctgct gatctacagc gccagcttca gatatgccgg cgtgcccgat 180

agattttccg gctctggcag cggcaccgac ttcaccctta caatcagttc cctgcaggcc 240

gaggacgtgg ccgtgtacta ttgtcagcag cactacagcc tgttcacctt cggctccggc 300

accaagctgg aaatcaag 318

<210> 57

<211> 318

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 light chain variable region humanized nucleotide sequence

<400> 57

gacatcgtga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60

atcaattgca aggccagcca ggacgtgtcc atcgacgtgg catggtatca gcagaagcct 120

ggccagcctc ctaagctgct gatctacagc gccagctgga gatatgccgg cgtgcccgat 180

agattttccg gctctagaag cggcaccgac tataccctta caatcagttc cctgcaggcc 240

gaggacgtgg ccgtgtacta ttgtcagcag cactacaacc tgttcacctt cggctccggc 300

accaagctgg aaatcaag 318

<210> 58

<211> 639

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 light chain humanized nucleotide sequence

<400> 58

gacatcctga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60

atcaattgca aggccagcca ggacgtgtcc atcgacgtgg catggtatca gcagaagcct 120

ggccagcctc ctaagctgct gatctacagc gccagcttca gatatgccgg cgtgcccgat 180

agattttccg gctctggcag cggcaccgac ttcaccctta caatcagttc cctgcaggcc 240

gaggacgtgg ccgtgtacta ttgtcagcag cactacagcc tgttcacctt cggctccggc 300

accaagctgg aaatcaagcg tacggtggct gcaccatctg tcttcatctt cccgccatct 360

gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctacccc 420

agagaagcca aagtgcagtg gaaggtggac aacgccctgc agagcggaaa cagccaggaa 480

agcgtgacag agcaggattc caaggattcc acatacagcc tgagcagcac actgacactg 540

tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgacaca ccagggactg 600

tcctcccctg tgacaaagag cttcaacaga ggagaatgc 639

<210> 59

<211> 1344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE2 heavy chain humanized nucleotide sequence

<400> 59

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60

acctgtaccg tgtccggcta cagcatcacc agcgactacg cctggaactg gatcagacag 120

cctcctggca atggactgga atggatgggc tacatcagct acagcggcag caccagatac 180

aaccccagcc tgaagtcccg gatcaccatc agcagagaca ccagcaagaa ccagttctcc 240

ctgaagctga gcagcgtgac agccgccgat accgccgtgt acttttgtgt gtacggcagc 300

agccagcggt tcttcgatta ttggggccag ggcaccctgg tcaccgtttc ttctgcgtcg 360

accaaaggcc cctccgtgtt tcctctggcc ccctcctcca agtctacctc cggcggtacc 420

gccgccctcg gttgtttggt caaagactac ttccccgagc ccgtgaccgt ctcctggaac 480

tccggcgccc tgacctccgg cgtgcacaca ttccccgccg tcctgcagtc ctccggcctg 540

tactctctgt cttccgtggt gaccgtgccc tccagctctt taggcacaca gacctacatc 600

tgcaacgtga accacaagcc ctccaatacc aaggtggaca agaaggtgga gcccaagtct 660

tgtgacaaga cacacacttg tcctccttgt cccgctcccg aactgctggg aggccctagc 720

gtgtttttat tcccccctaa acctaaggac accctcatga tcagccgtac ccccgaagtg 780

acttgtgtgg tggtggacgt gtcccacgag gaccccgaag tcaagttcaa ctggtacgtg 840

gacggcgtgg aggtgcataa tgccaagacc aagccccggg aggagcagta taatagcacc 900

tatagggtgg tgtccgtgct gaccgtgctg caccaagatt ggctgaacgg caaggagtac 960

aagtgtaagg tgtccaacaa ggctttaccc gctcccatcg agaagaccat ctccaaggct 1020

aagggacagc ctcgtgagcc tcaagtttat actttacccc cttctcgtga cgagctgacc 1080

aagaaccaag tttctttaac atgtttagtg aagggcttct acccctccga catcgccgtg 1140

gagtgggaga gcaatggaca gcccgagaac aactacaaga ccacaccccc cgttttagac 1200

agcgacggct ccttcttttt atactccaag ctcaccgtgg ataagtcccg gtggcagcaa 1260

ggtaacgtct tctcttgttc cgtgatgcac gaagctttac acaaccatta cacccagaag 1320

tctttatctt taagccccgg caag 1344

<210> 60

<211> 639

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 light chain humanized nucleotide sequence

<400> 60

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcacctgca aggccagtca ggatgtgagt attgatgtag cctggtatca acagaaacca 120

ggacaatctc ctaaactact gatttactcg gcttcgtggc ggtacgctgg agtccctgat 180

cgcttcactg gcagtcgatc tgggacggat tatactctca ccatcagcag tgtgcaggct 240

gaagacctgg cagtttatta ctgtcagcaa cattataatt tattcacgtt cggctcgggg 300

acaaagttgg aaataaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 360

gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctacccc 420

agagaagcca aagtgcagtg gaaggtggac aacgccctgc agagcggaaa cagccaggaa 480

agcgtgacag agcaggattc caaggattcc acatacagcc tgagcagcac actgacactg 540

tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgacaca ccagggactg 600

tcctcccctg tgacaaagag cttcaacaga ggagaatgc 639

<210> 61

<211> 1344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SE5 heavy chain humanized nucleotide sequence

<400> 61