Camel-derived nanoantibodies that bind to human immunoglobulin E

Abstract

The invention discloses a camel-derived nano antibody which binds to human immunoglobulin E (IgE). Specifically, the invention discloses an anti-IgE (Immunoglobulin E) specific nano antibody and a fusion protein thereof. The invention also discloses a coding sequence for coding the VHH chain of the nano antibody, a corresponding expression vector, a host cell and a method for producing the nano antibody. The nano antibody fusion protein provided by the invention has high affinity and high specificity, and can be used for diagnosing and / or treating IgE-mediated allergic diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biopharmaceuticals, and in particular relates to a camelid nanobody binding to human immunoglobulin E (IgE). BACKGROUND

[0002] Human immunoglobulin E (IgE) plays an important role in type I hypersensitivity. When the body is allergic, IgE antibodies recognize and bind to allergens, and IgE bound to allergens binds to the IgE high-affinity receptor I (FcεRI) on the surface of mast cells or basophils, leading to cross-linking of FcεRI. Cross-linking of FcεRI can trigger mast cells and basophils to degranulate and release histamine and other substances, which in turn mediate immediate hypersensitivity reactions, leading to a series of allergic symptoms in the body, such as vasodilation, bronchial constriction, etc. Antibodies targeting human IgE have been shown to be effective in treating allergies, such as omalizumab, but its price is expensive and its application is limited to a few allergic symptoms.

[0003] Compared with conventional antibodies, nanobodies have unique advantages: 1) nanobodies have simple structure and small molecular weight, which are more conducive to expression and use; 2) small size allows it to recognize epitopes that cannot be approached by traditional antibodies; 3) as a single-domain antibody, it has only one binding site, which has better penetration, specificity and detection linearity as a diagnostic reagent; 4) it is easier to engineer and couple with various fusion proteins or be labeled with various markers; 5) it is easier to prepare bifunctional antibodies, which is more conducive to the development of targeted drugs and cell target-oriented transport; 6) as a drug development, it has little immunogenicity to humans and is less likely to cause immune rejection. In addition, nanobodies can be directly delivered to the lungs after being atomized, or even directly administered through the nose, which can alleviate the shortage of medical resources.

[0004] Therefore, it is necessary to develop new nanobodies targeting human IgE, which will be suitable for more allergic diseases. SUMMARY

[0005] The present application provides a new nanobody targeting human IgE.

[0006] In a first aspect of the present application, a nanobody against human IgE is provided, the nanobody having three complementarity determining regions (CDRs) derived from a VHH chain as shown in the following amino acid sequence:

[0007] wherein the CDRs are CDR1, CDR2 and CDR3 determined by any one of Kabat rules, IMGT rules, Chothia rules, AbM rules or Contact rules.

[0008] In another preferred embodiment, the CDR1, CDR2 and CDR3 are selected from the group consisting of:

[0009] (a) CDRs determined based on the Kabat convention:

[0010] a CDR1 of amino acids as set forth in SEQ ID NO: 5,

[0011] a CDR2 of amino acids as set forth in SEQ ID NO: 6, and

[0012] a CDR3 of amino acids as set forth in SEQ ID NO: 7;

[0013] (b) CDRs determined based on the IMGT convention:

[0014] a CDR1 of amino acids as set forth in SEQ ID NO: 8,

[0015] a CDR2 of amino acids as set forth in SEQ ID NO: 9, and

[0016] a CDR3 of amino acids as set forth in SEQ ID NO: 10.

[0017] In another preferred embodiment, the amino acid sequence is SEQ ID NO: 4.

[0018] In another preferred embodiment, the Nanobody specifically binds to the Cε4 domain of IgE-Fc 2-4 .

[0019] In another preferred embodiment, the CDR regions of the VHH chain of the Nanobody comprise an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0020] In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence optionally having at least one amino acid added, deleted, modified and / or substituted, and capable of retaining IgE binding affinity.

[0021] In another preferred embodiment, the number of amino acids added, deleted, modified and / or substituted is 1-3, preferably 1-2, more preferably 1.

[0022] In another preferred embodiment, the VHH chain of the Nanobody further comprises a framework region (FR).

[0023] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0024] In another preferred embodiment, the framework region FR is of human or camel origin.

[0025] In another preferred embodiment, the antibody is a heavy chain antibody comprising a heavy chain constant region CH2 and CH3 (Fc fragment).

[0026] In another preferred embodiment, the heavy chain constant region is derived from an Fc fragment of IgG, preferably of human IgG.

[0027] In another preferred embodiment, the VHH chain of the anti-IgE nanobody has an amino acid sequence with a homology of > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, or > 99% to the amino acid sequence shown in SEQ ID NO: 4.

[0028] In another preferred embodiment, the VHH chain of the anti-IgE nanobody has one or more of the amino acid sequences shown in SEQ ID NO: 4.

[0029] In another preferred embodiment, the anti-human IgE nanobody comprises monomers, dimers (bivalent antibodies), tetramers (tetravalent antibodies), and / or multimers (multivalent antibodies).

[0030] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of the sequences shown in any one of SEQ ID NO: 4.

[0031] In a second aspect of the application, a fusion protein of an anti-human IgE nanobody is provided, the fusion protein comprising:

[0032] (i) a sequence of a VHH chain of an anti-human IgE nanobody as described in the first aspect of the application; and

[0033] (ii) an Fc fragment.

[0034] In another preferred embodiment, the VHH chain of the anti-human IgE nanobody is linked to the Fc fragment by a linker sequence.

[0035] In another preferred embodiment, the Fc fragment is a human IgGl Fc.

[0036] In another preferred embodiment, the fusion protein has the structure shown in formula I from N- to C-terminus:

[0037] Z1— L — Z2 (formula I)

[0038] wherein,

[0039] Z1 is a VHH chain of an anti-human IgE nanobody as described in the first aspect of the application;

[0040] L is a linker sequence;

[0041] Z2 is an Fc fragment of an immunoglobulin.

[0042] In another preferred embodiment, the amino acid sequence of the Fc fragment is set forth in SEQ ID NO: 11.

[0043] In another preferred embodiment, the amino acid sequence of the linker sequence is set forth in SEQ ID NO: 12.

[0044] In another preferred embodiment, the fusion protein further comprises:

[0045] (iii) an optional tag sequence that facilitates expression and / or purification.

[0046] In another preferred embodiment, the recombinant protein specifically binds to human IgE.

[0047] In another preferred embodiment, the nanobody specifically binds to the Cε4 domain of IgE-Fc 2-4 In another preferred embodiment, the nanobody specifically binds to the Cε4 domain of IgE-Fc

[0048] In a third aspect of the present application, there is provided a nucleotide molecule encoding the nanobody of the first aspect of the present application or the fusion protein of the second aspect of the present application.

[0049] In another preferred embodiment, the nucleotide molecule comprises DNA or RNA.

[0050] In a fourth aspect of the present application, there is provided an expression vector comprising the nucleotide molecule of the third aspect of the present application.

[0051] In a fifth aspect of the present application, there is provided a host cell comprising the expression vector of the fourth aspect of the present application, or having integrated into its genome the nucleotide molecule of the third aspect of the present application.

[0052] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0053] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell, a bacteriophage, or a combination thereof.

[0054] In a sixth aspect of the present application, there is provided a method of producing an anti-human IgE nanobody, comprising the steps of:

[0055] (a) culturing the host cell of the fifth aspect of the present application under conditions suitable for the production of the nanobody, thereby obtaining a culture comprising the anti-human IgE nanobody; and

[0056] (b) isolating or recovering said anti-human IgE Nanobody from said culture.

[0057] In another aspect of the application, a method for producing a fusion protein of an anti-human IgE Nanobody is provided, comprising the steps of:

[0058] (a) culturing the host cell of the fifth aspect of the application under conditions suitable for production of the Nanobody fusion protein, thereby obtaining a culture comprising the fusion protein of said anti-human IgE Nanobody; and

[0059] (b) isolating or recovering said fusion protein of the anti-human IgE Nanobody from said culture.

[0060] In a seventh aspect of the application, an immunoconjugate is provided, said immunoconjugate comprising:

[0061] (a) the anti-human IgE Nanobody of the first aspect of the application; and

[0062] (b) a conjugating moiety selected from the group consisting of: a detectable label, a drug.

[0063] In another preferred embodiment, said (a) moiety is conjugated to said conjugating moiety via a chemical bond or a linker.

[0064] In another preferred embodiment, said immunoconjugate comprises: a multivalent (e.g. bivalent) anti-human IgE Nanobody of the first aspect of the application. By multivalent is meant that the amino acid sequence of said immunoconjugate comprises multiple repeats of the anti-human IgE Nanobody of the first aspect of the application.

[0065] In another preferred embodiment, said multivalent is meant that the amino acid sequence of said immunoconjugate comprises multiple repeats of the anti-human IgE Nanobody of the first aspect of the application.

[0066] In an eighth aspect of the application, the use of a Nanobody of the first aspect of the application or a fusion protein of the second aspect of the application for the manufacture of a medicament, a reagent, an assay plate or a kit is provided.

[0067] Said reagent, assay plate or kit is used for: detecting IgE in a sample.

[0068] Said medicament is used for the prevention, diagnosis and / or treatment of an IgE-mediated allergic disease.

[0069] In another preferred embodiment, said IgE-mediated allergic disease is allergy.

[0070] In a ninth aspect of the application, a pharmaceutical composition is provided, said pharmaceutical composition comprising:

[0071] (i) the anti-human IgE Nanobody according to the first aspect of the application, or the fusion protein according to the second aspect of the application; and

[0072] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0073] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of an injection, a lyophilized agent.

[0074] In a tenth aspect of the application, a method for detecting IgE in a sample is provided, the method comprising the steps of:

[0075] (1) contacting the sample with the Nanobody according to the first aspect of the application;

[0076] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of IgE protein in the sample.

[0077] In another preferred embodiment, the IgE is a human IgE protein.

[0078] In an eleventh aspect of the application, a detection reagent for IgE is provided, the detection reagent comprising:

[0079] (i) the anti-human IgE Nanobody according to the first aspect of the application, or the fusion protein according to the second aspect of the application; and

[0080] (ii) a detection reagent.

[0081] In another preferred embodiment, the conjugating moiety of the immunoconjugate is a diagnostic isotope.

[0082] In another preferred embodiment, the pharmaceutically acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0083] In another preferred embodiment, the detection reagent is one or more agents selected from the group consisting of an isotope tracer, a contrast agent, a flow cytometry reagent, a cellular immunofluorescence reagent, a nano-magnetic particle and an imaging agent.

[0084] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0085] In another preferred embodiment, the dosage form of the detection reagent is a liquid or a powder (such as an aqueous agent, a needle agent, a lyophilized powder, a tablet, a buccal agent, an inhalation agent).

[0086] In a twelfth aspect of the application, a kit is provided, the kit comprising the detection reagent according to the eleventh aspect of the application.

[0087] In a thirteenth aspect of the invention, a method for treating IgE-mediated allergic diseases is provided, the method comprising administering to a desired subject the nanobody described in the first aspect of the invention or the fusion protein described in the second aspect of the invention.

[0088] In another preferred embodiment, the object includes mammals, such as humans.

[0089] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0090] Figure 1 The monoclonal Phage ELISA was used to detect the nanobody CA20 and IgE-Fc. 2-4 The combination of.

[0091] Figure 2 The results of Protein A column purification of the human IgG1 Fc fusion protein CA20-Fc from the nanobody CA20 are shown.

[0092] Figure 3 The SDS-PAGE electrophoresis results of the human IgG1 Fc fusion protein CA20-Fc of the nanobody CA20 are shown.

[0093] Figure 4 This demonstrates the ELISA detection of CA20-Fc and IgE-Fc. 2-4 The result of affinity.

[0094] Figure 5 The results show the detection of CA20-Fc and IgE-Fc by ELSIA. 2-4 The combination of different structural domains.

[0095] Figure 6 This demonstrates competitive ELISA detection of CA20-Fc inhibition of IgE-Fc. 2-4 The effect of binding receptor FcεRⅠα.

[0096] Figure 7 The nanobody CA20 showed inhibition of IgE-Fc 2-4 A schematic diagram illustrating the mechanism of binding to receptor FcεRⅠα. Detailed Implementation

[0097] Through extensive and in-depth research and screening, this invention unexpectedly discovered an anti-human IgE nanobody (CA20). This nanobody or its fusion protein can be used without binding to IgE-Fc. 2-4the Cε3 domain of IgE-Fc 2-4 binds to FcεRIα. The experimental results show that the anti-human IgE nanobody-human IgG1 Fc fusion protein CA20-Fc of the present application can bind to the Fc segment of human IgE antibody (IgE-Fc 2-4 ) with high affinity, and mainly specifically binds to the Cε4 domain of IgE-Fc 2-4 , and can effectively inhibit the binding of IgE-Fc 2-4 to FcεRIα. On this basis, the present application is completed.

[0098] The term

[0099] In order that the disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically identified otherwise, each of the following terms shall have the meaning given below.

[0100] The term "about" can mean a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0101] The term "administering" means physically introducing the product of the present application into a subject using any of a variety of methods and delivery systems known to those in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0102] The term "EC50" means the concentration for 50% of maximal effect (EC50), which refers to the concentration that elicits 50% of maximal effect.

[0103] The term "IC50" means the half maximal inhibitory concentration of the antagonist being measured.

[0104] IgE

[0105] IgE is the abbreviation of immunoglobulin E, which belongs to a type of immunoglobulin. IgE is mainly produced by plasma cells in the lamina propria of respiratory and digestive mucosa, and its content in the blood of normal people is extremely low, accounting for about 0.002% of total immunoglobulin (Ig) in serum. It is a cytophilic antibody, which mainly mediates type I allergic reaction.

[0106] When the human body is stimulated by allergens, IgE binds to the Fc receptor (FcεRI) on the surface of mast cells and basophils, causing these cells to degranulate and release histamine, leukotrienes and other inflammatory mediators, thereby triggering an allergic reaction. Among them, FcεR Iα is the α chain of FcεR I, containing 222 amino acid residues, with a molecular weight of 25 kDa. The extracellular region of FcεR Iα is the main part of IgE binding. When allergens first invade the body, they can induce B lymphocytes to produce antigen-specific IgE. These IgE subsequently bind to the surface of FcεR I on target cells (such as basophils and mast cells), making the target cells in a sensitized state.

[0107] Changes in the level of IgE have important clinical significance and are commonly used for the diagnosis of allergic diseases and the determination of allergens. Elevated IgE is mainly seen in parasitic diseases and allergic diseases, such as allergic rhinitis, allergic urticaria, allergic asthma and allergic conjunctivitis, etc.

[0108] The Fc segment of human IgE antibody (106-428 amino acid residues) includes three domains:

[0109] Cε2 domain (106-210 amino acid residues):

[0110] SRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQ VMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFED STKKCA (SEQ ID NO: 1);

[0111] Cε3 domain (211-318 amino acid residues):

[0112] DSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRAS GKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRA LMRSTTKTS (SEQ ID NO: 2); and

[0113] Cε4 domain (319-428 amino acid residues):

[0114] GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQ LPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQ TVQRAVSVNPGK (SEQ ID NO: 3).

[0115] Currently, antibodies targeting human IgE have been proven to be effective in treating allergy, and antibodies targeting human IgE mainly inhibit IgE-Fc 2-4 binding to FcεRIα by binding to the Cε3 domain of IgE-Fc 2-4 .

[0116] The nanobody of the present invention

[0117] As used herein, the terms "the nanobody of the present invention", "the anti-human IgE nanobody of the present invention", "the IgE-targeting single-domain antibody of the present invention", "the single-domain antibody of the present invention", "the IgE single-domain antibody of the present invention" are used interchangeably, and all refer to a nanobody that specifically recognizes and binds to IgE, including human IgE. Particularly preferred is a nanobody whose amino acid sequence of the VHH chain is set forth in SEQ ID NO: 4.

[0118] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons having the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a plurality of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light and heavy chains.

[0119] As used herein, the terms "single-domain antibody (VHH)", "nanobody" have the same meaning, which refers to the variable region of the heavy chain of the cloned antibody, and the single-domain antibody (VHH) composed of only one heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single-domain antibody (VHH) composed of only one heavy chain variable region.

[0120] As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence among antibodies and are responsible for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light chain and the heavy chain variable regions. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely β-sheet in structure, connected by three CDRs that form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, form the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0121] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules with the antibodies or fragments thereof of the present application. The present application also includes cell surface markers or antigens conjugated to the anti-IgE antibodies or fragments thereof.

[0122] As used herein, the term "heavy chain variable region" is used interchangeably with "V H H."

[0123] As used herein, the term "variable region" is used interchangeably with "complementarity determining region" (CDR).

[0124] In a preferred embodiment of the present application, the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2, and CDR3.

[0125] In a preferred embodiment of the present application, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region.

[0126] In the present application, the terms "antibody of the present application," "protein of the present application," or "polypeptide of the present application" are used interchangeably and refer to a polypeptide that specifically binds to an IgE protein, such as a protein or polypeptide having a heavy chain variable region. They can or can not contain the initial methionine.

[0127] The present application also provides other proteins or fusion expression products having the antibodies of the present application. In particular, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugates and fusion expression products) having a heavy chain comprising a variable region, provided that the variable region is identical to or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibodies of the present application.

[0128] Generally, the antigen binding properties of an antibody can be described by three specific regions of the variable region of the heavy chain, called the complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs) whose amino acid sequences are relatively conserved and do not directly participate in binding interactions. The CDRs form loops or "hot spots" on the surface of the variable region, and are in close proximity to each other through the FRs that form a beta sheet structure in space. The CDRs on the heavy chain and the corresponding CDRs on the light chain form the antigen binding site of the antibody. Which amino acids constitute the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same class.

[0129] The variable regions of the heavy chains of the antibodies of the present application are of particular interest because at least some of them are involved in binding the antigen. Thus, the present application includes those molecules having the variable region of the heavy chain of the antibody with the CDRs, provided that the CDRs are at least 90% homologous, preferably at least 95% homologous, and most preferably at least 98% homologous to the CDRs identified herein.

[0130] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusion proteins of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0131] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. The polypeptide fragments, derivatives, or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions (preferably conservative amino acid substitutions) of the amino acid residues, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence (such as a leader or secretion sequence, or a sequence or protein for purification of the polypeptide, or a proprotein sequence, or a fusion protein with a 6His tag). These fragments, derivatives, and analogs are within the scope of one skilled in the art in light of the teachings herein.

[0132] The term "antibody" of the present application refers to a polypeptide having IgE protein binding activity, which includes the above-mentioned CDR regions. The term also includes variants of polypeptides having the same function as the antibody of the present application, which include the above-mentioned CDR regions. These variants include, but are not limited to, deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or identical amino acids usually does not change the function of the protein. For another example, addition of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibody of the present application.

[0133] The variants of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA hybridizing to the DNA encoding the antibody of the present application under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present application.

[0134] The present application also provides other polypeptides, such as fusion proteins comprising a nanobody or a fragment thereof. In addition to the almost full-length polypeptides, the present application also includes fragments of the nanobody of the present application. Usually, the fragment has at least about 50 consecutive amino acids of the antibody of the present application, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids.

[0135] In the present application, "conservative variants of the antibody of the present application" refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by similar or identical amino acids compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table 1.

[0136] Table 1

[0137]

[0138]

[0139] Polynucleotides, vectors and host cells

[0140] The present application also provides polynucleotide molecules encoding the above-mentioned antibody or fragment thereof or fusion protein thereof. The polynucleotide of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0141] Polynucleotides encoding the mature polypeptides of the present application include: coding sequences that encode only the mature polypeptide; coding sequences that encode the mature polypeptide and various additional coding sequences; coding sequences that encode the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0142] The term "polynucleotide encoding a polypeptide" can be a polynucleotide that includes only coding sequence that encodes the polypeptide, or a polynucleotide that includes additional coding and / or non-coding sequences.

[0143] The present application also relates to polynucleotides that hybridize to the sequences described above and that have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization in the presence of a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0144] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0145] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in an isolated form.

[0146] At present, it is possible to obtain the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0147] The present application also relates to vectors comprising the appropriate DNA sequences described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express the proteins.

[0148] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples of such host cells are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0149] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the CaCl2method, which is well known in the art. Another method is the use of MgCl2. If necessary, the transformation can also be performed by electroporation. When the host is a eukaryote, the DNA can be transferred into the host cell using a DNA transfer method such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0150] The transformants obtained can be cultured in conventional media using conventional procedures to express the polypeptides encoded by the genes of the present application. The medium used to culture the host cell is dependent on the host cell used and can be selected from various conventional media. Culturing is performed under conditions suitable for growth of the host cell. When the host cell has reached an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0151] The recombinant polypeptides in the above methods can be expressed intracellularly, on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified from the cell using their physical, chemical, and other properties by various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0152] The antibodies of the present application can be used alone or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of these.

[0153] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.

[0154] Therapeutic agents that can be conjugated or coupled to the antibodies of the present application include, but are not limited to, 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2 and the like; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolyzing enzyme-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles and the like.

[0155] Immunoconjugates

[0156] The present application also provides immunoconjugates (ADC) based on the antibodies of the present application, preferably nanobody-drug conjugates (NDC).

[0157] Typically, the antibody-drug conjugate comprises the antibody, and an effector molecule, which is conjugated, and preferably chemically conjugated, to the antibody. The effector molecule is preferably a therapeutically active drug. Further, the effector molecule can be one or more of a toxin protein, a chemotherapeutic drug, a small molecule drug, an agonist small molecule (STING, TLR7, TLR8, etc.), or a radionuclide.

[0158] The antibody of the present application can be conjugated to the effector molecule via a conjugating agent. Examples of the conjugating agent can be any one or several of a non-selective conjugating agent, a conjugating agent utilizing a carboxyl group, a peptide chain, a conjugating agent utilizing a disulfide bond. The non-selective conjugating agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde and the like. The conjugating agent utilizing a carboxyl group can be any one or several of a conjugating agent of aconitic anhydride (e.g., aconitic anhydride), a conjugating agent of an acylhydrazone (conjugation site: acylhydrazone).

[0159] Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach a variety of functional groups, including imaging agents (e.g., chromophoric groups and fluorescent groups), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (e.g., a drug, a detection agent, a stabilizing agent) is conjugated (covalently linked) to the antibody. The functional agent can be linked to the antibody directly, or indirectly via a linker.

[0160] A single domain antibody can be conjugated to a drug to form an antibody drug conjugate (NDC). Typically, an NDC comprises a linker between the drug and the antibody. The linker can be a degradable linker or a non-degradable linker. A degradable linker is typically susceptible to degradation in the intracellular environment, e.g., the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptide-based linkers that are degradable by intracellular proteases, e.g., lysosomal proteases or endosomal proteases, or saccharide linkers, e.g., glucuronide linkers that are degradable by glucuronidases. Peptide-based linkers can include, for example, dipeptides, e.g., valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH less than 5.5, e.g., hydrazone linkers) and linkers that are degradable under reducing conditions (e.g., disulfide linker). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.

[0161] The linker has a reactive functional group capable of reacting with certain amino acid residues prior to attachment to the antibody, and the attachment is achieved through the reactive functional group. Mercapto-specific reactive functional groups are preferred and include, for example, maleimides, haloamides (e.g., iodo, bromo, or chloro); haloesters (e.g., iodo, bromo, or chloro); halo-methyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-bis-(mercurymethyl) dioxane, and counterions are acetate, chloride, or nitrate; and polymethylene dimethylsulfide sulfonate. The linker can include, for example, a maleimide attached to the antibody through a thio-butyrimide.

[0162] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker attaches the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug can have an amino, carboxyl, mercapto, hydroxyl, or keto group that can bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive functional group prior to attachment to the antibody.

[0163] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubulin inhibitors, typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[l,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0164] The immunoconjugate drugs of the application can also be radionuclide conjugate drugs (RDCs), which are comprised of an antibody of the application conjugated to a radionuclide.

[0165] In the present application, the drug-linker can be used to form the NDC in a single step. In other embodiments, a bifunctional linker compound can be used to form the NDC in a two- or multi-step process. For example, a cysteine residue is reacted with a reactive portion of the linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug to form the NDC.

[0166] In general, the functional groups on the linker are chosen to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkyne. Other useful functional groups include, for example, ketones and aldehydes (for reaction with hydrazides and alkyl-oxyl amines), phosphines (for reaction with azides); isocyanates and isothiocyanates (for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimidyl esters (for reaction with amines and alcohols). These and other ligation strategies, such as those described in Bioconjugate Techniques, 2ndEdition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of the drug moiety and the linker, each member of a complementary pair of reactive functional groups can be used on either the linker or the drug when the complementary pair is chosen.

[0167] The present application also provides methods of making an NDC, which can further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0168] In certain embodiments, the methods of the present application comprise: combining an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present application further comprise: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0169] In some embodiments, the immunoconjugate, preferably a single-domain antibody drug conjugate NDC, has the following formula:

[0170]

[0171] wherein:

[0172] nAb is an IgE-targeting single-domain antibody, an IgE-targeting heavy-chain antibody, or a multi-specific antibody as described above,

[0173] LU is a linker / linker;

[0174] D is a drug;

[0175] and subscript p is a value selected from 1 to 10.

[0176] Pharmaceutical compositions

[0177] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration.

[0178] The pharmaceutical composition of the present application can be directly used in combination with IgE protein molecules, and thus can be used in the treatment of allergy. In addition, other therapeutic agents can also be used simultaneously.

[0179] The pharmaceutical composition of the present application comprises a safe and effective amount (e.g. 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the nanobody (or conjugate thereof) as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as needle, solution should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0180] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account the administration route, the patient's health status, etc., which are within the skill of the skilled physician.

[0181] Labeled nanobody

[0182] In a preferred embodiment of the present application, the nanobody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label, or a fluorescent label.

[0183] The colloidal gold labeling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present application, the anti-IgE nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.

[0184] The anti-human IgE nanobody of the present application has good specificity and high titer.

[0185] Phage display technology

[0186] The principle of phage display technology is that a foreign gene is inserted into a proper position of a coat protein structural gene of a phage, and under the condition that the reading frame is normal and the normal function of the coat protein is not affected, the foreign gene is expressed along with the expression of the coat protein, so that the polypeptide or protein is displayed on the surface of the phage in the form of a fusion protein. The displayed protein can maintain a relatively independent spatial structure and biological activity, which is conducive to the binding of the target protein, and thus the phage display antibody library can be quickly screened by using the target protein.

[0187] After the display library is constructed, the target protein is used as a stationary phase, and the display library is incubated for a period of time, and then the unbound phage is washed away, and the adsorbed phage is eluted by using a competitive receptor. The eluted phage infects host bacteria for propagation and amplification, and then the next round of elution is performed.

[0188] After 2-5 rounds of "adsorption-elution-amplification" (for some antibodies with weak affinity, more rounds of elution are required), a highly enriched phage that can specifically bind to the target protein can be obtained.

[0189] Detection method

[0190] The present application also relates to a method for detecting IgE protein. The steps of the method are as follows: obtaining a cell and / or tissue sample; dissolving the sample in a medium; detecting the level of IgE protein in the dissolved sample.

[0191] In the detection method of the present application, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.

[0192] Kit

[0193] The present application also provides a kit containing the antibody (or fragment thereof) or detection plate of the present application. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, etc.

[0194] The present application also provides a detection kit for detecting the level of IgE, which comprises an antibody recognizing IgE protein, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0195] Application

[0196] As mentioned above, the Nanobodies of the present application have a wide range of biological and clinical applications, which involve diagnosis and treatment of IgE related diseases, basic medical research, biological research and many other fields. One preferred application is for clinical diagnosis and targeted therapy against IgE.

[0197] The main advantages of the present application include:

[0198] (a) The Nanobody (CA20) of the present application, human IgGl Fc fusion protein CA20-Fc, can bind to human IgE antibody Fc fragment (IgE-Fc 2-4 ) with high affinity.

[0199] (b) The Nanobody (CA20) of the present application, human IgGl Fc fusion protein CA20-Fc, mainly binds to the Cε4 domain of IgE and can inhibit IgE-Fc 2-4 binding to FcεRIα, but not to the Cε3 domain.

[0200] (c) The Nanobody of the present application can be used to develop an allergy therapeutic antibody targeting human IgE, which will be suitable for the diagnosis and / or treatment of allergic diseases.

[0201] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0202] Example 1 Screening of Nanobodies

[0203] 1.1 Construction of IgE immunization library

[0204] The Fc fragment of purified human IgE antibody (UNIPROT: P01854, aa 106-428) was mixed with Freund's adjuvant and subcutaneously injected into a camel at a dose of 0.1 mg / time for 4 times with an interval of 2 weeks. Two weeks after the fourth immunization, the immune cells in the blood were separated by intravenous blood collection.

[0205] Total RNA was extracted using the RNA extraction kit of Omega Company, and genomic DNA was removed using DNAase. The total RNA was reverse transcribed into cDNA using the PrimeScript TMII 1st Strand cDNA Synthesis Kit to reverse transcribe RNA into cDNA.

[0206] The coding gene fragment of VHH was obtained by PCR amplification using the above cDNA as a template and camel VHH specific primers. The amplified VHH sequence was cloned into the Ncol and Notl sites of phagemid pR2 (MRC Laboratory of Molecular Biology) using the Gibson Assembly method. The Gibson Assembly product obtained was the initial nanobody phagemid library.

[0207] The E. coli TG1 (MRC Laboratory of Molecular Biology) competent cells were prepared by 10% glycerol washing method, and then the Gibson Assembly product was electrotransformed into the TG1 competent cells. Five 150 mm 2YT plates containing 2% glucose and 50 μg / mL ampicillin were used to amplify the phagemid library. After scraping the plates, an appropriate amount of bacterial solution was inoculated into 200 mL 2YT containing 2% glucose and 50 μg / mL ampicillin to grow to the logarithmic phase. Then, 10 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) was added at 37°C for 45 min. Then, 50 mL of bacterial solution was centrifuged, and the bacterial cells were resuspended in 100 mL of 2YT containing 0.1% glucose, 50 μg / mL ampicillin, and 50 μg / mL kanamycin. The solution was incubated at 25°C for 20 hours to amplify the phage displaying nanobodies. The phage was concentrated by PEG precipitation, and finally resuspended in PBS and stored on ice.

[0208] 1.2 Screening of Nanobodies

[0209] The IgE-Fc 2-4 protein was diluted to 0.1 mg / mL with PBS, and 100 μL was added to one well of a 96-well enzyme-labeled plate, and coated at room temperature for 2 hours. Meanwhile, one antigen-free control well was set. PBS was used for washing 3 times, 300 μL of MPBS (PBS containing 5% skim milk) was added to each well, and blocked at room temperature for 2 hours. PBS was used for washing 3 times, and 1 x 10 11pfu of phage library phage (in 100 μL MPBS) was incubated at 60 rpm for 1 hour at room temperature. Wash 20 times with PBST (0.1% Tween-20). Add 100 μL trypsin at a concentration of 0.5 mg / mL per well and incubate for 1 hour at room temperature. The phage bound to the well is eluted, which is the first round of eluted phage.

[0210] The eluted phage was used to infect 2 mL of TG1 bacteria in the log phase of growth at 37°C for 45 minutes in a water bath and spread on a 150 mm2YT plate (containing 2% glucose and 50 μg / mL ampicillin) and incubated at 37°C overnight. Add 4 mL of 2YT to the above 150 mm plate, scrape the colonies and mix the bacterial solution and inoculate 100 μL into 100 mL of 2YT (containing 2% glucose and 50 μg / mL ampicillin) and incubate until the log phase of growth. Add KM13 to infect to complete the amplification of the first round of eluted phage.

[0211] Subsequently, IgE-Fc 2-4 Dilute with PBS to 0.01 mg / mL and add 100 μL to one well of a 96-well immunoplate and incubate overnight at 4°C. Set up one well without antigen as a control. Wash 3 times with PBS and add 300 μL MPBS (PBS containing 5% skim milk) per well and incubate for 2 hours at room temperature. Wash 3 times with PBS and add 1 x 10 10 pfu of the first round of eluted phage amplified (in 100 μL MPBS) was incubated at 60 rpm for 1 hour at room temperature. Wash 20 times with PBST (0.2% Tween-20). Add 100 μL trypsin at a concentration of 0.5 mg / mL per well and incubate for 1 hour at room temperature. The phage bound to the well is eluted, which completes the second round of screening.

[0212] The eluted phage was used to infect TG1 and spread on a 2YT plate (containing 2% glucose and 50 μg / mL ampicillin) and incubated for 16 hours to form colonies.

[0213] Randomly pick 80 single clones from the eluted phage clones of the first and second rounds of screening and inoculate into a 96-well cell culture plate containing 100 μL of 2YT medium (containing 2% glucose and 50 μg / mL ampicillin) per well, one clone per well, and incubate at 37°C, 250 rpm for 12 hours. Transfer 5 μL of the above bacterial solution to a new 96-well plate containing 200 μL of 2YT medium (containing 2% glucose and 50 μg / mL ampicillin) per well for incubation (the remaining bacterial solution is added with 15% glycerol to a final concentration and stored at -80°C). Incubate at 37°C, 250 rpm for 1.5 hours to an OD of 0.2-0.3. 600For about 0.5, 100 μL of bacterial solution was removed from each well. 50 μL of 4 x 10 8 pfu KM13 was added to each well, mixed, and incubated at 37°C for 45 min. The mixture was centrifuged at 3500 g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 200 μL of 2YT medium (containing 0.1% glucose, 50 μg / mL ampicillin, and 50 μg / mL kanamycin) and cultured at 25°C with shaking at 220 rpm for 20 h. The mixture was centrifuged at 3500 g for 10 min, and 75 μL of the supernatant was transferred to each well of a 96-well plate containing 225 μL of MPBS. The plate was mixed and stored at 4°C until use. Thus, the preparation of the monoclonal phage was completed.

[0214] The IgE-Fc 2-4 protein was diluted with PBS to 2 μg / mL, and 100 μL / well was added to each well of a 96-well enzyme-labeled plate. A blank control (PBS well) was also set up. The plate was coated at 4°C overnight. The plate was washed with PBS three times, 300 μL of MPBS was added to each well, and the plate was blocked at room temperature for 2 h. 100 μL of the prepared phage MPBS mixture was added to each well, and the plate was incubated at room temperature for 1 h. The plate was washed with PBST four times. The HRP-anti M13 antibody (Beijing Yiqiao God State) was diluted with MPBS, and 100 μL was added to each well of the above immunized plate, which was incubated at room temperature for 1 h. The plate was washed with PBST four times. 100 μL of TMB color developing substrate (Biyun Tian) was added to each well, the plate was wrapped with aluminum foil to protect it from light, and the plate was allowed to react at room temperature for 5 min. 50 μL of 1 M H2SO4 was added to each well to stop the reaction, and the OD 450 nm value was measured.

[0215] All positive clones with an OD 450 nm value greater than 1 were sent to a company for sequencing. The sequencing results were analyzed and compared, and the CA20 nanobody was obtained. The results of the two rounds of monoclonal phage ELISA are shown in Figure 1 From Figure 1 the results of the second round of screening, it can be seen that the positive rate in the monoclonal phage ELISA results is higher than that in the first round of screening. This indicates that the phage binding to IgE-Fc 2-4 was well enriched after two rounds of screening.

[0216] The amino acid sequence of the nanobody CA20 is as follows:

[0217] QVQLVESGGGSVQAGGSLRLSCVASGYHHS SYCMG WFRQAPGKERER VA DIDKDGSTNYADSVKG RFTISKDNAKNTLYLQMNSLKPEDTAMYYCAA DLGRAYNCYSGSWYRREFDNFWGQGTQVTVSS (SEQ ID NO: 4)

[0218] The underlined part is the CDR determined by the Kabat rule.

[0219] The CDR sequences of the nanobody are shown in Table 1 below.

[0220] Table 1

[0221]

[0222] Preparation of human IgGl Fc fusion protein of nanobody CA20 of Example 2

[0223] The primer was designed to fuse the gene sequence of the nanobody CA20 at the N-terminus with the signal peptide of IFNα protein to guide the secretion expression, and to fuse the human IgGl Fc at the C-terminus, while introducing a TEV enzyme cutting site between them, and to clone into the mammalian expression vector pTT5 (NRC Biotechnology Research Institute). The constructed vector was transiently transfected into mammalian cells HEK293F (ATCC) using PEI, respectively, and the supernatant was collected after 3 days of culture.

[0224] The fusion protein in the supernatant was purified using a Protein A column, and the results are shown in Figure 2 The CA20 nanobody with IgGl Fc tag can be expressed in HEK 293F cells and purified; SDS-PAGE electrophoresis was performed, and the results are shown in Figure 3 The CA20-Fc fusion protein with high purity was obtained from the supernatant, and the molecular weight was about 42 kDa.

[0225] The amino acid sequence of the human IgGl Fc protein is as follows:

[0226] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV

[0227] SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

[0228] KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV

[0229] KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[0230] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

[0231] The amino acid sequence of the Linker containing TEV enzyme cleavage site connecting the Nanobody and human IgGl Fc protein is as follows:

[0232] SRGSENLYFQGSGS (SEQ ID NO: 12).

[0233] Example 3 IgE binding affinity characterization of the Fc fusion protein CA20-Fc of the Nanobody CA20

[0234] The binding of the Fc fusion protein of the Nanobody CA20 to IgE-Fc 2-4 was characterized by ELISA: IgE-Fc 2-4 was diluted with PBS to 2 μg / mL, 100 μL per well was added to the immunoplate for coating, after washing and blocking, 1:4 gradient diluted CA20-Fc fusion protein solution was added, incubated at room temperature for 1 hour. After washing, HRP conjugated anti-human IgGl Fc secondary antibody (Beijing Yiqiao God) was added. Incubate for 1 hour, wash, add 100 μL TMB for color development, and add 50 μL 1M sulfuric acid to stop, detect OD 450 nm 450 nm The results are shown in Figure 4 , CA20-Fc can bind IgE-Fc 2-4 with high affinity, the EC 50 value is (2.941 ± 0.582) x 10 -10 M.

[0235] Example 4 Binding of the Fc fusion protein CA20-Fc of the Nanobody CA20 to different domains of IgE

[0236] The binding of the Fc fusion protein of the Nanobody CA20 to different domains of IgE-Fc 2-4 was characterized by ELISA: IgE-Fc 2-4 ​The three domains Cε2, Cε3, Cε4 of IgE-Fc2, IgE-Fc3, IgE-Fc4 were diluted to 2 μg / mL with PBS, 100 μL of each was added to the immunoplates for coating, after washing and blocking, 100 nM of CA20-Fc fusion protein solution was added, and incubated at room temperature for 1 hour. After washing, HRP conjugated anti-human IgG1 Fc secondary antibody (Beijing Yiqiao God) was added. Incubate for 1 hour, wash, add 100 μL TMB for color development, and add 50 μL 1M sulfuric acid to stop, detect OD 450 nm The results are shown in Figure 5 Figure 4, CA20-Fc mainly binds to the Cε4 domain of IgE-Fc 2-4 .

[0237] Example 5 Effect of Fc fusion protein CA20-Fc of nanobody CA20 on inhibition of IgE binding to receptor FcεR Iα

[0238] The Fc fusion protein of nanobody CA20 was characterized by competitive ELISA to inhibit IgE-Fc 2-4 binding to receptor FcεR Iα: FcεR Iα protein was diluted to 2 μg / mL with PBS, 100 μL of each was added to the immunoplates for coating, after washing and blocking, CA20-Fc fusion protein solution containing 20 nM biotin-labeled IgE-Fc 2-4 protein (1:4 gradient dilution) was added, and incubated at room temperature for 1 hour. After washing, horseradish peroxidase conjugated Streptavidin antibody (Shanghai Sangon) was added. Incubate for 1 hour, wash, add 100 μL TMB for color development, and add 50 μL 1M sulfuric acid to stop, detect OD 450 nm The results are shown in 450 nm The OD 2-4 values and concentrations were analyzed by fitting.

[0239] The results are shown in Figure 6 Figure 6, CA20-Fc can effectively inhibit IgE-Fc 2-4 binding to FcεR Iα.

[0240] Discussion

[0241] According to existing technology, IgE bound to allergens binds to IgE high-affinity receptor I (FcεRI) on the surface of mast cells or basophils, leading to a cross-linking reaction of FcεRI. This cross-linking can trigger degranulation of mast cells and basophils, releasing substances such as histamine, thereby mediating an immediate-type hypersensitivity reaction and causing a series of allergic symptoms, such as vasodilation and bronchoconstriction. Currently, antibodies targeting human IgE have been proven to be effective in treating allergies, and these antibodies primarily bind to IgE-Fc receptors. 2-4 The Cε3 structural domain.

[0242] This invention provides a high-affinity camel-derived nanobody targeting human immunoglobulin E (IgE), named CA20, whose human IgG1 Fc fusion protein CA20-Fc binds to human IgE-Fc. 2-4 EC 50 The value was (2.941±0.582)×10⁻¹⁰ M. CA20-Fc mainly and specifically binds to the Cε4 domain of IgE and can effectively inhibit IgE-Fc. 2-4 It binds to the receptor FcεRIα.

[0243] Therefore, based on the experimental results of this invention, it can be reasonably inferred that the nanobody or fusion protein of this invention inhibits IgE-Fc. 2-4 A novel mechanism for binding to receptor FcεRⅠα. For example... Figure 7 As shown, IgE-Fc combined with FcεRⅠα 2-4 The Cε3 domain is in an open conformation, and the Cε2 domain bends towards the Cε3 domain, providing space for the binding of FcεRIα. When the CA20 nanobody binds to IgE-Fc... 2-4 When the Cε4 domain is formed, it creates steric hindrance between itself and the curved Cε2 domain, causing the Cε2 domain to extend upwards. This creates steric hindrance between the Cε2 domain and FcεRIα, and may also cause the Cε3 domain to change from an open conformation to a closed conformation, thereby inhibiting IgE-Fc 2-4 It binds to the receptor FcεRⅠα.

[0244] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A Nanobody against human IgE, characterized in that, The nanobody has three complementarity determining regions (CDRs) derived from a VHH chain as shown in the amino acid sequence of SEQ ID NO: 4; The CDRs are CDR1, CDR2 and CDR3 determined by any one of Kabat convention, IMGT convention, Chothia convention, AbM convention or Contact convention.

2. The Nanobody of claim 1, wherein The CDR1, CDR2 and CDR3 are selected from the group consisting of: (a) CDRs determined based on Kabat convention: CDR1 of amino acids as shown in SEQ ID NO: 5, CDR2 of amino acids as shown in SEQ ID NO: 6, and CDR3 of amino acids as shown in SEQ ID NO: 7; (b) CDRs determined based on IMGT convention: CDR1 of amino acids as shown in SEQ ID NO: 8, CDR2 of amino acids as shown in SEQ ID NO: 9, and CDR3 of amino acids as shown in SEQ ID NO:

10.

3. The nanobody of claim 1, wherein The nanobody has the amino acid sequence as shown in SEQ ID NO: 4, or has an amino acid sequence with homology of ≥ 85%, ≥ 90%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% to the amino acid sequence as shown in SEQ ID NO:

4.

4. A fusion protein of a Nanobody against human IgE, characterized in that, The fusion protein comprises: (i) the sequence of the VHH chain of the anti-human IgE nanobody as claimed in claim 1; and (ii) a Fc fragment.

5. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the nanobody as claimed in claim 1 or the fusion protein as claimed in claim 4.

6. An expression vector, characterized by, The expression vector contains the nucleotide molecule as claimed in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector as claimed in claim 6, or has the nucleotide molecule as claimed in claim 5 integrated on its genome.

8. An immunoconjugate, comprising, The immunoconjugate contains: (a) the anti-human IgE nanobody as claimed in claim 1; and (b) a conjugated moiety selected from the group consisting of: a detectable label, a drug.

9. Use of a Nanobody according to claim 1 or a fusion protein according to claim 4, characterized in that, for preparing a medicament, a reagent, an assay plate or a kit; the reagent, assay plate or kit is used for: detecting IgE in a sample; the medicament is used for preventing, diagnosing and / or treating an IgE-mediated allergic disease.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (i) the anti-human IgE nanobody as claimed in claim 1, or the fusion protein as claimed in claim 4; and (ii) a pharmaceutically acceptable carrier.

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