Shark-derived nanoantibodies that bind to human immunoglobulin E

Abstract

The invention discloses a shark-derived nano antibody binding 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 or the fusion protein thereof 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 shark-derived 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 and bronchial constriction. Antibodies targeting human IgE have been shown to be effective in treating allergies, such as omalizumab, but are expensive and have limited applicability to allergic symptoms.

[0003] Chondrichthyan animals (nurse sharks, striped bamboo sharks, etc.) can produce nanobodies (Nbs), which have the smallest variable antigen-binding fragment, known as VNAR domains, with a molecular weight of only 13-15 kDa. Compared with conventional antibodies, nanobodies have unique advantages: 1) nanobodies have a simple structure and small molecular weight, which are more conducive to expression and use; 2) small size allows it to recognize epitopes that traditional antibodies cannot approach; 3) as a single-domain antibody with only one binding site, it 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 are 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 produce immune rejection. In addition, nanobodies can be directly delivered to the lungs after being aerosolized, or even administered directly 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 applicable to more allergic diseases. SUMMARY

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

[0006] In a first aspect of the present application, a heavy chain variable region VNAR that specifically binds to human IgE is provided, the VNAR comprising the following complementarity determining regions CDRs:

[0007] CDR1 as shown in SEQ ID NO: 5; and CDR3 as shown in SEQ ID NO: 6.

[0008] In another preferred embodiment, the heavy chain variable region further comprises a framework region (FR).

[0009] In another preferred embodiment, the VNAR has an amino acid sequence as set forth in SEQ ID NO: 4, or an amino acid sequence having a sequence identity of > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, or > 99% to the amino acid sequence as set forth in SEQ ID NO: 4.

[0010] In another preferred embodiment, any one of the above-mentioned amino acid sequences further comprises a derivative sequence which has at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified and / or substituted, and which retains the ability to specifically bind to IgE.

[0011] In another preferred embodiment, the VNAR has an amino acid sequence as set forth in SEQ ID NO: 4.

[0012] In another preferred embodiment, the derivative sequence which has at least one amino acid added, deleted, modified and / or substituted, and which retains the ability to specifically bind to IgE, is an amino acid sequence having a homology or sequence identity of at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0013] In a second aspect of the present application, there is provided a shark nanobody against human IgE, said nanobody comprising a heavy chain variable region VNAR as described in the first aspect of the present application.

[0014] In another preferred embodiment, the nanobody comprises or consists of a sequence as set forth in SEQ ID NO: 4.

[0015] In another preferred embodiment, the nanobody comprises a humanized antibody, a shark-derived antibody, a chimeric antibody.

[0016] In another preferred embodiment, the nanobody is a Triakis scabbarius antibody.

[0017] In another preferred embodiment, the nanobody further comprises a hinge region and / or a constant region.

[0018] In another preferred embodiment, the nanobody maintains more than 50% activity at 80°C.

[0019] In another preferred embodiment, the nanobody maintains 80% activity at a pH range of 2.0-9.0.

[0020] In a third aspect of the application, there is provided an antibody against human IgE, said antibody comprising one or more heavy chain variable regions as described in the first aspect of the application.

[0021] In another preferred embodiment, the antibody against IgE can be a monomeric, bivalent, and / or multivalent antibody.

[0022] In another preferred embodiment, the antibody against IgE is a bivalent antibody.

[0023] In another preferred embodiment, the antibody is a monospecific or multispecific antibody.

[0024] In a fourth aspect of the application, there is provided a chimeric antigen receptor (CAR), said CAR comprising an extracellular domain, said extracellular domain comprising a heavy chain variable region as described in the first aspect of the application.

[0025] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0026] L-Nb-H-TM-C-CD3ζ (Ia)

[0027] wherein,

[0028] L is nothing or a signal peptide sequence;

[0029] Nb is a specific binding domain targeting IgE, comprising a heavy chain variable region as described in the first aspect of the application;

[0030] H is nothing or a hinge region;

[0031] TM is a transmembrane domain;

[0032] C is a costimulatory signaling domain;

[0033] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or a mutant / modified thereof);

[0034] said “-” is a connecting peptide or a peptide bond.

[0035] In another preferred embodiment, the L is a signal peptide selected from the group consisting of CD8, GM-CSF, CD4, CD28, CD137, or a mutant / modified thereof, or a combination thereof.

[0036] In another preferred embodiment, the H is a hinge region selected from the group consisting of CD8, CD28, CD137, IgG, or a combination thereof.

[0037] In another preferred embodiment, the TM is selected from the transmembrane region of the following group of proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or a mutant / modified version thereof, or a combination thereof.

[0038] In another preferred embodiment, the C is selected from the costimulatory domain of the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or a mutant / modified version thereof, or a combination thereof.

[0039] In a fifth aspect of the present application, there is provided a recombinant protein having:

[0040] (i) the heavy chain variable region as defined in the first aspect of the present application, the shark nanobody as defined in the second aspect of the present application, or the antibody as defined in the third aspect of the present application; and

[0041] (ii) an optional tag sequence to assist expression and / or purification.

[0042] In another preferred embodiment, the tag sequence comprises a Fc tag, a HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0043] In another preferred embodiment, the recombinant protein specifically binds IgE.

[0044] In another preferred embodiment, the recombinant protein comprises a fusion protein.

[0045] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0046] In another preferred embodiment, the tag sequence is a Fc tag.

[0047] In a sixth aspect of the present application, there is provided a shark nanobody fusion protein against human IgE, the fusion protein comprising:

[0048] (a) the shark nanobody variable region VNAR as defined in the first aspect of the present application or the shark nanobody as defined in the second aspect of the present application; and

[0049] (b) an Fc fragment.

[0050] In another preferred embodiment, the Fc fragment is an IgG Fc fragment.

[0051] In another preferred embodiment, the Fc fragment is a human IgG1 Fc fragment.

[0052] In another preferred embodiment, the amino acid sequence of the VNAR is set forth in SEQ ID NO: 4.

[0053] In another preferred embodiment, the VHH chain of the anti-human IgE nanobody is linked to the Fc fragment via a linker sequence.

[0054] In another preferred embodiment, the fusion protein has a structure as set forth in Formula I from N-terminus to C-terminus:

[0055] Z1--L-Z2(Formula I)

[0056] In the formula,

[0057] Z1is a heavy chain variable region as described in the first aspect of the present application;

[0058] L is a linker sequence;

[0059] Z2is a human IgG1 Fc fragment.

[0060] In another preferred embodiment, the amino acid sequence of the Fc fragment is set forth in SEQ ID NO: 7.

[0061] In another preferred embodiment, the amino acid sequence of the linker sequence is set forth in SEQ ID NO: 8.

[0062] In a seventh aspect of the present application, a conjugate is provided, the conjugate comprising:

[0063] (a) a heavy chain variable region as described in the first aspect of the present application, a shark nanobody as described in the second aspect of the present application, an antibody as described in the third aspect of the present application, a recombinant protein as described in the fifth aspect of the present application, or a fusion protein as described in the sixth aspect of the present application; and

[0064] (b) a conjugating moiety selected from the group consisting of: a detectable label, a drug.

[0065] In another preferred embodiment, the (a) moiety is conjugated to the conjugating moiety via a chemical bond or a linker.

[0066] In another preferred embodiment, the drug is a drug for treating allergy.

[0067] In another preferred embodiment, the conjugate comprises: a multivalent (e.g., bivalent) heavy chain variable region as described in the first aspect of the present application.

[0068] In another preferred embodiment, the polyvalency refers to the presence of multiple repeats of the same or different heavy chain variable region as described in the first aspect of the present application in the amino acid sequence of the conjugate.

[0069] In an eighth aspect of the present application, there is provided a nucleotide molecule encoding the heavy chain variable region as described in the first aspect of the present application, the Nanobody as described in the second aspect of the present application, or the fusion protein as described in the sixth aspect of the present application.

[0070] In another preferred embodiment, the nucleotide molecule is RNA, DNA or cDNA.

[0071] In a ninth aspect of the present application, there is provided an expression vector comprising the nucleotide molecule as described in the eighth aspect of the present application.

[0072] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof. Preferably, the expression vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0073] In another preferred embodiment, the expression vector further comprises a promoter, a transcriptional enhancer element WPRE, a long terminal repeat LTR, and the like.

[0074] In a tenth aspect of the present application, there is provided a host cell comprising the expression vector as described in the ninth aspect of the present application, or having integrated into the genome the nucleotide molecule as described in the eighth aspect of the present application.

[0075] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0076] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cell, mammalian cell.

[0077] In an eleventh aspect of the present application, there is provided an engineered immune cell expressing the chimeric antigen receptor as described in the fourth aspect of the present application.

[0078] In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

[0079] (i) chimeric antigen receptor αβ T cell (CAR-T cell);

[0080] (ii) chimeric antigen receptor γδ T cell (CAR-T cell);

[0081] (iii) chimeric antigen receptor NKT cell (CAR-NKT cell);

[0082] (iv) chimeric antigen receptor NK cells (CAR-NK cells).

[0083] In another preferred embodiment, the engineered immune cell comprises an autologous or allogeneic αβ T cell, γδ T cell, NKT cell, NK cell, or a combination thereof.

[0084] In another preferred embodiment, the engineered immune cell is a CAR-T cell.

[0085] In a twelfth aspect of the present application, a method for producing the nanobody of the second aspect of the present application or the fusion protein of the sixth aspect of the present application is provided, comprising the steps of:

[0086] (a) culturing the host cell of the tenth aspect of the present application under conditions suitable for production of the shark nanobody or fusion protein thereof, thereby obtaining a culture comprising the anti-human IgE shark nanobody or fusion protein thereof;

[0087] (b) isolating and / or recovering the anti-human IgE shark nanobody or fusion protein thereof from the culture; and

[0088] (c) optionally, purifying and / or modifying the anti-human IgE shark nanobody or fusion protein thereof obtained in step (b).

[0089] In a thirteenth aspect of the present application, use of the heavy chain variable region of the first aspect of the present application, the shark nanobody of the second aspect of the present application, the recombinant protein of the fifth aspect of the present application, or the fusion protein of the sixth aspect of the present application, for the manufacture of a medicament, reagent, detection plate or kit is provided.

[0090] The reagent, detection plate or kit is used for detecting IgE in a sample.

[0091] The medicament is used for preventing, diagnosing and / or treating an IgE-mediated allergic disease.

[0092] In a fourteenth aspect of the present application, a pharmaceutical composition is provided, comprising:

[0093] (i) the heavy chain variable region of the first aspect of the present application, the shark nanobody of the second aspect of the present application, the antibody of the third aspect of the present application, or the fusion protein of the sixth aspect of the present application; and

[0094] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0095] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of an injection, a lyophilized agent.

[0096] In a fifteenth aspect of the present application, there is provided a method for detecting human IgE protein in a sample, said method comprising the steps of:

[0097] (1) contacting the sample with a Nanobody according to the second aspect of the present application;

[0098] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of human IgE protein in the sample.

[0099] In a sixteenth aspect of the present application, there is provided a detection reagent for human IgE, said detection reagent comprising:

[0100] (i) an anti-IgE Nanobody according to the second aspect of the present application, or a fusion protein according to the sixth aspect of the present application; and

[0101] (ii) a detectably acceptable carrier.

[0102] In another preferred embodiment, the conjugated moiety of the immunoconjugate is a diagnostic isotope.

[0103] In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0104] In another preferred embodiment, the detection reagent is one or more reagents 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.

[0105] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0106] In another preferred embodiment, the detection reagent is in a dosage form selected from the group consisting of a liquid and a powder (e.g. an aqueous solution, a syringe, a lyophilized powder, a tablet, a buccal, and an inhalation aerosol).

[0107] In a seventeenth aspect of the present application, there is provided a kit comprising a detection reagent according to the sixteenth aspect of the present application.

[0108] In an eighteenth aspect of the present application, there is provided a method for treating an IgE-mediated allergic disease, said method comprising administering to a subject in need thereof a Nanobody according to the second aspect of the present application or a fusion protein according to the sixth aspect of the present application.

[0109] In another preferred embodiment, the subject is a mammal, such as a human.

[0110] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the Examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. Attached Figure Description

[0111] Figure 1 The monoclonal Phage ELISA was used to detect the nanobody SA14 and IgE-Fc. 2-4 The combination of.

[0112] Figure 2 The results of Protein A column purification of human IgG1 Fc fusion protein SA14-Fc of nanobody SA14 are shown.

[0113] Figure 3 The SDS-PAGE electrophoresis results of the human IgG1 Fc fusion protein SA14-Fc of the nanobody SA14 are shown.

[0114] Figure 4 This demonstrates the ELISA detection of SA14-Fc and IgE-Fc. 2-4 The result of affinity.

[0115] Figure 5 The results show the detection of SA14-Fc and IgE-Fc by ELSIA. 2-4 The combination of different structural domains.

[0116] Figure 6 This demonstrates competitive ELISA detection of SA14-Fc inhibiting IgE-Fc. 2-4 The effect of binding receptor FcεRⅠα.

[0117] Figure 7 Flow cytometry analysis showed that SA14-Fc inhibited IgE-Fc. 2-4 The effect of binding to the FcεRIα receptor on the surface of KU812 cells.

[0118] Figure 8 The nanobody SA14 showed inhibition of IgE-Fc 2-4 A schematic diagram illustrating the mechanism of binding to receptor FcεRⅠα. Detailed Implementation

[0119] Through extensive and in-depth research and screening, the inventors unexpectedly obtained a shark nanobody (SA14) against human IgE. The shark nanobody or its fusion protein (human IgG1 Fc fusion protein of nanobody SA14) of this invention can effectively bind primarily to the Cε2 domain of IgE, rather than primarily through binding to IgE-Fc. 2-4 The Cε3 domain can effectively suppress IgE-Fc 2-4 By binding to the FcεRIα receptor on the surface of KU812 cells, allergies can be effectively treated or suppressed. Based on this, the present invention was completed.

[0120] Definitions

[0121] To facilitate a better understanding of the present disclosure, certain terms are defined first. As used in this application, unless specifically stated otherwise, each of the following terms has the meaning given below.

[0122] The term“about” can refer to a value or a composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0123] The term“administering” refers to physical introduction of a product of the present application into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0124] The term“EC50” refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that elicits 50% of the maximal effect.

[0125] The term“IC50” refers to the half maximal inhibitory concentration of an antagonist being measured.

[0126] IgE

[0127] 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 the total immunoglobulin (Ig) in serum. It is a cytophilic antibody that mainly mediates type I allergic reactions.

[0128] When the human body is stimulated by allergens, IgE will bind 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 allergic reactions. Among them, FcεR Iα is the α chain of FcεR I, which contains 222 amino acid residues and has a molecular weight of 25 kDa. The extracellular region of FcεR Iα is the main site for binding to IgE. 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.

[0129] The level change of IgE has important clinical significance, and is often used for the diagnosis of allergic diseases and the determination of allergens. The increase of IgE is mainly seen in parasitic diseases and allergic diseases, such as allergic rhinitis, allergic urticaria, allergic asthma and allergic conjunctivitis, etc.

[0130] The Fc segment (106-428 amino acid residues) of human IgE antibody includes three domains:

[0131] Cε2 domain (106-210 amino acid residues):

[0132] SRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQ VMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFED STKKCA (SEQ ID NO: 1);

[0133] Cε3 domain (211-318 amino acid residues):

[0134] DSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRAS GKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRA LMRSTTKTS (SEQ ID NO: 2); and

[0135] Cε4 domain (319-428 amino acid residues):

[0136] GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQ LPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQ TVQRAVSVNPGK (SEQ ID NO: 3).

[0137] At present, antibodies targeting human IgE have been proved to be effective in treating allergy, and the antibodies targeting human IgE mainly inhibit the binding of IgE-Fc 2-4 to FcεRIα by binding to the Cε3 domain of IgE-Fc 2-4 .

[0138] Shark nanobody

[0139] Single-domain antibody (Nb), full name heavy chain variable region heavy chain antibody (VHH). VHH crystal is 2.5 nm long 4 nm, so it is also called single-domain antibody or nanobody. In 1993, Hamers-casterman et al. found a L chain missing "heavy chain antibody" in the peripheral blood (PBMC) of camelids, which only contains a heavy chain variable region (VHH) and two heavy chains (CH2 and CH3 regions). VHH retains all antigen binding ability, is the smallest antibody that retains complete antigen binding fragments, is called single-domain antibody (Single-domain antibodies), also known as nanobody. The biggest advantage of nanobody is its small size, with a molecular weight of only 15 KD, which is one tenth of the traditional antibody (150 KD).

[0140] In 1995, Flajnik discovered a natural heavy chain antibody-antibody new antigen receptor (IgNAR) in cartilaginous fishes. IgNAR is a unique antibody that has been found in several different types of sharks, including nurse sharks, striped dogfish, bamboo sharks, striped dogfish and horned sharks. IgNAR is different from traditional antibodies, which are composed of two heavy chains, each consisting of five constant regions, a hinge region and a variable region.

[0141] The variable region of IgNAR, also known as VNAR, contains only two complementarity determining regions (CDRs), CDR1 and CDR3. Due to the absence of CDR2, VNAR is the smallest immunoglobulin naturally occurring so far, with a molecular weight of about 12 kDa. VNAR can also contain two hypervariable loops (Hypervariable loop, HV) HV2 and HV4. Through the groove on the hypervariable loop and the smaller size, VNAR can enter the groove that general antibodies cannot enter, and has strong antigen affinity.

[0142] The sequence of shark nanobody is FR1, FR2, FR3a, FR3b and FR4, HV2, HV4, CDR1 and CDR3, wherein FR1 and FR4 are fixed amino acid sequences, and CDR1 and CDR3 are complementarity determining regions of antibodies, which determine binding to different antigens.

[0143] VNAR has the advantages of strong tissue penetration, higher stability, recognition of hidden epitopes of antigens, and development of new targets for tumor treatment. Therefore, based on the above advantages, VNAR has great prospects for drug research and development when combined with antibodies, cytokines, CAR-NK, etc.

[0144] Shark nanobodies (VNARs, Variable New Antigen Receptors) exhibit high thermal stability and high acid stability due to their unique structure and physiological properties, making them have application potential in multiple special indications. For example, they can be applied in gastrointestinal diseases, for example, shark nanobodies can remain stable in gastric acid environment, so they are suitable for treating gastrointestinal infections caused by pathogens such as Helicobacter pylori. In inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, nanobodies can directly act on the intestine through oral administration, reducing systemic side effects.

[0145] Shark nanobodies can be applied in the treatment of infectious diseases. In viral infections, high stability allows shark nanobodies to still function in harsh environments, making them suitable for developing treatments and diagnostic tools for viral infections (such as enterovirus, influenza virus, etc.). In bacterial infections, shark nanobodies can remain active in high-temperature or acidic environments, making them suitable for difficult-to-treat bacterial infections, especially drug-resistant strains.

[0146] Shark nanobodies can be applied in cancer treatment. In the tumor microenvironment, the inside of the tumor is often acidic, and shark nanobodies can remain active in this environment for targeted and destruction of tumor cells. High thermal stability allows shark nanobodies to be combined with hyperthermia to enhance treatment effect.

[0147] Shark nanobodies can be applied in the treatment of oral diseases. In periodontal disease and dental caries, the oral environment is complex, with large pH changes, and the acid stability of shark nanobodies makes them suitable for treating and preventing oral bacterial infection and inflammation.

[0148] Shark nanobodies can be applied in the treatment of skin diseases. Stability in high-temperature and different pH environments makes shark nanobodies suitable for developing topical drugs for treating skin infections and inflammation, such as eczema, psoriasis, etc.

[0149] In addition, due to its stability, shark nanobodies can be used to develop environmental and food safety detection tools that can still accurately detect pollutants or pathogens under high-temperature or acidic conditions. In enzyme stabilizers, in biological catalysis and industrial enzyme preparations, shark nanobodies can be used as stabilizers to maintain the activity and function of enzymes. In biosensors, shark nanobodies can be used to develop biosensors for extreme conditions for use in environmental monitoring, medical diagnosis, etc.

[0150] Overall, the high thermal stability and high acid stability of shark nanobodies make them have application potential in multiple fields, especially those involving harsh environments in medical and biotechnology applications. These unique properties make shark nanobodies a powerful tool for solving complex medical problems and developing new treatment options.

[0151] Shark nanobodies of the invention

[0152] As used herein, the terms "antibody of the invention", "single domain antibody of the invention", "shark antibody", "shark single domain antibody", "single domain antibody against IgE", "shark single domain antibody against IgE", and the like have the same meaning and are used interchangeably to refer to a single domain antibody (nanobody) obtained by immunizing a shark that specifically recognizes and binds to IgE protein, including human IgE protein.

[0153] The nanobody of the invention comprises a heavy chain variable region that specifically binds IgE, said VNAR comprising the following complementarity determining regions CDRs:

[0154] CDR1 as set forth in SEQ ID NO: 5; and CDR3 as set forth in SEQ ID NO: 6.

[0155] In another preferred embodiment, the amino acid sequence of said VNAR is as set forth in SEQ ID NO: 4.

[0156] The term "CDR3" refers to the region of the TCR that directly contacts the antigen, the CDR3 sequence of a shark single domain antibody is longer than that of IgG, typically 8-18 amino acids.

[0157] The present invention includes not only the intact antibody, but also fragments of the antibody that are immunologically active or fusion proteins of the antibody with other sequences. Thus, the present invention also includes fragments, derivatives and analogs of the antibody.

[0158] As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the antibody of the invention. A polypeptide fragment, derivative or analog of the invention can be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) a polypeptide having a substituent group at one or more amino acid residues, or (iii) a polypeptide formed by fusing the mature polypeptide to another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence or protein for purifying the polypeptide or a proteinogen sequence, or a fusion protein with a 6His tag). These fragments, derivatives and analogs are within the scope of those of ordinary skill in the art in light of the teachings herein.

[0159] The term "antibody" of the present application refers to a polypeptide having IgE binding activity comprising the CDR regions described above. The term also includes variants of polypeptides comprising the CDR regions described above which have the same function as the antibodies of the present application. These variants include, but are not limited to, deletions from, and / or insertions into, and / or substitutions of, one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids of a polypeptide disclosed herein. The present application also includes a polypeptide in which a conservative substitution of an amino acid is made, with the exception of cysteine residues not involved in disulfide bonding. Conservative substitution of amino acids can be performed by known methods in the art. The term also includes fragments and derivatives of the antibodies of the present application which are active.

[0160] The variants of the polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA hybridizing to the DNA encoding the antibodies of the present application under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present application.

[0161] The present application also provides other polypeptides, such as fusion proteins comprising a nanobody or fragment thereof. In addition to the substantially full-length polypeptides, the present application also includes fragments of the nanobodies of the present application. Typically, the fragments have at least about 50 contiguous amino acids of the antibodies of the present application, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0162] In the present application, "conservative variants of the antibodies of the present application" refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids of the amino acid sequence of the antibodies of the present application are replaced by amino acids of similar or analogous properties. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0163] Table 1

[0164]

[0165]

[0166] Polynucleotides, vectors, and host cells

[0167] The present application also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof, or chimeric antigen receptors comprising the antibodies. The polynucleotides 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 a coding strand or a non-coding strand.

[0168] The polynucleotides encoding the mature polypeptides of the present application include: a coding sequence encoding only the mature polypeptide; a coding sequence encoding the mature polypeptide and various additional coding sequences; a coding sequence encoding the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0169] The term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising a coding sequence encoding the polypeptide, or a polynucleotide further comprising additional coding and / or non-coding sequences.

[0170] The present application also relates to polynucleotides that hybridize to the above-mentioned sequences and 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" refer to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2x SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs 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.

[0171] The nucleotide full-length sequence of the antibody of the present application or fragments thereof can generally 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 relatively short. Generally, a long fragment of the sequence 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.

[0172] 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 transforming 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 in isolated form.

[0173] At present, it is possible to obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present application by chemical synthesis completely. Then the DNA sequence can be introduced into various existing DNA molecules (or as a vector) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0174] The present application also relates to a vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0175] 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 are: bacterial cells of 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.

[0176] The 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 DNA can be introduced into the host by treatment of competent cells with the DNA following the exponential growth phase, using the CaCl2method, the steps of which are well known in the art. Alternatively, MgCl2can be used. If desired, the transformation can also be performed by electroporation. When the host is a eukaryote, the DNA can be introduced into the host using a method of DNA transfection such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0177] The transformants obtained can be cultured using conventional methods to express the polypeptide encoded by the gene of the present application. Depending on the host cells used, the culture medium used in the culture can be selected from various conventional media. The culture is performed under conditions suitable for the growth of the host cells. When the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0178] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. 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, ultra-treatment, ultra-centrifugation, 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.

[0179] Pharmaceutical composition

[0180] 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.

[0181] The pharmaceutical composition of the present application can be directly used to bind IgE protein molecules, and thus can be used to treat IgE overexpression diseases, such as allergic diseases such as asthma. In addition, other therapeutic agents can also be used simultaneously.

[0182] 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 the needle and the 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.

[0183] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the 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.

[0184] Labeled nanobody

[0185] 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.

[0186] The colloidal gold labeling can be performed 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.

[0187] The anti-IgE nanobodies of the present application have good specificity and high potency.

[0188] Phage display technology

[0189] 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. In the case that the reading frame is normal and the normal function of the coat protein is not affected, the foreign gene will be 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.

[0190] 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. Then, the unbound phages are washed away, and the adsorbed phages are eluted by using a competitive receptor. The eluted phages are used to infect host bacteria for propagation and expansion, and then the next round of elution is performed.

[0191] After several rounds of "adsorption-elution-expansion" (for some antibodies with weak affinity, more rounds of elution are required), a high enrichment of phages that can specifically bind to the target protein can be obtained.

[0192] Detection method

[0193] 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; and detecting the level of IgE protein in the dissolved sample.

[0194] 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.

[0195] Kit

[0196] 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, an instruction manual, a buffer, etc.

[0197] 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.

[0198] The main advantages of the present application include:

[0199] ​(a) The human IgGl Fc fusion protein of the nanobody SA14 described in the present application can bind to the Fc fragment of human IgE antibody (IgE-Fc 2-4 ) with high affinity.

[0200] (b) The human IgGl Fc fusion protein of the nanobody SA14, SA14-Fc, mainly binds to the Cε2 domain of IgE, rather than the Cε3 domain, i.e., it can inhibit the binding of IgE-Fc 2-4 to the FcεRl receptor on the surface of KU812 cells.

[0201] (c) Compared with traditional monoclonal antibodies, VNAR has a simple structure, is expressed by a single gene, and can be produced on a large scale by various expression systems, which is conducive to reducing costs.

[0202] (d) The molecular weight of shark single-domain antibody is smaller, has strong tissue penetration and strong stability, can be used for oral or inhalation preparation to regulate the immune system of the body, and can be administered by the respiratory tract, providing a new method for the prevention and treatment of allergies.

[0203] (e) Nanobody can be used to develop an allergic therapeutic antibody targeting human IgE, which will be suitable for the diagnosis and / or treatment of allergic diseases.

[0204] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0205] Example 1 Screening of Nanobody

[0206] 1.1 Construction of IgE immune library

[0207] The Fc fragment of human IgE antibody (UNIPROT: P01854, aa 106-428) expressed and purified by HEK293F cells (ATCC, CBP60437) was mixed with Freund's adjuvant, and the shark was immunized 4 times at a dose of 0.05 mg / time, with an interval of 2 weeks. Two weeks after the fourth immunization, blood was taken and immune cells in the blood were separated.

[0208] 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 RTase (RTase) Kit of TAKARA Company.TM II 1st Strand cDNA Synthesis Kit to reverse transcribe RNA into cDNA. Using shark VNAR specific primers, the cDNA above was used as template to amplify the coding gene fragment of VNAR by PCR. The amplified VNAR sequence was cloned into the Ncol and Notl sites of phagemid pR2 (MRC Laboratory of Molecular Biology) by Gibson Assembly method. The Gibson Assembly product was the initial nanobody phagemid library.

[0209] E. coli TG1 (MRC Laboratory of Molecular Biology) competent cells were prepared by 10% glycerol wash method. Then the Gibson Assembly product above was electroporated into TG1 competent cells and plated into 5 150mm 2YT (containing 2% glucose and 50μg / mL ampicillin) plates to amplify the phagemid library. After scraping the plates, an appropriate amount of bacteria was inoculated into 200mL 2YT (containing 2% glucose and 50μg / mL ampicillin) to grow to the logarithmic phase. Then 10 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) was added to infect for 45 minutes at 37°C. 50mL bacteria were centrifuged and the bacterial cells were resuspended in 100mL 2YT (containing 0.1% glucose, 50μg / mL ampicillin and 50μg / mL kanamycin). The bacteria were incubated at 25°C for 20 hours to amplify the phage displaying nanobodies. The phage was concentrated by PEG precipitation method and finally resuspended in PBS and stored on ice.

[0210] 1.2 Screening of nanobody SA14

[0211] IgE-Fc 2-4 protein was diluted to 0.1mg / mL with PBS and 100μL was added to one well of a 96-well enzyme-labeled plate. The plate was coated at room temperature for 2 hours. Meanwhile, a control well without antigen was set up. The plate was washed 3 times with PBS and 300μL MPBS (PBS containing 5% skim milk) was added to each well to block at room temperature for 2 hours. The plate was washed 3 times with PBS and 1x10 11pfu above. The phage (dissolved in 100 μL MPBS) was incubated at room temperature for 1 hour at 60 rpm. The plate was washed 20 times with PBST (0.1% Tween-20). 100 μL of trypsin at a concentration of 0.5 mg / mL was added to each well, and the phage bound to the well was eluted by incubation at room temperature for 1 hour. The eluted phage was used to infect TG1, and the plate was incubated at 37°C for 16 hours to form colonies.

[0212] Eighty single clones were randomly selected from the eluted phage clones from the screening, and were inoculated 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 were incubated at 37°C for 12 hours with shaking at 250 rpm. Five μL of the bacterial solution was transferred to a new 96-well plate containing 200 μL of 2YT medium (containing 2% glucose and 50 μg / mL ampicillin) per well, and was incubated at 37°C for 1.5 hours with shaking at 250 rpm (the remaining bacterial solution was stored at -80°C after being added with glycerol at a final concentration of 15%). The OD 600 was about 0.5, 100 μL of the bacterial solution was removed from each well. 50 μL of 4 x 10 8 pfu of KM13 was added to the 2YT, and the mixture was incubated at 37°C for 45 minutes. The mixture was centrifuged at 3500 g for 10 minutes, and the supernatant was discarded. The precipitate was resuspended with 200 μL of 2YT medium (containing 0.1% glucose, 50 μg / mL ampicillin, and 50 μg / mL kanamycin), and was incubated at 25°C for 20 hours with shaking at 220 rpm. The mixture was centrifuged at 3500 g for 10 minutes, and 75 μL of the supernatant was transferred to a well of a 96-well plate containing 225 μL of MPBS per well, and was mixed. The plate was stored at 4°C until use. Thus, the preparation of the single clone phage was completed.

[0213] The IgE-Fc 2-4 protein was diluted with PBS to a concentration of 2 μg / mL, and 100 μL was added to each well of a 96-well enzyme-labeled plate. A blank control (PBS well) was also set up. The plate was incubated at 4°C overnight. The plate was washed 3 times with PBS, and 300 μL of MPBS was added to each well. The plate was incubated at room temperature for 2 hours. 100 μL of the phage MPBS mixture prepared above was added to each well, and the plate was incubated at room temperature for 1 hour. The plate was washed 4 times with PBST. The HRP-anti M13 antibody (Beijing Yiqiao God State) was diluted with MPBS, and 100 μL was added to each well of the above-mentioned plate. The plate was incubated at room temperature for 1 hour. The plate was washed 4 times with PBST. 100 μL of TMB color developing substrate (Biyun Tian) was added to each well, and the plate was wrapped with aluminum foil to protect from light. The plate was incubated at room temperature for 5 minutes. 50 μL of 1 M H2SO4 was added to each well to stop the reaction, and the OD 450 nm values were measured. The OD450 nm Positive clones with value greater than 1 were sent to the company for sequencing, and the sequencing results were analyzed and aligned to obtain the SA14 nanobody, the monoclonal phage ELISA results of which are shown in Figure 1 It can be seen from Figure 1 that most of the phage monoclonals can bind IgE-Fc 2-4 protein.

[0214] The amino acid sequence of the nanobody SA14 is as follows:

[0215] TQRVEQTPTTTTKEAGESLTINCVL RDSRC AL ENT Y WYFTKKGATKKE SLSDGGRYAETVNKASKSFSLRISDLRVEDSGTYYC KAWGPAGCYRNSI EG GGTILTVK(SEQ ID NO:4)

[0216] The two antigen complementarity determining regions (CDR1 and CDR3) of the nanobody SA14 are shown in the underlined part above, specifically:

[0217] CDR1: RDSRCALENTY (SEQ ID NO: 5)

[0218] CDR3: KAWGPAGCYRNSI (SEQ ID NO: 6)

[0219] Example 2 Preparation of human IgG1 Fc fusion protein of nanobody SA14

[0220] The primer was designed to fuse the signal peptide of IFNα protein at the N-terminal of the gene sequence of the nanobody SA14 to guide the secretion expression, and to fuse the human IgG1 Fc at the C-terminal, 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. The fusion protein in the supernatant was purified using a Protein A column, and the results are shown in Figure 2 The SA14 nanobody with IgG1 Fc tag can be expressed in HEK 293F cells and purified; SDS-PAGE electrophoresis was performed, and the results are shown in Figure 3 From the supernatant, a high-purity SA14-Fc fusion protein was obtained.

[0221] The amino acid sequence of the human IgG1 Fc protein is as follows:

[0222] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

[0223] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[0224] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS

[0225] DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

[0226] HEALHNHYTQKSLSLSPGK(SEQ ID NO:7)

[0227] The amino acid sequence of the linker containing the TEV restriction site that connects this nanobody to the human IgG1 Fc protein is as follows:

[0228] SRGSENLYFQGSGS(SEQ ID NO:8)

[0229] Example 3: Affinity characterization of human IgG1 Fc fusion protein SA14-Fc binding to IgE.

[0230] The Fc fusion protein of the SA14 nanobody and IgE-Fc were characterized by ELISA. 2-4 Binding status: IgE-Fc 2-4 Dilute to 2 μg / mL with PBS, add 100 μL to each well of the immunoplate for coating. After washing and blocking, add serially diluted SA14-Fc fusion protein solution (1:4 ratio) and incubate at room temperature for 1 hour. After washing, add HRP-conjugated anti-human IgG1Fc secondary antibody (Beijing Yiqiao Shenzhou). Incubate for 1 hour, wash, add 100 μL of TMB for color development, and stop with 50 μL of 1M sulfuric acid. Detect OD. 450 nm Value, for OD 450 nm Fit analysis was performed on the values ​​and concentrations, and the results are as follows: Figure 4 As shown, SA14-Fc can bind to IgE-Fc with high affinity. 2-4 Combined with EC 50 The value is (1.683±0.400)×10 -10 M.

[0231] Example 4: Binding of the Fc fusion protein SA14-Fc of the nanobody SA14 to different domains of IgE.

[0232] The Fc fusion protein of the SA14 nanobody and IgE-Fc were characterized by ELISA. 2-4 The combination of different structural domains: IgE-Fc 2-4 The three domains Cε2, Cε3, and Cε4 of the protein, namely IgE-Fc2, IgE-Fc3, and IgE-Fc4, were diluted to 2 μg / mL with PBS. 100 μL of each domain was added to each well of an immunoplate for coating. After washing and blocking, 100 nM SA14-Fc fusion protein solution was added, and the plate was incubated at room temperature for 1 hour. After washing, HRP-conjugated anti-human IgG1 Fc secondary antibody (Beijing Yiqiao Shenzhou) was added. After incubation for 1 hour, followed by washing, 100 μL of TMB was added for color development, and 50 μL of 1M sulfuric acid was added to stop the reaction. OD was then measured. 450 nm Value, result as Figure 5 As shown, SA14-Fc mainly binds to IgE-Fc. 2-4 The Cε2 structural domain.

[0233] Example 5: Effect of the Fc fusion protein SA14-Fc of the nanobody SA14 on inhibiting the binding of IgE to receptor FcεRIα.

[0234] The Fc fusion protein of the SA14 nanobody was characterized by its ability to inhibit IgE-Fc using competitive ELISA. 2-4 Binding to receptor FcεRIα: FcεRIα protein was diluted to 2 μg / mL with PBS, and 100 μL was added to each well for coating. After washing and blocking, IgE-Fc containing 20 nM biotin was added. 2-4 SA14-Fc fusion protein solutions (1:4 serial dilutions) were incubated at room temperature for 1 hour. After washing, horseradish peroxidase-conjugated streptavidin antibody (Shanghai Sangon Biotech) was added. After incubation for 1 hour, followed by washing, 100 μL of TMB was added for color development, and 50 μL of 1M sulfuric acid was added to stop the reaction. OD was then measured. 450 nm Value, for OD 450 nm Fit analysis was performed on the values ​​and concentrations, and the results are as follows: Figure 6 As shown, SA14-Fc can effectively inhibit IgE-Fc. 2-4 It binds to the receptor FcεRⅠα.

[0235] Example 6: Effect of human IgG1 Fc fusion protein SA14-Fc nanobody on inhibiting IgE binding to FcεRI receptor.

[0236] The Fc fusion protein of the nanobody SA14 was detected by flow cytometry to inhibit IgE-Fc 2-4 binding to FcεRlα receptor on the surface of KU812 cells (human basophilic leukemia cells): using IgE-Fc 2-4 protein to treat KU812 cells for 24 hours to promote the expression of FcεRl receptor on the surface of the cells and to make the FcεRl receptor on the surface of KU812 cells fully bind to IgE-Fc 2-4 protein. The SA14-Fc fusion protein solution was added in a gradient dilution of 1:5, incubated at 37°C for 1 hour, the IgE protein on the surface of the cells was labeled with PE anti-human IgE antibody, the IgE level on the surface of the cells was detected by flow cytometry, and the results are shown in Figure 7 Fig. 2. The Fc fusion protein of SA14 can effectively reduce the IgE level bound to the surface of KU812 cells.

[0237] Discussion

[0238] It is known according to the prior art that IgE combined with allergens binds to the IgE high-affinity receptor I (FcεRI) on the surface of mast cells or basophils, resulting in cross-linking of FcεRI. Cross-linking of FcεRI can trigger degranulation of mast cells and basophils and release of histamine and other substances, thereby mediating immediate hypersensitivity and causing a series of allergic symptoms in the body, such as vasodilation, bronchial constriction, etc. The antibodies targeting human IgE have been confirmed to be effective in treating allergies, and the antibodies targeting human IgE mainly bind to the Cε3 domain of IgE-Fc 2-4 .

[0239] The present application provides a camel-derived nanobody targeting human immunoglobulin E (IgE) with high affinity, named SA14, and its human IgG1 Fc fusion protein CA20-Fc binds to the Cε2 domain of IgE-Fc 2-4 with an EC 50 value of (2.941±0.582)×10-10M, and CA20-Fc specifically binds to the Cε2 domain of IgE and can effectively inhibit IgE-Fc 2-4 binding to the receptor FcεRIα.

[0240] Therefore, based on the experimental results of the present application, it can be reasonably speculated that the nanobody or fusion protein of the present application has a new mechanism of inhibiting IgE-Fc 2-4 binding to the receptor FcεRlα. As shown in Figure 8 ,

[0241] IgE-Fc 2-4the Cε3 domain is in an open conformation, and the Cε2 domain is bent towards the Cε3 domain, providing space for the binding of FcεRI α. When the SA14 nanobody binds to the Cε2 domain of IgE-Fc 2-4 , it creates steric hindrance between the Cε2 and Cε3 domains, causing the Cε2 domain to stretch upwards, thus creating steric hindrance between the Cε2 domain and FcεRI α, and possibly causing the Cε3 domain to change from an open to a closed conformation, thus inhibiting the binding of IgE-Fc 2-4 to the receptor FcεRI α.

[0242] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that these equivalents ype within the scope of the appended claims.

Claims

1. A heavy chain variable region VNAR that specifically binds human IgE, characterised in that, The VNAR comprises the following complementarity determining regions (CDRs): CDR1 as set forth in SEQ ID NO: 5; and CDR3 as set forth in SEQ ID NO:

6.

2. The VNAR of claim 1, wherein, The amino acid sequence of the VNAR is as set forth in SEQ ID NO: 4, or has >85%, >90%, >95%, >96%, >97%, >98%, or >99% sequence identity thereto.

3. A shark nanobody against human IgE, characterized in that, The nanobody comprises the heavy chain variable region of claim 1.

4. An antibody against human IgE, characterized in that, The antibody comprises one or more heavy chain variable regions of claim 1.

5. A shark nanobody fusion protein against human IgE, characterized in that, The fusion protein comprises: (a) the heavy chain variable region of claim 1 or the shark nanobody of claim 2; and (b) an Fc fragment.

6. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the heavy chain variable region of claim 1, the shark nanobody of claim 3, or the fusion protein of claim 5.

7. An expression vector, characterized by, The expression vector contains the nucleotide molecule of claim 6.

8. A host cell, characterized in that, The host cell contains the expression vector of claim 7, or has integrated into its genome the nucleotide molecule of claim 6.

9. Use of the heavy chain variable region according to claim 1, the shark nanobody according to claim 3, the fusion protein according to claim 5, characterized in that, for preparing a medicament, reagent, assay plate, or 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 heavy chain variable region of claim 1, the shark nanobody of claim 3, or the fusion protein of claim 5; and (ii) a pharmaceutically acceptable carrier.

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