Nano-antibody antiFc epsilon RIa-D11 targeting Fc epsilon RIalpha protein extracellular domain and application of nano-antibody antiFc epsilon RIa-D11

Abstract

The invention provides a nano antibody antiFc [epsilon] RIa-D11, the nano antibody is competitively combined with lgE to effectively block aggregation of lgE on the surface of Fc [epsilon] RIalpha protein, and the invention further provides application of the nano antibody antiFc [epsilon] RIa-D11 in intervention of allergic diseases, development of desensitization drugs and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceuticals, and particularly relates to a nanobody antiFcεRIa-D11 targeting the extracellular segment of FcεRIα protein and application thereof. BACKGROUND

[0002] As the sixth largest chronic disease in the world, 40% of people have some kind of allergic disease. The occurrence of rhinitis, asthma, and even anaphylactic shock caused by allergic reactions brings great trouble to many people's life, and has been listed by WTO as one of the three major diseases to be researched and prevented in the 21st century. Among them, the frequency of emergency medical treatment and hospitalization of moderate or severe patients is high, which is the main reason for the increase of asthma treatment cost and disability and death.

[0003] Blocking the binding of IgE to FcεRI has improved the effect on various allergic diseases. At present, due to the limitations of anti-antibody drug development, there is still a lack of anti-allergic program targeting FcεRI.

[0004] The FDA-approved anti-IgE monoclonal antibody Omalizumab is the first approved targeted therapy for moderate to severe asthma in the world, and is widely used for the treatment of asthma and chronic spontaneous urticaria. Omalizumab can reduce the number of FcεRI on the surface of effector cells by blocking the binding of IgE to FcεRI, thereby exerting the effect of inhibiting degranulation and subsequent allergic reactions. However, Omalizumab also has the defects of slow onset and incomplete prevention.

[0005] Therefore, for moderate or severe asthma and other serious allergic reactions, there is an urgent need in the art to provide new therapeutic drugs. SUMMARY

[0006] The present application aims to provide a nanobody antiFcεRIa-D11 targeting the extracellular segment of FcεRIα protein and application thereof.

[0007] In a first aspect of the present application, a nanobody antiFcεRIa-D11 binding to FcεRIα protein is provided, the nanobody antiFcεRIa-D11 comprising three antigen complementarity determining regions of CDR1, CDR2 and CDR3,

[0008] The CDR1 comprises an amino acid sequence shown as SEQ ID NO: 1;

[0009] The CDR2 comprises an amino acid sequence shown as SEQ ID NO: 2;

[0010] The CDR3 comprises an amino acid sequence shown as SEQ ID NO: 3.

[0011] In another preferred embodiment, the CDR sequences are annotated using the IMGT numbering scheme.

[0012] In another preferred embodiment, the CDR1 comprises an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

[0013] In another preferred embodiment, the CDR1 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 1.

[0014] In another preferred embodiment, the CDR2 comprises an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2.

[0015] In another preferred embodiment, the CDR2 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 2.

[0016] In another preferred embodiment, the CDR3 comprises an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3.

[0017] In another preferred embodiment, the CDR3 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 3.

[0018] In another preferred embodiment, the one or more preferably refers to preferably 1-3, more preferably 1-2, most preferably 1.

[0019] In another preferred embodiment, the mutation preferably is a substitution, insertion or deletion of an amino acid.

[0020] In another preferred embodiment, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 1 ;

[0021] the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 2;

[0022] The amino acid sequence of said CDR3 is shown in SEQ ID NO: 3.

[0023] In another preferred embodiment, said Nanobody antiFcsRIa-D11 is a single domain antibody comprising only a heavy chain variable region.

[0024] In another preferred embodiment, said Nanobody antiFcsRIa-D11 is a heavy chain antibody.

[0025] In another preferred embodiment, said Nanobody antiFcsRIa-D11 does not have a CH1 region between the heavy chain variable region and the hinge region.

[0026] In another preferred embodiment, said Nanobody antiFcsRIa-D11 comprises the amino acid sequence shown in SEQ ID NO: 4.

[0027] In another preferred embodiment, said Nanobody antiFcsRIa-D11 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 4.

[0028] In another preferred embodiment, said Nanobody antiFcsRIa-D11 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 4.

[0029] In another preferred embodiment, said one or more is preferably 1-3, more preferably 1-2, most preferably 1.

[0030] In another preferred embodiment, said mutation is preferably a substitution, insertion or deletion of an amino acid.

[0031] In another preferred embodiment, said Nanobody antiFcsRIa-D11 binds to the EC 50 with a value of 0.01-10 nM, preferably 0.1-5 nM, most preferably 0.2-3 nM.

[0032] In another preferred embodiment, said Nanobody antiFcsRIa-D11 is the amino acid sequence shown in SEQ ID NO: 4.

[0033] In another preferred embodiment, said Nanobody antiFcsRIa-D11 blocks the binding of FcsRI protein to IgE.

[0034] In another preferred embodiment, the blocking means that the nanobody anti-FcsRIa-D11 and IgE compete for binding to the FcsRI protein

[0035] In another preferred embodiment, the blocking means that the nanobody anti-FcsRIa-D11 and IgE bind to the same epitope of the FcsRI protein.

[0036] In another preferred embodiment, the nanobody anti-FcsRIa-D11 blocks the binding of FcsRI protein to IgE with an IC 50 value of 1-500 nM, preferably 100-400 nM, and most preferably 200-300 nM.

[0037] In another preferred embodiment, the nanobody anti-FcsRIa-D11 binds to the same epitope of FcsRIa as IgE.

[0038] In another preferred embodiment, the nanobody anti-FcsRIa-D11 further comprises a Fc domain, preferably an IgG Fc domain, more preferably a human IgGl Fc domain.

[0039] In another preferred embodiment, the human IgGl Fc domain has an amino acid sequence as set forth in SEQ ID NO: 5.

[0040] In another preferred embodiment, the nucleotide sequence encoding the human IgGl Fc domain is set forth in SEQ ID NO: 6.

[0041] In a second aspect of the present application, there is provided an antibody that binds to FcsRIa protein, wherein the antibody is a monomer, a bivalent antibody and / or a multivalent antibody, and wherein the antibody comprises one or more of the nanobodies anti-FcsRIa-D11 of the first aspect of the present application.

[0042] In another preferred embodiment, the antibody binds to FcsRIa protein with an EC 50 value of 0.01-10 nM, preferably 0.1-5 nM, and most preferably 0.2-3 nM.

[0043] In another preferred embodiment, the antibody blocks the binding of FcsRI protein to IgE.

[0044] In another preferred embodiment, the antibody blocks the binding of FcsRI protein to IgE with an IC 50 value of 1-500 nM, preferably 100-400 nM, and most preferably 200-300 nM.

[0045] In a third aspect of the present application, there is provided a polynucleotide encoding the Nanobody antiFcsRIa-D11 of the first aspect of the present application.

[0046] In another preferred embodiment, the polynucleotide sequence is set forth in SEQ ID NO: 7.

[0047] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA, RNA, cDNA or a combination thereof

[0048] In a fourth aspect of the present application, there is provided a vector comprising the polynucleotide of the third aspect of the present application.

[0049] In another preferred embodiment, the vector is an expression vector.

[0050] In another preferred embodiment, the vector is a mammalian expression vector.

[0051] In a fifth aspect of the present application, there is provided a host cell comprising the vector of the fourth aspect of the present application.

[0052] In another preferred embodiment, the host cell is a host cell for expression of a foreign protein.

[0053] In another preferred embodiment, the host cell includes, but is not limited to, a bacterium, a yeast, an insect cell or a mammalian cell.

[0054] In another preferred embodiment, the host cell is a HEK293F cell.

[0055] In a sixth aspect of the present application, there is provided an immunoconjugate comprising:

[0056] (a) the Nanobody antiFcsRIa-D11 of the first aspect of the present application; and / or

[0057] (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a cytokine nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0058] In another preferred embodiment, the detectable label includes a radionuclide, a gold nanoparticle / nanorod.

[0059] In another preferred embodiment, the radionuclide includes a diagnostic isotope and / or a therapeutic isotope.

[0060] In a seventh aspect of the present application, there is provided a recombinant protein having:

[0061] (i) the Nanobody antiFcsRIa-Dl 1 according to the first aspect of the application; and / or

[0062] (ii) an optional tag sequence to facilitate expression and / or purification.

[0063] In an eighth aspect of the application, a pharmaceutical composition comprising the Nanobody antiFcsRIa-Dl 1 according to the first aspect of the application, the polynucleotide according to the third aspect of the application, the vector according to the fourth aspect of the application, or the host cell according to the fifth aspect of the application is provided.

[0064] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0065] In a ninth aspect of the application, a kit comprising the Nanobody antiFcsRIa-Dl 1 according to the first aspect of the application, the polynucleotide according to the third aspect of the application, the vector according to the fourth aspect of the application, the host cell according to the fifth aspect of the application, the immunoconjugate according to the sixth aspect of the application, or the pharmaceutical composition according to the eighth aspect of the application is provided.

[0066] In a tenth aspect of the application, use of the Nanobody antiFcsRIa-Dl 1 according to the first aspect of the application, the polynucleotide according to the third aspect of the application, the vector according to the fourth aspect of the application, the host cell according to the fifth aspect of the application, or the immunoconjugate according to the sixth aspect of the application for:

[0067] (Z1) the preparation of a kit for the detection of IgE; and / or

[0068] (Z2) the preparation of a medicament for the treatment of a disease associated with FcsRIa-lgE binding.

[0069] In another preferred embodiment, the IgE is from a mammal.

[0070] In another preferred embodiment, the kit is an enzyme-linked immunoassay kit for the detection of IgE.

[0071] In another preferred embodiment, the disease associated with FcsRIa-lgE binding is an allergic disease.

[0072] In another preferred embodiment, the allergic disease includes, but is not limited to, rhinitis, asthma, or anaphylactic shock.

[0073] In another preferred embodiment, the medicament for the treatment of a disease associated with FcsRIa-lgE binding is a broad spectrum desensitization medicament.

[0074] In another preferred embodiment, the treatment comprises: treating and / or ameliorating.

[0075] In another preferred embodiment, the treatment / amelioration refers to: alleviating symptoms, alleviating the disease course, restoring health, improving quality of life, and / or preventing complications.

[0076] In another preferred embodiment, the administration of the pharmaceutical composition comprises, but is not limited to: subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, nasal administration or aerosol administration.

[0077] In an eleventh aspect of the present application, a method for producing a Nanobody binding to FcεRIα protein is provided, comprising the steps of:

[0078] (S1) culturing the host cell of the fifth aspect of the present application under conditions suitable for production of Nanobodies, thereby obtaining a culture comprising Nanobodies binding to FcεRIα protein;

[0079] (S2) isolating and / or recovering the Nanobodies binding to FcεRIα protein from the culture; and

[0080] (S3) optionally purifying and / or modifying the Nanobodies binding to FcεRIα protein obtained in step (S2).

[0081] In another preferred embodiment, the Nanobody binding to FcεRIα protein blocks the binding of FcεRIα protein and IgE.

[0082] 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 will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 The amino acid sequence of the Nanobody antiFcεRIa-D11 of the present application is shown, and the sequences marked with different underlines are CDR sequences marked with different rules.

[0084] Figure 2 ELISA results of the screening of monoclonal phages specifically binding to human FcεRIα protein are shown.

[0085] Figure 3 The purification and SDS-PAGE analysis results of the Nanobody of the present application are shown. Among them, Figure 3 A shows the elution chart of the Nanobody of the present application purified by Protein A column, Figure 3 B shows the SDS-PAGE result chart of the Nanobody of the present application.

[0086] Figure 4 The binding of the nanobodies of the present application to FcεRIa was analyzed by ELISA.

[0087] Figure 5 The ability of the nanobodies of the present application to compete with IgE for binding to FcεRIa protein, i.e. the blocking ability of the nanobodies of the present application, was analyzed by ELISA.

[0088] Figure 6 Other antibodies with affinity to FcεRIa screened are shown.

[0089] Figure 7 The ability of the other antibodies with affinity to FcεRIa screened to block the binding of FcεRIa and IgE was analyzed.

[0090] Figure 8 The results of the structural prediction analysis of the complex formed by the binding of the nanobodies of the present application to FcεRIa were analyzed by the AlphaFold tool software, wherein, Figure 8 A and Figure 8 B are the structural prediction figures from two perspectives. DETAILED DESCRIPTION

[0091] The present inventors, through extensive and in-depth research, for the first time accidentally discovered a nanobody anti-FcεRIa-D11 that can compete with IgE protein for binding to FcεRIa protein, which can effectively block the binding of FcεRIa and IgE and can be used for the preparation of allergy-related drugs. The nanobody of the present application has high affinity for the extracellular segment of human FcεRIa protein, has low immunogenicity, and is also easy to realize engineering production or modification, and has great application value in the development of allergy drugs.

[0092] FcεRIa protein

[0093] FcεRI (Fc epsilon Receptor I) is a transmembrane protein composed of αβγ2 four subunits, wherein the extracellular segment of the α subunit bears the function of binding to IgE and is composed of two 85-amino-acid immunoglobulin-like domains.

[0094] In type I hypersensitivity, multiple specific IgE aggregates bound to allergens and cross-linked on the surface of mast cells and basophils bind to the extracellular segment of the α subunit of the high-affinity receptor FcεRI protein, thereby causing aggregation of FcεRI protein, activating downstream messenger protein kinase phosphorylation, activating the reaction pathway, and ultimately leading to the degranulation reaction of effector cells, releasing a large amount of enzymes, cytokines and vasoactive mediators, causing various clinical manifestations of allergic reactions, including respiratory tract obstruction, skin rash, etc.

[0095] Crystal structure of the complex of FcεRIα ectodomain and IgE-Fc shows that FcεRIα ectodomain binds to Cε3-Cε4 domains of IgE-Fc in a 1:1 ratio and locks IgE-Fc in an open conformation, avoiding its binding to CD23.

[0096] Therefore, in addition to traditional histamine receptor antagonists and glucocorticoid drugs, antibodies that can block the interaction of IgE with the receptor FcεRI can also be used as drugs to treat allergic diseases.

[0097] In theory, antibodies that specifically bind FcεRI can also block the cross-linking of IgE with FcεRI and have an inhibitory effect, and previous studies at home and abroad have proven that this is indeed the case. Studies have also shown that anti-FcεRI antibodies can replace specific allergens as a broad-spectrum desensitizing agent.

[0098] Therefore, the specific FcεRIa nanobody provided by the present application can be used as a therapeutic drug for treating and diagnosing diseases related to FcεRIa, especially allergic diseases, and also has the potential to be used as a desensitizing drug.

[0099] Nanobody

[0100] Nanobodies (Nbs), also known as single-domain antibodies, are small antibodies produced by camelids (camels, llamas, alpacas). Nanobodies have the smallest variable antigen-binding fragment, known as the VHH domain, with a molecular weight of only 13-15 kDa. Compared with conventional antibodies, nanobodies have a simple structure, a small molecular weight, and are more conducive to expression and use; at the same time, the small size allows it to recognize epitopes that cannot be approached by traditional antibodies. Nanobodies can be delivered to the lungs by nebulization, or can be administered through the nasal cavity.

[0101] Nanobody of the present application

[0102] The nanobody of the present application refers to the nanobody antiFcεRIa-D11 provided by the present application, which can compete with lgE protein to bind to FcεRIa protein, and the nanobody can effectively block the binding of FcεRIa and lgE. Specifically, the nanobody antiFcεRIa-D11 comprises three antigen complementarity determining regions CDR1, CDR2 and CDR3,

[0103] the amino acid sequence of CDR1 is GFTSGRT (SEQ ID NO: 1);

[0104] the amino acid sequence of CDR2 is IYRERNGSA (SEQ ID NO: 2);

[0105] The amino acid sequence of the CDR3 is ATVSCSEVFNPYY (SEQ ID NO: 3).

[0106] In another preferred embodiment, the CDR sequences are annotated using IMGT numbering.

[0107] In another preferred embodiment, the amino acid sequence of the Nanobody anti-FcsRIa-D11 is as follows:

[0108] QLQLVESGGGSVQAGGSLKLSCAASGFTSGRTCMGWFRQAPGKEREGV AMIYRERNGSANYADSVKGRFTISRDNPKNTVYLQMNSLKPEDTAMYYCAT VSCSEVFNPYYFGLGTQVTVSS (SEQ ID NO: 4).

[0109] In another preferred embodiment, the CDR sequences of the Nanobody anti-FcsRIa-D11 are annotated using IMGT numbering.

[0110] In another preferred embodiment, the CDR sequences of the Nanobody anti-FcsRIa-D11 are annotated using Kabat numbering.

[0111] In another preferred embodiment, the CDR sequences of the Nanobody anti-FcsRIa-D11 are annotated using Contact numbering.

[0112] In another preferred embodiment, the CDR sequences annotated using IMGT numbering, Kabat numbering, Contact numbering are as shown in Figure 1

[0113] In another preferred embodiment, the Nanobody anti-FcsRIa-D11 further comprises a Fc domain.

[0114] In another preferred embodiment, the Fc domain is a human lgG1 Fc domain.

[0115] In another preferred embodiment, the amino acid sequence of the human lgG1 Fc domain is as shown below:

[0116] ​PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5).

[0117] In another preferred embodiment, the nucleotide sequence encoding the human lgG1 Fc domain is as follows:

[0118] CCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 6)

[0119] In another preferred embodiment, the nucleotide sequence encoding said Nanobody anti-FcsRIa-D11 is as follows:

[0120] CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAAACTCTCCTGTGCAGCCTCTGGATTCACCTCCGGTCGCACCTGTATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCAATGATCTATAGGGAGCGGAATGGCAGCGCAAACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACCCCAAGAATACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCAACAGTGAGCTGCTCCGAGGTGTTTAATCCTTACTACTTTGGCCTGGGGACCCAGGTCACCGTCTCCTCA (SEQ ID NO: 7)

[0121] The main advantages of the present application include:

[0122] (1) The nanobody of the present application is derived from natural camel heavy chain antibody, has high affinity to the extracellular segment of human FcεRla protein, can effectively block the combination of FcεRla and IgE, and can be used for the development of broad-spectrum desensitization drugs.

[0123] (2) The nanobody of the present application has simple structure and small molecular weight, is beneficial to expression and use, has small human immunogenicity, and is not easy to produce immune rejection.

[0124] (3) The nanobody of the present application is convenient to express in E. coli and various eukaryotic systems in large amount, thus is easy to produce and prepare, and has high yield.

[0125] (4) Since the nanobody of the present application has only one binding site and belongs to single domain antibody, it has better permeability, specificity and detection linearity as a detection and diagnosis reagent;

[0126] (5) The nanobody of the present application is convenient to couple with various fusion proteins or is easier to be labeled by various markers, thus is easier to be engineered; in addition, it is easier to prepare bifunctional antibody, thus is more beneficial to the development of targeted drugs and the directional transportation of cell targets.

[0127] The application will be further described in connection with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0128] Example 1: Preparation of anti-FcεRIa-D11

[0129] 1) For immunization of single-humped camel (Camelus dromedarius), the first immunization used Freund's complete adjuvant (Sigma, F5881), and the following two times used Freund's incomplete adjuvant (Sigma, F5506). The protein dosage was 500 μg each time, and the FcεRIa protein extracellular end protein (obtained by expression using 293F mammalian cell system) was mixed with the adjuvant at a volume ratio of 1:1. The first two injections were subcutaneous injections, and the last two injections were intramuscular injections. The camel was immunized for a total of 4 times, with a frequency of once every 3 weeks.

[0130] 2) At the 14th week, the peripheral blood lymphocytes were isolated by intravenous blood collection and Ficoll separation.

[0131] 3) The total RNA was extracted using the RNA extraction kit of Omega Biotek, and the genomic DNA was removed.

[0132] 4) The RNA was reverse transcribed into cDNA using the PrimeScript TM II First-strand cDNA synthesis kit of Takara.

[0133] 5) Construction of the nanobody phage display library: Using the above cDNA as a template, the coding sequence of the nanobody was obtained by PCR amplification using specific camel VHH primers (forward primer sequence as shown in SEQ ID NO:8: GCTGCACAGCCTGCTATGGCACAGKTGCAGCTCGTGGAGTCTGGGGG; reverse primer sequence as shown in SEQ ID NO:9: GAGTTTTTGTTCGGCTGCTGCTGAGGAGACGGTGACCTGGGTCCCC). The amplified nanobody sequence was then inserted into the NcoI and NotI sites of the phage particle pR2 (MRC Laboratory of Molecular Biology) using Gibson assembly (Yisheng Biotechnology Co., Ltd., 10911ES20). The resulting Gibson assembly product was the initial nanobody phage library, and the product was recovered.

[0134] 6) *E. coli* T1 (MRC Laboratory of Molecular Biology) competent cells were prepared using the 10% glycerol washing method. Activated TG1 cells were cultured in 250 mL of 2×YT medium (formulation 1L: 16 g tryptone (OXOID), 10 g yeast extract (OXOID), 5 g NaCl (Sinopharm Chemical Reagent Co., Ltd.; all inorganic salts used in subsequent examples were from the same source)) until OD500. 600 The sample was approximately 0.6-0.8. After centrifugation at 5000g for 15 minutes, it was washed three times with 250mL, 250mL, and 100mL of pre-cooled 10% glycerol (Sinopharm Chemical Reagent Co., Ltd.). Finally, it was resuspended with 1mL of 10% glycerol and dispensed into 500μL tubes.

[0135] 7) Transform *E. coli* TG1 competent cells using a BTX ECM 399 electroporator. The product recovered from Gibson assembly was mixed with 500 μL of TG1 competent cells and transferred to a 0.1 cm electroporation cuvette, followed by electroporation at 2.5 kV. The electroporated product was resuspended in 20 mL of 2× medium and incubated at 37°C and 220 rpm for 1 h. 10 μL of the bacterial culture was diluted to 990 μL of 2×YT, and then another 40 μL of the bacterial culture was diluted to 160 μL of 2×YT. A 100 μL plate was then plated and incubated overnight at 37°C. The next day, the library size was calculated by counting the cells (library size = count × 10⁻⁶). 5). The bacteria were spread on 5 150mm 2xYT plates supplemented with 100pg / ml ampicillin (Zhimeng Bio, 1146GR005) and 2% glucose (National Pharmaceutical Group Chemical Reagents Co., Ltd.) to amplify the phage library, incubated at 37°C overnight. Next, the transformed colonies were scraped from the plates, vortexed thoroughly with a final concentration of 25% glycerol, snap-frozen in 1 mL aliquots in liquid nitrogen and stored at -80°C, and the size of the phage library was calculated.

[0136] 8) Amplification of the Nanobody phage display library: To amplify the phage library of nanobodies, 0.2ml of the frozen library was thawed on ice, diluted into 200ml of 2xYT medium supplemented with 100pg / ml ampicillin and 2% glucose and incubated at 37°C, 220rpm. Next, 1x1011 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) was added to the culture and incubated at 37°C (water bath) for 45 minutes. The cell pellet was separated by high speed centrifugation and resuspended in 200ml of 2xYT medium supplemented with 0.1% glucose, 50pg / ml kanamycin (Zhimeng Bio, 1162GR025) and 100pg / ml ampicillin. The cells were incubated at 25°C, 220rpm for 20 hours to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate the phage particles. The precipitated phage particles were resolubilized in 1xPBS (formula: 10mmol / L Na2HPO4; 1.75mmol / L KH2PO4; 137mmol / L NaCl; 2.65mmol / L KCl; pH 7.2-7.6) and stored in 1ml aliquots at -80°C in the presence of 25% glycerol.

[0137] 9) Screening: The FcεRIa antigen was diluted in PBS to a final concentration of 0.1mg / ml and coated into one well of a 96-well immunoplate ELISA (Nunc maxsorp plate) and one well of the ELISA plate was left out as a negative control. After washing 3 times with 1xPBS, 300mL of MPBS (1xPBS containing 5% skimmed milk) was added to each well of the ELISA plate and incubated at room temperature for 2 hours to block unbound sites. Next, the plate was washed 3 times with 1xPBS and 1x1011 11pfu (diluted in 100 μΙ_ MPBS) phage library was added to each well and after 1 hour incubation at room temperature, the plate was washed 3 times with PBST (1 x PBS containing 0.1 % Tween 20). Phage displaying nanobodies specific for FcεRIa were eluted by incubation with trypsin at a final concentration of 0.5 mg / ml for 1 hour at room temperature. 10 μΙ_ of eluted phage was added to 1 mL of E. coli TG1 competent cells and incubated at 37°C (water bath) for 45 min to allow infection, then the bacterial culture was plated on 2 x YT supplemented with 100 μg / mL ampicillin and 2% glucose at 37°C overnight.

[0138] 10) Preparation of monoclonal phage: After one round of panning, 48 individual colonies were picked into a 96-well round bottom culture dish containing 100 μΙ_ of 2 x YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). This was incubated at 37°C, 180 rpm for 12 hours. Next, 5 μΙ_ of this culture was inoculated into a new 96-well round bottom culture dish containing 200 μΙ_ of 2 x YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). The freshly inoculated plate was incubated at 37°C, 250 rpm for 1.5 hours until the OD 260 was about 0.5. 50 μΙ_ of 2 x YT medium containing 4 x 10 8 pfu of KM13 helper phage was added to each well of the plate and incubated at 37°C for 45 min without shaking to allow infection. After infection, 150 μΙ_ of supernatant was discarded and the remaining part was centrifuged at 3500 g for 15 min, the supernatant was discarded and the bacterial pellet was resuspended in 200 mL of 2 x YT medium supplemented with 100 μg / mL ampicillin, 50 μg / mL kanamycin and 0.1 % glucose (w / v) and incubated at 25°C, 250 rpm overnight, for about 14-16 hours. The next day, the culture was centrifuged at 3500 g for 30 min and then 150 μΙ_ of supernatant was transferred to a new 96-well plate and stored at 4°C for screening of nanobodies.

[0139] 11) Phage ELISA test: 96-well round bottom immuno ELISA plates were coated with 100 μΙ_ of FcεRIa at a final concentration of 0.2 μg / mL using 1 x PBS for dilution and incubated at 4°C for 16 hours. Next, the ELISA plates were washed 3 times with 1 x PBS. The plates were blocked with MPBS (1 x PBS containing 5% skimmed milk) for 2 hours at room temperature. After completion, the wells were washed 4 times with PBS-0.1 % Tween 20. 100 μΙ_ of 1 x 10 11PFU phage was added to each well and incubated at room temperature for 1 hour. The ELISA plate was then washed 5 times with PBS-0.1% Tween 20. Next, 100 μL of HRP-KM13 (Beijing Yiqiao Shenzhou) diluted 1:8000 with MPBS was added to each well, and the plate was incubated at room temperature for 1 hour. The wells were then washed 4 times with PBS-0.1% Tween 20, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB, Beyotime) was added to each well. The plate was incubated in the dark for 7 minutes for color development, and then 50 μL of 1M H2SO4 was immediately added to stop the reaction. The OD was measured using a microplate reader. 450nm Value, result as Figure 2 As shown.

[0140] 12) OD 450nm Positive clones with a value greater than 1 were selected and cultured to screen for FcεRIa protein-specific positive phage nanobodies that have affinity for FcεRIa protein and the ability to block the binding of FcεRIa protein and IgE. The sequences were sequenced and analyzed to determine the sequence results, and the nanobodies were named antiFcεRIa-D11 (the amino acid sequence shown in SEQ ID NO:4). This nanobodies have a specific CDR region that binds to FcεRIa protein.

[0141] Example 2: Expression and purification of nanobody-Fc fusion protein

[0142] The secretion-guided peptide gene sequence was designed and fused to the N-terminus of the nanobody gene to ensure secretion after expression. Human IgG1 Fc was fused to the C-terminus of the nanobody gene, and the nanobody gene and human IgG1 Fc were recombined. Then, it was cloned into the mammalian expression vector pTT5.

[0143] The construct vector was transfected into HEK293F cells (density approximately 2.5 x 10⁻⁶) using polyethyleneimine (PEI, Yisheng Biotechnology Co., Ltd., 40816ES03). 6 Cells / ml, ATCC, CBP60437), in Freestyle TM Transfected mammalian cells were cultured in 293 expression medium (Yonglian Biotechnology) at 5% CO2, 150 rpm, and 37°C for 4 days. The cells were then centrifuged at 3000 rpm for 10 minutes to culture for 5 days. The supernatant of the mammalian cell culture was then collected by centrifugation at 2000 rpm for 10 minutes. The nanobody-Fc fusion protein was purified using a Protein A column, eluted with 0.1 M acetic acid, and analyzed by SDS-PAGE electrophoresis.

[0144] Electrophoresis results as follows Figure 3 As shown.

[0145] Figure 3 a indicates that Protein A column successfully enriched a large amount of target protein from cell culture supernatant and the target protein was successfully eluted from the column; Figure 3 B indicates that the obtained protein has high purity and few impurities, and therefore the results of this example demonstrate that a nanobody-Fc fusion protein with high purity is obtained from the supernatant.

[0146] Example 3: Analysis of the binding affinity of nanobody to FcεRIa protein

[0147] Immuno MaxiSorb plates (Nunc) were coated with 2 μg / mL of FcεRIa protein at room temperature for 2 h. After washing the ELISA plate with 1xPBS three times, 240 μL of MPBS was added per well at room temperature for 2 h to block unbound sites. Next, the nanobody-Fc was gradient diluted (starting at 50 nM, three consecutive gradient dilutions, 12 dilution gradients) with 5% milk diluted with PBST. 100 μL was added per well and incubated at room temperature for 1 h. After 1 h, the ELISA plate was washed with 1xPBST three times, and HRP-anti-human IgG Fc (Beijing Yiqiao God) was diluted with 5% milk diluted with PBST at a ratio of 1:10000 and incubated at room temperature for 1 h. After washing the ELISA plate with 1xPBST three times, 100 μL per well of TMB (Biyun Tian) was added, and the reaction was carried out at room temperature in the dark for 7 min. Finally, 50 μL of 1M H2SO4 was added to stop the reaction and the absorbance at 450 nm was detected.

[0148] The results of the analysis of the ELISA are shown in Figure 4 .

[0149] As can be seen from the figure, the EC 50 of FcεRIa protein and D11-IgG1 Fc is about 0.7289 nM. The results of this example show that the prepared nanobody has high binding affinity to FcεRIa protein.

[0150] Example 4: Analysis of the blocking ability of nanobody to FcεRIa protein ligand IgE protein

[0151] Immuno MaxiSorb plates (Nunc) were coated with 2 pg / mL of FcεRIa protein at room temperature for 2 h. After washing the ELISA plates with 1xPBS for three times, 240 pL of MPBS was added to each well at room temperature for 2 h to block the unbound sites. Next, the ligand IgE protein of FcεRIa protein was gradient diluted with 5% milk diluted in PBST (starting at 2000 nM, four-fold serial dilution, 12 dilutions). 100 pL was added to each well, and 20 nM of Nanobody-Fc fusion protein was added to each well, and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed with 1xPBST for three times, and HRP-anti-human IgG Fc (Beijing Yiqiao God) was diluted with 5% milk diluted in PBST at a ratio of 1:10000 and incubated at room temperature for 1 h. After washing the ELISA plates with 1xPBST for three times, 100 pL of TMB (Bi Yun Tian) was added to each well, and reacted at room temperature for 7 min in the dark. Finally, 50 pL of 1 M H2SO4 was added to stop the reaction, and the absorbance at 450 nm was detected.

[0152] The results of the analysis of the ELISA are shown in Figure 5

[0153] The results of this example show that the Nanobody fusion protein D11-IgG1 Fc prepared competes with the FcεRIa protein ligand IgE protein for binding, and the IC50 of the Nanobody of the application is 246.3 nM. 50 Thus, the Nanobody of the application can effectively block the binding of FcεRIa and IgE.

[0154] In addition, Figure 6 and Figure 7 The results of Example 4 and Example 5 show that among the several sequences screened in the same batch for affinity to the FcεRIa protein, only D11 has the blocking ability to the FcεRIa protein ligand IgE protein.

[0155] Example 5: Structural prediction of the binding site between the Nanobody and the FcεRIa protein

[0156] AlphaFold developed by DeepMind was used as an artificial intelligence tool for predicting structures. The amino acid sequences of the FcεRIa protein and the anti-FcεRIa-D11 Nanobody were provided, and the structure prediction was performed after selecting the database. The prediction model file provided by AlphaFold (usually stored in.pdb format) can be imported into visualization software (PyMOL) for intuitive observation and analysis of protein structures.

[0157] The results of the prediction are shown in Figure 8 Figure 8 ​​Figures 8A and 8B are structural diagrams of two different perspectives.

[0158] The results show that the binding epitope region of antiFcεRIa-D11 in the structural diagram is approximately at the 135-142, 155-160, and 175-185 amino acid residues of the FcεRIa antigen, and the binding site coincides with the binding region of IgE on FcεRIa.

[0159] In contrast, although the structures of A9, 2C, etc. can bind to the FcεRIa protein, the binding site does not have a competitive relationship with IgE, and thus does not have blocking ability.

[0160] The prediction results are consistent with the experimental results, confirming that antiFcεRIa-D11 can block the binding of IgE to FcεRIa.

[0161] All of the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and by equivalent means without departing from the scope of the appended claims.

Claims

1. A Nanobody binding to the FcεRIα protein, antiFcsRIa-Dl 1, characterized in that, The nanobody antiFcsRIa-D11 comprises three antigen complementarity determining regions, CDR1, CDR2 and CDR3, The CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1; The CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2; The CDR3 comprises an amino acid sequence as set forth in SEQ ID NO:

3.

2. The Nanobody of claim 1, antiFcsRIa-Dl 1, wherein The nanobody antiFcsRIa-D11 blocks the binding of FcsRI protein to IgE.

3. A polynucleotide, comprising, The polynucleotide encodes the nanobody antiFcsRIa-D11 of claim 1.

4. A vector, characterized in that, The expression vector comprises the polynucleotide of claim 3.

5. A host cell, characterized in that, The host cell comprises the vector of claim 4.

6. An immunoconjugate, characterized in that, The immunoconjugate comprises: (a) the nanobody antiFcsRIa-D11 of claim 1; and / or (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a cytokine nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

7. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the nanobody antiFcsRIa-D11 of claim 1 or 2, the polynucleotide of claim 3, the vector of claim 4, or the host cell of claim 5.

8. A kit characterized in that, The kit comprises the nanobody antiFcsRIa-D11 of claim 1 or 2, the polynucleotide of claim 3, the vector of claim 4, the host cell of claim 5, or the immunoconjugate of claim 6.

9. Use of the Nanobody anti-FcsRIa-D11 according to claim 1 or 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5 or the immunoconjugate according to claim 6, characterized in that, for the following uses: (Z1) the preparation of a kit for the detection of IgE; and / or (Z2) the preparation of a medicament for the treatment of a disease associated with FcsRIa-IgE binding.

10. A method of producing a Nanobody that binds to a FcεRI α protein, characterized in that, comprising the steps of: (S1) culturing the host cell of claim 5 under suitable conditions for the production of a nanobody, thereby obtaining a culture comprising nanobodies binding FcsRIa protein; (S2) isolating and / or recovering the nanobodies binding FcsRIa protein from the culture; and (S3) optionally purifying and / or modifying the nanobodies binding FcsRIa protein obtained in step (S2).

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