CN117551195B

CN117551195B - VHH nanobody targeting TSLP and application thereof

Info

Publication number: CN117551195B
Application number: CN202410043982.6A
Authority: CN
Inventors: 陈晖�; 路萍萍; 王晋; 闫条
Original assignee: Hangzhou Changxi Pharmaceutical Co ltd
Current assignee: Hangzhou Changxi Pharmaceutical Co ltd
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2024-04-09
Anticipated expiration: 2044-01-12
Also published as: CN117551195A

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a VHH nanobody targeting TSLP and application thereof, wherein the nanobody specifically comprises a complementarity determining region CDR1-3 of a VHH chain shown in SEQ ID NO. 4. Further, the VHH chain in the nanobody contains a CDR1 having the sequence shown in SEQ ID NO.1, a CDR2 having the sequence shown in SEQ ID NO.2, and a CDR3 having the sequence shown in SEQ ID NO. 3. The TSLP nanobody disclosed by the invention can be combined with TSLP antigens of human and monkey with high specificity. And is capable of blocking TSLP and its receptor binding, thereby blocking downstream signaling pathways, blocking TSLP stimulating PBMCs to secrete cofactors. It is hopeful to develop a novel inhalation preparation targeting TSLP.

Description

VHH nanobody targeting TSLP and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a VHH nanobody targeting TSLP and application thereof.

Background

The incidence of bronchial asthma and allergies (allergies) has increased in recent 30 years. Allergic progress (atopic march) is a natural phenomenon of allergy, manifested by atopic dermatitis (atopic dermatitis, AD) in young age, and often food allergy, allergic rhinitis (allergic rhinitis, AR) and even asthma in adults. Asthma is a heterogeneous disease that exhibits great variability in the severity of the disease, the type of airway inflammation, and the therapeutic agents, and is caused by a variety of pathophysiological mechanisms or endotypes. In severe asthma, these features not only reduce responsiveness to standard drugs, but also require more specific replacement therapies for reasons of existing disease severity because of the need for personalized treatment of individual pathogenesis of different asthmatics, and targeted biological agents bring new promise for asthmatics.

TSLP (thymic interstitial lymphopoietin, thymic stromal lymphopoietin) is a pleiotropic cytokine belonging to the short-chain tetra-alpha helix bundle type i IL-2 family. More and more research results indicate that TSLP may be a key factor in initiating asthma and allergy and mediate the development of allergic processes. Furthermore, TSLP gene polymorphism is closely related to allergies such as AD (eczema), food allergies, AR, and asthma. Numerous studies have demonstrated that about 2/3 of severe asthma manifests as overexpression of Th2 cytokines, TSLP being an important factor in causing Th2 cytokine overexpression. TSLP-TSLPR (Thymic stromal lymphopoietin receptor) function is accomplished primarily through the JAK-STAT signaling pathway. It is thought that upregulation of TSLP, which binds to TSLPR on DC cells, causes JAK activation, recruits the transcription factor STAT5, causes downstream signal transduction, ultimately leading to activation of DC cells. DC cell activation exhibits up-regulation of expression of costimulatory molecules and secretion of chemokines, thereby providing an advantageous microenvironment for Th0 to Th2 cell differentiation, leading to Th2 cell-based inflammatory responses, and concomitant factor (IL-4, IL-13, IL-5) release.

Asthma and chronic obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD) are among the most common respiratory diseases, with 3 and 2.1 million patients worldwide, respectively. Pulmonary inhalation administration is the preferred mode of administration for the prevention and treatment of respiratory diseases such as asthma, COPD and the like. Inhalation therapy is an ancient method of treating respiratory diseases and is by far considered the most effective and safest therapy. Nanobodies are variable regions of heavy chain antibodies found in camelids with naturally deleted light chains and have a molecular weight of 12-15kDa. The nano antibody has the advantages of large difference in structure, small molecular weight, strong tissue penetrability, high affinity, strong stability and the like compared with the traditional antibody. These advantages allow nanobodies to be developed as inhaled antibody drugs.

Disclosure of Invention

The invention provides a nano antibody with strong binding force to TSLP, which can accurately detect TSLP and diagnose TSLP related diseases, and can be used for dry powder inhalation administration so as to meet the requirements of clinical treatment of TSLP related diseases. Specifically, the invention discloses the following technical scheme:

in a first aspect, the invention discloses a nanobody against TSLP (thymic interstitial lymphopoietin, thymic stromal lymphopoietin) comprising complementarity determining regions CDR1-3 of the VHH chain shown in SEQ ID NO. 4.

Further, the VHH chain in the nanobody contains a CDR1 having the sequence shown in SEQ ID NO.1, a CDR2 having the sequence shown in SEQ ID NO.2, and a CDR3 having the sequence shown in SEQ ID NO. 3.

In a specific embodiment of the invention, the nanobody further comprises a framework region, the structure of the VHH chain of which is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Further, the amino acid sequence of the nano antibody is shown as SEQ ID NO. 4.

Further, the nanobody includes a derivative sequence of at least 1, 2, 3 or more amino acids, optionally added, deleted, modified and/or substituted for the sequence shown in SEQ ID NO.4 and capable of retaining the ability to specifically bind TSLP, particularly including conservative amino acid substitutions. Conservative substitutions are substitutions that occur in a family of amino acids associated with a side chain.

Further, the nanobody includes an antibody having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID No. 4.

Further, the nanobody is effective to block the interaction of TSLP and TSLPR, thereby blocking PBMCs from being activated and releasing CCL17, CCL22.

The term TSLPR as used herein, the thymic interstitial lymphopoietin receptor (Thymic stromal lymphopoietin receptor), may also be referred to as TSLP receptor.

Further, the TSLP includes TSLPs of humans, mice, rats, or non-human primates (e.g., monkeys).

Further, the nanobody includes a humanized antibody.

Further, the nanobody includes a monomer, a bivalent antibody, and/or a multivalent antibody.

The bivalent or multivalent antibodies may be linked by any linking peptide, and in a specific embodiment of the present invention, the bivalent antibody is constructed by using (GGGGS) 3 as a linking peptide.

In another aspect, the invention also discloses a nucleic acid molecule encoding the nanobody described above.

The terms "polynucleotide," "oligonucleotide," and "nucleic acid molecule" are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule may be single-stranded or double-stranded.

In another aspect, the invention also discloses a recombinant vector comprising the nucleic acid molecule, wherein the recombinant vector comprises the nucleic acid molecule, or the recombinant vector expresses the nanobody.

Further, the recombinant vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, other gene transfer systems, or combinations thereof.

Further, the viral expression vector includes a lentiviral vector, an adenoviral vector, an adeno-associated viral expression vector, or other types of viral vectors.

In some embodiments, the recombinant vector may comprise a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the recombinant vector may further comprise an origin of replication. Recombinant vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein shells, but not just these.

In another aspect, the invention also discloses a host cell in which the nanobody described above is expressed, or which contains the nucleic acid molecule described above or the recombinant vector described above.

In some embodiments, the host cell may be a prokaryote, such as e.coli (e.coli); or it may be a eukaryotic organism, such as a single cell eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or a hybridoma. Exemplary host cells include CHO cells, 293 cells, CHO-K1 cells, HEK293 cells, caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, jurkat cells, hepG2 cells, heLa cells, HT-1080 cells, HCT-116 cells, hu-h7 cells, huvec cells, molt 4 cells, and the like.

It is to be understood that the term host cell refers not only to a particular individual cell, but also to the progeny or potential progeny of such a cell. Since certain changes may occur in subsequent generations due to mutation or environmental influences, the progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term host cell as used herein.

In another aspect, the invention also discloses a pharmaceutical composition comprising one or more of the aforementioned nanobody, nucleic acid molecule, recombinant vector, host cell, conjugate.

Preferably, the pharmaceutical composition contains the aforementioned nanobody.

Further, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

Further, the pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release formulations, aerosols, films, injections, intravenous drip agents, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, pellets, nasal formulations, pulmonary formulations, eye drops and the like, oral or parenteral formulations.

Further, the pharmaceutical compositions are aerosol inhalants (also known as nebulizers), metered dose inhalants (pMDI, also known as aerosols) and dry powder inhalants (DPI, also known as inhalation powders).

Further, the pharmaceutical composition is a powder spray.

Further, the pharmaceutical composition may be administered in any of the following ways: inhaled, oral, rectal, nasal, buccal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracardiac, intrasternal or intravenous administration.

Further, the pharmaceutical composition may be administered by inhalation.

Further, the pharmaceutical composition may be administered alone or in combination with other drugs.

Further, the other drugs include protein drugs and non-protein drugs.

In some embodiments, the pharmaceutical composition may contain a formulating material for modifying, maintaining or retaining the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or permeation of the composition. In particular, suitable formulating materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); an antimicrobial agent; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediamine tetraacetic acid (EDTA)); complexing agents (e.g. caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); a filler; a monosaccharide; disaccharides; and other carbohydrates (e.g., glucose, sucrose, mannose, or dextrins); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavors, and diluents; an emulsifying agent; hydrophilic polymers (e.g., polyvinylpyrrolidone); a low molecular weight polypeptide; salt forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride (benzalkonium chloride), benzoic acid, salicylic acid, thimerosal (thimerosal), phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); a suspending agent; surfactants or wetting agents (e.g., pluronic), PEG, sorbitan esters, polysorbates (e.g., polysorbate 20, polysorbate), triton, tromethamine, lecithin, cholesterol, tyloxapol (tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, mannitol, sorbitol), delivery vehicles, diluents, excipients, and/or pharmaceutical adjuvants.

The term "treating" encompasses alleviating or preventing at least one symptom or other aspect of a disorder, or reducing the severity of a disease, and the like. The pharmaceutical compositions do not require complete cure or elimination of each symptom or phenomenon of the disease to constitute effective therapeutic agents. As recognized in the relevant art, drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eliminate every phenomenon of the disease to be considered a useful therapeutic agent.

In another aspect, the invention also discloses a conjugate, which is the aforementioned nanobody with a detectable moiety label.

Further, the detectable moiety includes an enzyme, a fluorescent material, a luminescent material, a radioactive material, a positron emitting metal, and a non-radioactive paramagnetic metal ion.

In another aspect, the invention also discloses a method of producing a TSLP antibody, comprising culturing the aforementioned host cell;

alternatively, the method comprises a step of introducing the nucleic acid molecule or the recombinant vector into a cell and culturing the cell.

Further, the production method includes an operation of culturing the cells and obtaining the TSLP antibody from the culture.

Further, the TSLP antibodies are obtained from the culture by any one or more of the following methods: the medium and host cells are centrifuged, the cells are broken up by high pressure homogenization, the cell debris is removed by centrifugation or the antibody is purified by affinity chromatography.

In another aspect, the invention also discloses a method for detecting, enriching or purifying TSLP, comprising contacting the nanobody described above with a sample.

Further, the sample may contain TSLP.

Further, the detection is of non-diagnostic or diagnostic purpose.

In another aspect, the invention features a method of treating a TSLP-related disease, the method including administering to a subject one or more of the foregoing nanobodies, nucleic acid molecules, recombinant vectors, host cells, conjugates, pharmaceutical compositions.

On the other hand, the invention also discloses application of the nano antibody, the nucleic acid molecule, the recombinant vector, the host cell, the conjugate and the pharmaceutical composition in preparing the medicine for TSLP related diseases.

Further, the TSLP-related diseases include rhinitis, sinusitis, conjunctivitis, asthma, chronic Obstructive Pulmonary Disease (COPD), atopic Dermatitis (AD), eosinophilic esophagitis (EOE), rheumatoid Arthritis (RA), psoriasis (Psoriasis), hypersensitivity reactions, immune disorders, respiratory diseases, nasal polyps, urticaria (chronic urticaria), bronchial diseases, allergic reactions, fibrotic disorders.

Further, the TSLP-related diseases also include cancers, including specifically breast, colon, lung, ovarian and prostate, and epithelial cancers.

The term "allergic inflammation" as used herein refers to the phenomenon of an immunoglobulin E-related immune response. Allergic inflammation includes, in addition to inflammatory skin conditions (e.g., atopic dermatitis), also inflammatory diseases of the lung, such as allergic sinusitis, allergic asthma, allergic conjunctivitis.

The term "asthma" as used herein is a chronic inflammatory airway disorder characterized by airway eosinophilia, high levels of serum IgE and mast cell activation, which can promote airway hyperresponsiveness, epithelial damage and mucus hypersecretion.

The term "atopic dermatitis" as used herein is a chronic itching inflammatory skin disease characterized by skin lesions, as well as elevated serum total IgE, eosinophilia and increased histamine release from basophils and mast cells.

The term "fibrotic disorders" as used herein includes, but is not limited to, systemic and localized scleroderma, keloids and hypertrophic scars, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, cirrhosis of the liver, fibrosis due to chronic hepatitis B or C infection, kidney disease, heart disease due to scar tissue, and ocular diseases (e.g., macular degeneration, as well as retinopathy and vitreosis). Other fibrotic diseases include chemotherapy drug induced fibrosis, radiation induced fibrosis, and injuries and burns.

Although the above indications are preferred, other diseases, disorders or conditions are also suitable for treatment or prevention by administering the nanobodies of the invention to an individual. The diseases, disorders and conditions include, but are not limited to, inflammation, autoimmune diseases, cartilage inflammation, fibrotic diseases and/or bone degradation, arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathy arthritis, juvenile reactive arthritis, juvenile Lei Deshi Syndrome (juvenole Reiter's synthome), SEA Syndrome (seronegative, tendinopathy, joint disease Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, juvenile rheumatoid arthritis, polyarthritis rheumatoid arthritis, juvenile Lei Deshi Syndrome (juvenole Reiter's synthome) systemic rheumatoid arthritis, ankylosing spondylitis, enteropathy arthritis, reactive arthritis, lei Deshi Syndrome, SEA Syndrome (seronegative, tendinopathy, arthrosis Syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa Wegener's granulomatosis, arteritis, multiple myodynia, sarcoidosis, scleroderma, primary biliary sclerosis, sclerosing cholangitis, sjogren's Syndrome, psoriasis, plaque psoriasis, trichome psoriasis, skin fold psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, stills disease (SLE), myasthenia gravis, inflammatory Bowel Disease (IBD), crohn's disease (Crohn's disease), ulcerative colitis, celiac disease, multiple Sclerosis (MS), asthma, COPD, guillain-Barre disease (Guillain-Barre disease), type I diabetes, graves ' disease, addison's disease, raynaud's disease, autoimmune hepatitis, GVHD, and the like.

Further, the TSLP-related disease is asthma, chronic Obstructive Pulmonary Disease (COPD), atopic Dermatitis (AD), or the like.

Drawings

FIG. 1 is the results of detection of recombinant nanobody and human TSLP-His protein binding experiments.

FIG. 2 is the results of detection of ELISA binding experiments of recombinant nanobodies and monkey TSLP.

Fig. 3 is the result of terzepelumab blocking TSLP and TSLPR binding and blocking downstream STAT5 phosphorylation FACS detection.

FIG. 4 is the result of CSJ-117-FAB blocking TSLP and TSSLPR binding and blocking downstream STAT5 phosphorylation FACS detection.

FIG. 5 is a graph showing the results of blocking TSLP and TSSLPR binding by bivalent recombinant nanobody B04 and downstream STAT5 phosphorylation FACS detection

FIG. 6 is a result of recombinant nanobody B04 blocking TSLP and TSSLPR binding and blocking downstream STAT5 phosphorylation FACS detection

FIG. 7 is a graph showing the results of competition experiments on terzepelumab and CSJ-117-FAB epitopes.

FIG. 8 is a graph showing the results of competition experiments between recombinant nanobody B04 and CSJ-117-FAB epitope.

Fig. 9 is a graph of the results of a terzepelumab and recombinant nanobody B04 epitope competition experiment.

FIG. 10 is a graph showing the results of detection of the amount of CCL17 secretion by the TSLP-stimulated PBMC inhibited by recombinant nanobody B04.

FIG. 11 is a graph showing the results of detecting the amount of expression of CCL22 secreted by the TSLP-stimulated PBMC by the recombinant nanobody B04.

FIG. 12 is a graph showing the results of affinity detection of 113/154-2-B04.

FIG. 13 is a graph showing the results of affinity detection of CSJ-117-FAB.

FIG. 14 is a graph showing the results of affinity detection of CSJ-117-.

Fig. 15 is a graph of affinity detection results of terzepelumab (Tezepelumab).

Detailed Description

The present invention will be further described with reference to specific embodiments, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Screening and preparation of the Universal method-TSLP antibody B04

Step 1, preparation of human and monkey TSLP recombinant proteins

Human TSLP (UniProtKB: Q969D 9) and monkey TSLP (UniProtKB: A0A7N9CAT 7) sequence information are retrieved from the UniProt database, optimized according to human codon preference, and 6 XHis tag is added at the C end and subcloned into pcDNA3.4 vector to construct eukaryotic expression vector. Protein expression and purification were performed in the 293F cell expression system.

Step 2, immunization of alpaca, library construction and panning

The alpaca was immunized five times at 3 weeks intervals and blood was collected for immune titer detection after the third and fourth immunization. After the fifth immunization, 100mL of peripheral blood was collected, PBMCs were isolated using lymphocyte-separating medium, RNA was extracted, cDNA was prepared by reverse transcription, and VHH fragments were amplified. And (5) recovering PCR products and constructing a nanobody yeast display library. After the library construction was completed, panning of the yeast library was performed.

Step 3, yeast monoclonal flow detection

After sorting, the saccharomycete liquid is coated on an SDCAA plate, monoclonal culture is selected, after induction expression is carried out for 48 hours, the monoclonal antibody is incubated with Biotin-antigen, and after incubation is completed, flow detection is carried out by using PE-strepitavidin for the secondary antibody. Yeast clones bound to the target antigen were lysed using 0.2% SDS (10 min incubation at 95 ℃), centrifuged, and 0.5. Mu.L of the supernatant was used as template for PCR amplification (remaining stock at-20 ℃).

Step 4, candidate antibody expression and purification

And (3) constructing a vector according to a yeast monoclonal flow detection result, adding a 6 XHis tag at the C end, subcloning into a pcDNA3.4 vector, and constructing a eukaryotic expression vector. Antibody expression and purification work was performed in 293F cell lines.

The amino acid sequence of the VHH antibody (nanobody) B04 is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGSIFTHLIMGWYRQAPGKQRERIATIGIDGTTNTEDSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAGTFSDDGTNWLNYWGQGTQVTVSS（SEQ ID NO.4）

wherein, CDR1-3 are respectively: GSIFTHLI, IGIDGTT, NAGTFSDDGTNWLNY (SEQ ID NO. 1-3)

Example one, B04 antibody binding Activity assay to human TSLP protein

Human TSLP-His protein was coated using 96-well plates, 1ug/ml, 100 ul/well, 4℃overnight. The antigen coating was removed and washed 3 times with PBST (0.5% tween). Adding 100ul of antibody diluted in gradient, and incubating for 1 hour at room temperature, wherein the initial concentration is 10 mug/ml and the concentration is 3 times diluted for 7 points; the liquid in the wells was thrown off and washed 3 times with PBST; adding HRP-labeled anti-alpaca secondary antibody (diluted 1:10000) or goat anti-human Fc secondary antibody into an ELISA plate according to 100 mu L/hole, and incubating for 1 hour at room temperature; after the liquid in the wells was thrown off, the well plates were washed 3 times with PBST; adding 100 mu L/hole TMB color development liquid; incubating for 15 minutes at room temperature in a dark place; adding 50 mu L/Kong Zhongzhi liquid; OD450 values within wells were read using a microplate reader.

TABLE 1 antibodies and human TSLP binding Activity

As shown in FIG. 1, the B04 antibody has good binding with human TSLP (panel A), and the affinity of the B04 antibody with bivalent structure is obviously improved compared with that of the B04 antibody with monovalent structure (panel B). The bivalent structure is a VHH- (GGGGS) 3-VHH.

Example two, B04 antibody binding Activity assay for monkey TSLP protein

Candidate antibodies were coated using 96-well plates, 2 ug/ml, 100 ul/well, 4 ℃ overnight. The antigen coating was removed and washed 5 times with PBST (0.05% tween 20). The Biotin-cyno TSLP-his antigen was added in a gradient dilution, with an initial concentration of 10 ug/ml, 3-fold in 7 spots. Incubate at 37 degrees for 1 hour. The liquid in the wells was removed and washed 5 times with PBST (0.05% tween 20). Add 100. Mu.L/well HRP-strepitavdin antibody (1:10000 dilution), incubate for 1 hour at room temperature; after removing the liquid from the wells, the well plate was washed 5 times with PBST; adding 100 mu L/hole TMB color development solution, and incubating for 10-15 minutes at room temperature in a dark place; adding 50 mu L/Kong Zhongzhi liquid; OD450 in the wells was read using a microplate reader.

As shown in FIG. 2, the B04 antibody and the monkey TSLP have good binding (A diagram), and the affinity of the B04 antibody with a bivalent structure is obviously improved compared with that of the B04 antibody with a monovalent structure (B diagram).

Example three, candidate antibody function blocking detection

1) Positive control antibodies, terstuzumab (self-expression purified in 293 system), CSJ-117-FAB-His (self-expression purified in 293 system) and B04 were serially diluted in five-fold gradients from 200. Mu.g/ml to eight spots and seeded in 96-well plates at 2X 100. Mu.l. (antibody concentrations were 200. Mu.g/ml, 40. Mu.g/ml, 8. Mu.g/ml, 1.6. Mu.g/ml, 0.32. Mu.g/ml, 0.064. Mu.g/ml, 0.0128. Mu.g/ml, 0.00256. Mu.g/ml, 0.000512. Mu.g/ml, 0. Mu.g/ml; antibody final concentrations in 96 well plates were 100. Mu.g/ml, 20. Mu.g/ml, 4. Mu.g/ml, 0.8. Mu.g/ml, 0.16. Mu.g/ml, 0.032. Mu.g/ml, 0.0064. Mu.g/ml, 0.00128. Mu.g/ml, 0.000256. Mu.g/ml, 0. Mu.g/ml).

2) The Human TSLP was diluted to 0.4. Mu.g/mL (final concentration 0.1. Mu.g/mL) and inoculated with 50. Mu.l per well and incubated for 1h at 37 ℃.

3) After incubation, the 293F-IL7Ra-TSLPR-Luc cell line in logarithmic proliferation phase was taken and 1xPBS was washed twice and resuspended to 4x10 ⁵ Per ml, 50 μl/well: 20000 cells were cultured for 6h.

4) After the incubation, 20. Mu.l of One-Glo was added to each well and the bioluminescence values were read on-machine.

TABLE 2 maximum inhibition and IC50

The results of the assays for terzepelumab, CSJ-117-FAB-His and B04 are shown in FIGS. 3-6, in sequence, with the maximum inhibition and IC50 being counted in Table 2, and the results indicate that B04 is effective in inhibiting the binding of TSLP and its receptor, thereby blocking downstream STAT5 phosphorylation.

Example four, candidate antibody epitope competition experiments

Performing epitope competition experiments on B04 and a positive control antibody, and solidifying Biotin-TSLP-His by using an SA sensor for 150s; the buffer is PBST (PBS+0.02% tween 20), firstly binds to an AB1 antibody (CSJ-117 full antibody or terstuzumab), and after balancing, binds to an AB2 antibody (B04 and CSJ-117 full antibody or terstuzumab as a control of the same epitope); affinity detection: equilibrium is carried out for 60s, binding for 180s, dissociation for 180s, and detection temperature is 25 ℃.

The results of binding epitope competition experiments are shown in figures 7-9: the terzepelumab and CSJ-117 are different epitopes, B04 overlaps with part of the epitope of the positive antibody CSJ-117, and B04 is different from the positive antibody terzepelumab.

Example five, PBMC stimulation experiments

1) The TSLP recombinant protein was adjusted to 0.0128. Mu.g/mL with serum-free RPMI1640 medium, and 50. Mu.L of recombinant TSLP protein dilution (final TSLP concentration in wells 0.0032. Mu.g/mL) was added to each well;

2) Diluting the positive control antibody and the antibody to be detected by adopting a serum-free RPMI1640 culture medium, and continuously diluting 8 concentration gradient points from 200 mug/mL (the final concentration of the corresponding hole is 100 mug/mL); 100 mu L of diluted antibody is added into each well, and after gentle blowing and mixing, the well plate is placed at 37 ℃ and 5% CO ₂ Incubating in a cell incubator for 30 minutes; wells without antibody served as controls;

3) Resuspension of recovered PBMC cells with 1640+20% FBS medium, taking a 96-well cell culture plate, adding PBMC cells into a biosafety cabinet, and inoculating 50 μl (1×10≡6 cells/well) per well; culturing the cells for 24 hours;

4) The culture was removed from the 96-well plate overnight, centrifuged at 1000Xg for 5 minutes at room temperature, and the culture supernatant was collected and assayed for CCL22 and CCL17 protein expression levels in the culture using ELISA kit.

TABLE 3 PBMC stimulation experiment results

The result of CCL17 expression level detection is shown in FIG. 10, the result of CCL22 expression level detection is shown in FIG. 11, and the experimental result shows that TSLP can activate PBMC cells to release CCL17 and CCL22; antibody B04 was able to effectively block TSLP-activated PBMC cells from releasing CCL17 and CCL22.

Example six candidate antibody affinity detection

1) Biotin-TSLP (29-159) -His was immobilized using an SA sensor at a concentration of 5. Mu.g/ml and an immobilization time of 80s.

2) The buffer was PBST (PBS+0.02% tween 20) and the antibodies were diluted to 5, 2.5, 1.25, 0.625, 0.3125, 0nM.

3) Affinity detection: equilibrium is carried out for 60s, binding for 180s, dissociation for 180s, and detection temperature is 25 ℃.

TABLE 4 affinity assay results

The results of the assay are shown in FIGS. 12-15, which demonstrate that B04 binds to TSLP with a higher affinity.

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims

1. An anti-TSLP nano antibody, the amino acid sequence of CDR1 of the nano antibody is shown as SEQ ID NO.1, the amino acid sequence of CDR2 of the nano antibody is shown as SEQ ID NO.2, and the amino acid sequence of CDR3 of the nano antibody is shown as SEQ ID NO. 3.

2. The nanobody of claim 1, wherein the amino acid sequence of the nanobody is shown in SEQ ID No. 4.

3. A derivative product comprising the nanobody of claim 1, the derivative product selected from the group consisting of:

1) A nucleic acid molecule encoding the nanobody of claim 1;

2) A recombinant vector comprising thereon a nucleic acid molecule encoding the nanobody of claim 1, or, the recombinant vector expressing the nanobody of claim 1;

3) A host cell expressing the nanobody of claim 1, or containing a nucleic acid molecule encoding the nanobody of claim 1 or 2) the recombinant vector;

4) A pharmaceutical composition comprising the nanobody of claim 1, or at least one of 1), 2), 3).

4. The derivative product of claim 3, further comprising one or more pharmaceutically acceptable carriers.

5. A derivative product according to claim 3, wherein the pharmaceutical composition comprises a spray, aerosol or inhalation powder.

6. A method of producing a TSLP antibody, the method comprising culturing the host cell of claim 3;

alternatively, the method comprises the step of introducing the nucleic acid molecule of claim 3 or the recombinant vector of claim 3 into a cell and culturing the cell.

7. A method of detecting, enriching or purifying TSLP, the method comprising contacting the nanobody of claim 1 or 2 with a sample, the method being of non-diagnostic interest.

8. Use of the nanobody of claim 1 or the derivative of claim 3 in the manufacture of a medicament for the treatment of asthma, atopic dermatitis or chronic obstructive pulmonary disease.