Anti-TSLP nanobodies and uses thereof

Abstract

To provide an anti-TSLP nanobody having good blocking activity and good clinical treatment effect, and easy to produce.SOLUTION: The present invention provides an anti-TSLP Nanobody, and also provides a coding sequence encoding the Nanobody, a corresponding expression vector and a host cell capable of expressing the Nanobody, and a method for producing the Nanobody of the present invention. The Nanobodies of the invention have good TSLP / TSLPR blocking activity. The nanobody of the present invention is expressed in Pichia pastoris, and the expression yield in the fermentation tank reaches 17 23g / L.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical or biopharmaceutical technology, and more particularly to anti-TSLP nanobodies and uses thereof. [Background technology]

[0002] In recent years, treatment approaches for moderate to severe asthma have focused on attempts to suppress Th2 cell responses. Antibody drugs for treating severe asthma often target the Th2 pathway, including IgE (omalizumab), IL5 (mepolizumab, reslizumab), IL5R (benralizumab), and IL4R (dupilumab). All of these drugs have demonstrated effective control of eosinophilic asthma, and all are administered subcutaneously or intravenously. Approximately one-third of patients with severe asthma lack the hallmarks of Th2 inflammatory pathway activation. Currently, patients with non-Th2 asthma who are unable to respond to existing standard care and treatments have no biological treatment options, necessitating the development of novel therapeutic approaches.

[0003] Thymic stromal lymphopoietin (TSLP) is a short-chain cytokine with a four-helix bundle folding structure and belongs to the IL-2 cytokine family. TSLP is an epithelial cytokine produced in response to pro-inflammatory stimuli (e.g., allergens, viruses, and other pathogens in the lungs) and plays an important role in the development and maintenance of airway inflammation. TSLP downstream induces the release of Th2 cytokines, including IL-4, IL-5, and IL-13, leading to inflammation and asthma symptoms. TSLP also inhibits the proliferation of many types of cells involved in non-Th2 inflammation. TSLP can also activate inflammatory cytokines. Therefore, early upstream activation of TSLP in the inflammatory cascade has already made it a potential target in a broad range of asthma patients. Blocking TSLP prevents immune cell release of proinflammatory cytokines, thereby preventing asthma exacerbations, improving asthma control, and addressing related conditions such as chronic obstructive pulmonary disease.

[0004] Nanobodies (Nb), i.e., heavy chain nanoantibodies (VHHs), are heavy chain nanoantibodies consisting of a single heavy chain variable domain obtained by cloning the variable domain of a naturally occurring light chain-deficient heavy chain antibody (HCAb) found in camels. These nanobodies are the smallest currently available unit that is fully functional, stable, and capable of binding to antigens. Nanobodies are characterized by high stability, good water solubility, easy humanization, high targeting, and strong permeability, making them suitable for immunological experiments, diagnosis, and therapy. Nanoantibodies are gradually becoming a new generation of antibody therapy. Therefore, there is a strong need in the field to develop anti-TSLP nanoantibodies that have good blocking activity, good clinical therapeutic effects, and are easy to produce. Summary of the Invention [Problem to be solved by the invention]

[0005] The objective of the present invention is to provide an anti-TSLP nanobody that has good blocking activity, good clinical therapeutic effect, and is easy to produce. [Means for solving the problem]

[0006] In a first aspect, the present invention provides an anti-TSLP Nanobody, wherein the CDR regions, which are the complementarity determining regions of the VHH chain in said Nanobody, are one or more selected from the group consisting of: (1) CDR1 represented by SEQ ID NO: 1, CDR2 represented by SEQ ID NO: 2, and CDR3 represented by SEQ ID NO: 3; (2) CDR1 represented by SEQ ID NO: 14, CDR2 represented by SEQ ID NO: 15, and CDR3 represented by SEQ ID NO: 16; and (3) CDR1 represented by SEQ ID NO: 27, CDR2 represented by SEQ ID NO: 28, and CDR3 represented by SEQ ID NO: 29. In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0007] In another preferred embodiment, the VHH chain further comprises a framework region FR, wherein the framework region FR is one or more selected from the group consisting of: (1) FR1 represented by SEQ ID NO: 4, FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO: 7; (2) FR1 represented by SEQ ID NO: 10: FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO: 11; (3) FR1 represented by SEQ ID NO: 17: FR2 represented by SEQ ID NO: 18, FR3 represented by SEQ ID NO: 19, and FR4 represented by SEQ ID NO: 20; (4) FR1 represented by SEQ ID NO: 23, FR2 represented by SEQ ID NO: 18, FR3 represented by SEQ ID NO: 19, and FR4 represented by SEQ ID NO: 24; (5) FR1 represented by SEQ ID NO: 30, FR2 represented by SEQ ID NO: 31, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 33; and (6) FR1 represented by SEQ ID NO: 36, FR2 represented by SEQ ID NO: 31, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 37.

[0008] In another preferred embodiment, the CDR regions of the VHH chain of said Nanobody comprise an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with any of SEQ ID NOs: 1-3, 14-16, and 27-29.

[0009] In another preferred embodiment, the amino acid sequence of the CDR region of the VHH chain of said Nanobody comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any of SEQ ID NOs: 1-3, 14-16 and 27-29.

[0010] In another preferred example, any one of the above amino acid sequences further includes a derivative sequence that has undergone addition, deletion, modification, and / or substitution of at least one amino acid (e.g., 1 to 3, preferably 1 to 2, more preferably 1) while retaining the ability to specifically bind to TSLP.

[0011] In another preferred embodiment, the Nanobody is capable of specifically binding to TSLP.

[0012] In another preferred embodiment, the Nanobody can effectively block the interaction between TSLP and TSLPR.

[0013] In another preferred embodiment, the TSLP is a human or non-human mammalian TSLP.

[0014] In another preferred embodiment, the TSLP is human, mouse, rat, or non-human primate (eg, monkey) TSLP.

[0015] In another preferred embodiment, the Nanobodies include humanized antibodies, camelid-derived antibodies, and chimeric antibodies.

[0016] In another preferred embodiment, the amino acid sequence of the VHH chain of said Nanobody is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 38, or a combination thereof6.

[0017] In another preferred embodiment, the anti-TSLP Nanobody comprises a monomer, a dimer (bivalent antibody), a tetramer (tetravalent antibody), and / or a multimer (multivalent antibody).

[0018] In another preferred embodiment, the anti-TSLP Nanobody comprises a VHH chain having one or more of the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:34 or SEQ ID NO:38.

[0019] In another preferred embodiment, the anti-TSLP Nanobody comprises a VHH chain having the amino acid sequence set forth in two or more of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:34, SEQ ID NO:38.

[0020] In another preferred embodiment, the VHH chains are linked via a linking peptide.

[0021] In another preferred embodiment, the connecting peptide is selected from the following sequence: (GaSb)x, where a, b, and x=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a=4 and b=1, and x=4).

[0022] In another preferred embodiment, the sequence of the connecting peptide is GGGGSGGGGSGGGGSGGGGS.

[0023] In a second aspect of the invention, there is provided an anti-TSLP antibody, which is directed against a TSLP epitope and comprises an anti-TSLP nanobody according to the first aspect of the invention.

[0024] In another preferred embodiment, the anti-TSLP antibody comprises one or more anti-TSLP nanobodies.

[0025] In another preferred embodiment, the anti-TSLP antibody comprises a monomer, a dimer (bivalent antibody), a tetramer (tetravalent antibody), and / or a multimer (multivalent antibody).

[0026] In another preferred embodiment, the anti-TSLP antibody comprises a VHH chain having one or more of the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:34, or SEQ ID NO:38.

[0027] In another preferred embodiment, the anti-TSLP antibody comprises two VHH chains having the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:34, or SEQ ID NO:38.

[0028] In another preferred embodiment, the antibody is capable of specifically binding to TSLP.

[0029] In another preferred embodiment, the antibody is directed specifically against a TSLP protein having a precise spatial structure.

[0030] In another preferred embodiment, the antibody can effectively block the interaction between TSLP and TSLPR.

[0031] In another preferred embodiment, the affinity (KD value) of the antibody for TSLP is less than 3.77 nM.

[0032] In another preferred embodiment, the target antibody has excellent TSLP / TSLPR blocking activity, and the blocking activity is significantly better than that of the control antibody tezepelumab, wherein the control antibody tezepelumab is available from AstraZeneca or Amgen.

[0033] In another preferred embodiment, the antibody can effectively inhibit the proliferation of BaF3 / TSLPR-IL7R cells, and its inhibitory activity is better than that of the control antibody tezepelumab.

[0034] In another preferred embodiment, the antibody is a nanobody.

[0035] In a third aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of an anti-TSLP nanobody according to the first aspect of the invention or an anti-TSLP antibody according to the second aspect of the invention.

[0036] In another preferred embodiment, the polynucleotides are in a combinatorial form.

[0037] In another preferred embodiment, the polynucleotide comprises one or more of the sequences set forth in SEQ ID NOs: 9, 13, 22, 26, 35 or 39.

[0038] In another preferred embodiment, the polynucleotide comprises RNA, DNA, or cDNA.

[0039] In a fourth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the third aspect of the invention.

[0040] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0041] In another preferred embodiment, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, or retrovirus.

[0042] In a fifth aspect of the present invention, there is provided a host cell which comprises an expression vector according to the fourth aspect of the invention or has incorporated into its genome a polynucleotide according to the third aspect of the invention.

[0043] In another preferred embodiment, the host cell comprises a prokaryotic or eukaryotic cell.

[0044] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cells, mammalian cells, phages, or combinations thereof.

[0045] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus acidophilus, Streptomyces, Proteus mirabilis, or a combination thereof.

[0046] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0047] In another preferred embodiment, the host cell is Pichia pastoris.

[0048] In a sixth aspect of the present invention, there is provided a method for producing an anti-TSLP Nanobody, the method comprising the steps of: (a) obtaining a culture comprising an anti-TSLP Nanobody by culturing a host cell according to the fifth aspect of the invention under conditions suitable for the production of said Nanobody; (b) isolating or recovering the anti-TSLP nanobody from the culture; and (c) Optionally, purifying and / or modifying the anti-TSLP nanobody obtained in step (b).

[0049] In a seventh aspect of the present invention, there is provided an immunoconjugate comprising: (a) an anti-TSLP nanobody according to the first aspect of the invention, or an anti-TSLP antibody according to the second aspect of the invention; and (b) a conjugate moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a magnetic nanoparticle, a viral coat protein or VLP, or a combination thereof.

[0050] In another preferred embodiment, the radionuclide includes: (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0051] In another preferred embodiment, the conjugated moiety is a drug or toxin.

[0052] In another preferred embodiment, the drug is a cytotoxic drug.

[0053] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0054] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, and typical cytotoxic drugs include, for example, auristatin, camptothecin, duocarmycin, etoposide, maytansine and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines, or benzodiazepine-containing drugs. Benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vinca alkaloids, or combinations thereof.

[0055] In another preferred embodiment, the toxin is auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoid, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, The toxin is selected from the group consisting of vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-octastaphylococcus, gelonin, mitogellin, restrictocin, phenomycin, enomycin, chrysin, crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitor, glucocorticoid, or a combination thereof.

[0056] In another preferred embodiment, the conjugated moiety is a detectable marker.

[0057] In another preferred embodiment, the conjugated moiety is a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme that produces a detectable product, a radionuclide, a biotoxin, a cytokine (such as IL-2), an antibody, an antibody Fc fragment, an antibody scFv fragment, gold nanoparticles / nanorods, a virus particle, a liposome, a magnetic nanoparticle, a prodrug-activating enzyme (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL) or The nanoparticles are selected from the group consisting of nanoparticles in any form.

[0058] In an eighth aspect of the invention, there is provided a multispecific antibody comprising an anti-TSLP nanobody according to the first aspect of the invention or an anti-TSLP antibody according to the second aspect of the invention.

[0059] In another preferred embodiment, the multispecific antibody further comprises an Fc fragment of an antibody.

[0060] In a ninth aspect of the present invention, there is provided a recombinant protein comprising: (i) an anti-TSLP nanobody according to the first aspect of the invention, or an anti-TSLP antibody according to the second aspect of the invention; and (ii) An optional tag sequence to aid in expression and / or purification.

[0061] In another preferred embodiment, the tag sequences include an Fc tag, an HA tag, and a 6His tag.

[0062] In another preferred embodiment, the recombinant protein specifically binds to the TSLP protein.

[0063] In a tenth aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) an anti-TSLP nanobody according to the first aspect of the invention, or an anti-TSLP antibody according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention, or a multispecific antibody according to the eighth aspect of the invention, or a recombinant protein according to the ninth aspect of the invention; and (ii) A pharmaceutically acceptable carrier.

[0064] In another preferred embodiment, the conjugated moiety of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0065] In another preferred embodiment, the pharmaceutical composition further comprises a drug for treating other immune system diseases or tumor diseases.

[0066] In another preferred embodiment, the drug for treating other immune system diseases or tumor diseases is selected from the group consisting of budesonide, fluticasone, beclomethasone, mometasone furoate, albuterol, theophylline, formoterol, tiotropium bromide, sulfasalazine, methotrexate, cyclophosphamide, fluorouracil, bleomycin, anastrozole, or a combination thereof.

[0067] In another preferred embodiment, the pharmaceutical composition is used for the manufacture of a medicament for preventing and / or treating a disease or condition associated with TSLP.

[0068] In another preferred embodiment, the TSLP-associated disease or condition comprises an immune system disease or a tumor disease.

[0069] In another preferred embodiment, the immune system disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergy, ulcerative colitis, Crohn's disease, rhinitis, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis, allergic pneumonitis, allergic granulomatous vasculitis, nasal polyps, or a combination thereof.

[0070] In another preferred embodiment, the tumor disease is selected from the group consisting of breast cancer, pancreatic adenocarcinoma, cervical cancer, multiple myeloma, colorectal cancer, lung cancer, thyroid cancer, ovarian cancer, liver cancer, or a combination thereof.

[0071] In an eleventh aspect of the invention, there is provided the use of an anti-TSLP nanoantibody according to the first aspect of the invention, an anti-TSLP antibody according to the second aspect of the invention, an immune complex according to the seventh aspect of the invention, or a multispecific antibody according to the eighth aspect of the invention, or a recombinant protein according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention, for (a) the manufacture of a medicament for the prevention and / or treatment of a TSLP-associated disease, and / or (b) the manufacture of a reagent, detection plate, or kit for detecting TSLP.

[0072] In another preferred embodiment, the TSLP-associated disease or condition comprises an immune system disease or a tumor disease.

[0073] In another preferred embodiment, the immune system disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergy, ulcerative colitis, Crohn's disease, rhinitis, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis, allergic pneumonitis, allergic granulomatous vasculitis, nasal polyps, or a combination thereof.

[0074] In another preferred embodiment, the tumor disease is selected from the group consisting of breast cancer, pancreatic adenocarcinoma, cervical cancer, multiple myeloma, colorectal cancer, lung cancer, thyroid cancer, ovarian cancer, liver cancer, or a combination thereof.

[0075] In another preferred embodiment, the TSLP is human TSLP.

[0076] In another preferred embodiment, the reagent is a diagnostic reagent.

[0077] In another preferred embodiment, the diagnostic reagent is a contrast agent.

[0078] In another preferred embodiment, the reagent is for detecting a TSLP protein or a fragment thereof in a sample.

[0079] In another preferred embodiment, the detection includes detection by flow cytometry and detection by cellular immunofluorescence.

[0080] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0081] In a twelfth aspect of the present invention, there is provided a method for detecting TSLP protein in a sample, the method comprising the steps of: (1) contacting a sample with an anti-TSLP nanoantibody according to the first aspect of the invention, or an anti-TSLP antibody according to the second aspect of the invention, or an immune complex according to the seventh aspect of the invention, or a multispecific antibody according to the eighth aspect of the invention, or a recombinant protein according to the ninth aspect of the invention; (2) detecting the formation of an antigen-antibody complex, where the formation of the complex indicates the presence of TSLP protein in the sample;

[0082] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0083] In a thirteenth aspect of the present invention, there is provided a detection reagent for TSLP protein, the detection reagent comprising: (i) an anti-TSLP nanobody according to the first aspect of the invention, or an anti-TSLP antibody according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention, or a multispecific antibody according to the eighth aspect of the invention, or a recombinant protein according to the ninth aspect of the invention; and (ii) A metrologically acceptable carrier.

[0084] In another preferred embodiment, the conjugated moiety of the immunoconjugate is a diagnostic isotope.

[0085] In another preferred embodiment, the metrically acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0086] 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 detection reagent, a cellular immunofluorescence detection reagent, a magnetic nanoparticle, and an imaging agent.

[0087] In another preferred embodiment, the detection reagent is for in vivo detection.

[0088] In another preferred embodiment, the detection reagent is in the form of a liquid or powder (eg, an aqueous solution, an injection, a lyophilized powder, a tablet, a lozenge, or an aerosol inhalant).

[0089] In a fourteenth aspect of the present invention, there is provided a kit for detecting TSLP protein, the kit comprising an immune complex according to the seventh aspect of the invention or a detection reagent according to the thirteenth aspect of the invention, and instructions.

[0090] In another preferred embodiment, the instructions state that the kit is for non-invasively detecting TSLP expression in a subject.

[0091] In a fifteenth aspect of the present invention, there is provided the use of an immunoconjugate according to the seventh aspect of the present invention for the manufacture of an imaging agent for detecting TSLP protein in the body.

[0092] In another preferred embodiment, the detection is for the diagnosis or prognosis of a disease or condition associated with TSLP.

[0093] In a sixteenth aspect of the invention, there is provided a method of treating a disease, the method comprising administering to a subject in need thereof an anti-TSLP nanobody according to the first aspect of the invention, an anti-TSLP antibody according to the second aspect of the invention, an immunoconjugate according to the seventh aspect of the invention, or a multispecific antibody according to the eighth aspect of the invention, or a recombinant protein according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention.

[0094] In another preferred embodiment, the subject comprises a human or non-human mammal.

[0095] In another preferred example, the non-human mammal includes rodents (eg, mice, rabbits) and non-human primates (eg, monkeys). Of course, it is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Brief explanation of the drawings]

[0096] [Figure 1A] Figure 1A shows the results of flow cytometry detection of the blocking activity of 31 candidate antibodies. The results show that the blocking activity of 14 of the 31 candidate antibodies was significantly better than that of the control antibody tezepelumab. [Figure 1B] Figure 1B shows the results of flow cytometry detection of the blocking activity of 31 candidate antibodies. The results show that the blocking activity of 14 of the 31 candidate antibodies was significantly better than that of the control antibody tezepelumab. [Figure 2] Figure 2 shows the binding kinetics of 14 blocking TSLP nanoantibodies. [Figure 3]Figure 3 shows the SDS-PAGE results of the expression supernatants of the bivalent single domain antibodies Bi-HuNb5-31, Bi-HuNb7-54, and Bi-HuNb10-63 from shaking cultures in Pichia pastoris. The yields were 475 μg / mL, 310 μg / mL, and 510 μg / mL, respectively. [Figure 4] Figure 4 shows the results of detecting the blocking activity of the humanized bivalent antibodies by ELISA. The results show that the blocking activity of the three humanized bivalent antibodies was significantly better than that of the control antibody tezepelumab. [Figure 5] Figure 5 shows the results of the growth inhibitory effect of the humanized bivalent antibodies on BaF3 / TSLPR-IL7R cells. The results show that two of the humanized antibodies had a stronger inhibitory effect on the pSTAT5 signaling pathway in BaF3 / TSLPR-IL7R cells than the control antibody tezepelumab. DETAILED DESCRIPTION OF THE INVENTION

[0097] For the first time, we have unexpectedly discovered an anti-TSLP nanobody. Experimental results show that the nanobody of the present invention can effectively block the interaction between TSLP and TSLPR, with significantly better blocking activity than the control antibody tezepelumab, and can effectively inhibit the proliferation of BaF3 / TSLPR-IL7R cells with significantly better inhibitory activity than the control antibody tezepelumab. Fermentation tank expression in Pichia pastoris yielded 17-23 g / L, significantly higher than the industry standard. Based on this, the present inventors have completed the present invention.

[0098] term As used herein, the terms "Nanobody of the invention", "Nanobody of the invention", "anti-TSLP Nanobody of the invention", "TSLP Nanobody of the invention", "anti-TSLP Nanobody", and "TSLP Nanobody" have the same meaning and can be used interchangeably, and all refer to a Nanobody that specifically recognizes and binds to TSLP (including human TSLP).

[0099] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with similar structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the number of disulfide bonds between heavy chains depends on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end followed by a constant region at the other end. The light chain constant region faces the first constant region of the heavy chain, and the light chain variable region faces the variable region of the heavy chain. Special amino acid residues form an interface between the light and heavy chain variable regions.

[0100] As used herein, the terms "single domain," "VHH," "nanobody," and "heavy chain antibody" (single domain antibody, sdAb, or nanobody) have the same meaning and can be used interchangeably to refer to the variable region of the heavy chain of a cloned antibody. A nanobody (VHH) consisting of only one heavy chain variable region is constructed and is the smallest fully functional antigen-binding fragment. Typically, an antibody is first obtained that lacks the natural light chain and heavy chain constant region 1 (CH1), and then the heavy chain variable region of the antibody is further cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0101] As used herein, the term "variable" refers to the fact that certain portions of the variable regions of antibodies differ in sequence, thereby determining the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. Relatively conserved portions of the variable regions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions, each with a primarily β-sheet structure, connected by three CDRs that form connecting loops, and sometimes with a partial β-sheet structure. The CDRs in each chain are closely spaced by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits different effector functions, such as participating in antibody-dependent cellular toxicity of the antibody.

[0102] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of the antibodies of the invention or fragments thereof conjugated to drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules. Additionally, the invention includes cell surface markers or antigens conjugated to the anti-TSLP antibodies or fragments thereof.

[0103] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0104] As used herein, the terms "variable region" and "complementarity determining region (CDR)" can be used interchangeably.

[0105] In one preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises three complementarity determining regions: CDR1, CDR2, and CDR3.

[0106] In one preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and heavy chain constant region described above.

[0107] As used herein, the terms "antibody of the invention," "protein of the invention," and "polypeptide of the invention" are used interchangeably and refer to polypeptides that specifically bind to TSLP protein, such as proteins or polypeptides having a heavy chain variable region, which may or may not contain an initial methionine.

[0108] The present invention also provides other proteins or fusion expression products of the antibodies of the present invention. Specifically, the present invention includes any protein or protein complex and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region similar to or at least 90%, preferably at least 95%, homologous to the heavy chain variable region of an antibody of the present invention.

[0109] Generally, the antigen-binding properties of an antibody are characterized by three specific regions located in the heavy chain variable region, called the variable region (CDR), which are divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conserved and are not directly involved in binding reactions. These CDRs form a ring structure, which is spatially close together by the beta sheet formed by the FRs. The CDRs in the heavy chain and the corresponding CDRs in the light chain constitute the antigen-binding site of the antibody. Comparison of the amino acid sequences of similar antibodies can determine which amino acids constitute the FR or CDR regions.

[0110] The heavy chain variable region of the antibody of the present invention is of particular interest because at least a portion of it is involved in antigen binding. Therefore, molecules having a heavy chain variable region of a CDR-containing monoclonal antibody are included in the present invention as long as their CDRs share 90% or more (preferably 95% or more, and most preferably 98% or more) homology with the CDRs identified herein.

[0111] The present invention includes not only complete antibodies, but also immunologically active antibody fragments or fusion proteins consisting of antibodies and other sequences. Thus, the present invention further includes fragments, derivatives and analogs of said antibodies.

[0112] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain essentially the same biological function or activity as an antibody of the present invention. Fragments, derivatives, and analogs of the polypeptides of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, whether or not such substituted amino acid residues are encoded by the genetic code; (ii) polypeptides in which one or more amino acid residues have been substituted; (iii) polypeptides in which the mature polypeptide has been fused to another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides in which an additional amino acid sequence has been fused to the polypeptide (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a protein precursor sequence, or a fusion protein formed with a 6His tag). Based on the disclosure herein, these fragments, derivatives, and analogs are within the knowledge of those skilled in the art.

[0113] The antibody of the present invention refers to a polypeptide containing the above-described CDR region and having TSLP-binding activity. The term also encompasses mutated forms of the polypeptide containing the above-described CDR region that have the same function as the antibody of the present invention. These mutations include, but are not limited to, deletion, insertion, and / or substitution of one or more amino acids (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10) and addition of one or more amino acids (usually 20 or less, preferably 10 or less, and more preferably 5 or less) to the C-terminus and / or N-terminus. For example, substitution with amino acids with close or similar functions is generally not considered to alter the function of the protein. Furthermore, addition of one or more amino acids to the C-terminus and / or N-terminus also generally does not alter the function of the protein. The term also encompasses active fragments and active derivatives of the antibody of the present invention.

[0114] Variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, naturally occurring mutants, induced mutants, proteins encoded by DNA capable of hybridizing with the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained with antisera against the antibody of the present invention.

[0115] The present invention further includes fusion proteins of other polypeptides, such as Nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the Nanobodies of the invention. Typically, such fragments comprise at least about 50 contiguous amino acids of an antibody of the invention, 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.

[0116] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which, compared to the amino acid sequence of an antibody of the present invention, 10 or fewer, preferably 8 or fewer, more preferably 5 or fewer, and most preferably 3 or fewer amino acids have been substituted with amino acids having similar or close properties. These conservatively mutated polypeptides are preferably generated by amino acid substitutions as shown in Table A.

[0117] [Table A]

[0118] Furthermore, the present invention provides a polynucleotide molecule encoding the above-mentioned antibody, or a fragment thereof, or a fusion protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

[0119] Polynucleotides encoding mature polypeptides of the present invention include coding sequences encoding only the mature polypeptide, coding sequences for the mature polypeptide and various additional coding sequences, coding sequences for the mature polypeptide (and any additional coding sequences) and non-coding sequences.

[0120] The term "polynucleotide encoding a polypeptide" may be a polynucleotide that encodes the polypeptide, or may further include additional coding and / or non-coding sequences.

[0121] The present invention further relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% homology between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In this context, "stringent conditions" refers to (1) hybridization and elution at low ionic strength and high temperature, e.g., 0.2×SSC, 0.1% SDS, and 60°C, or (2) the presence of a denaturing agent during hybridization, e.g., 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll at 42°C, or (3) hybridization only occurs when the homology between the two sequences is at least 90%, preferably 95%. Furthermore, the polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0122] The full-length nucleotide sequences or fragments of the antibodies of the present invention can typically be obtained by PCR amplification, recombinant techniques, or artificial synthesis. Synthetic techniques are applicable, particularly when the fragments are short in length, to synthesize the relevant sequences. Typically, multiple smaller fragments are first synthesized and then concatenated to obtain longer fragments of the sequence. Alternatively, the coding sequence for the heavy chain can be fused together with an expression tag (e.g., 6His) to form a fusion protein.

[0123] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant techniques, typically by cloning the sequence into a vector, introducing it into cells, and then isolating the relevant sequence from host cells grown by conventional methods. The biological molecules (nucleic acids, proteins, etc.) of the present invention include biological molecules that exist in isolated form.

[0124] Currently, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis. Furthermore, these DNA sequences can be introduced into various known DNA molecules (or vectors, etc.) or cells well known in the art. Mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.

[0125] Furthermore, the present invention relates to vectors containing the above-described appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform appropriate host cells so as to express the proteins.

[0126] Host cells may be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells, including bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium, fungal cells such as yeast, insect cells such as fruit fly S2 or Sf9, and animal cells such as CHO, COS7, and 293 cells.

[0127] Transformation of host cells by DNA recombination may be carried out using conventional techniques familiar to those skilled in the art. When the host is a prokaryotic cell, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the procedures of which are well known in the art. Another method uses MgCl2. If necessary, transformation can be performed by electroporation. When the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate precipitation, microinjection, conventional mechanical methods such as electroporation, and lipofection can be used.

[0128] The resulting transformant can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Conventional culture media may be selected depending on the host cells used. The cells are cultured under conditions suitable for the growth of the host cells. Once the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are further cultured.

[0129] The recombinant polypeptides in the above methods can be expressed intracellularly or at the cell membrane, or can be secreted extracellularly. If necessary, the recombinant proteins can be isolated and purified using various isolation methods based on their 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 treatments, treatment with protein precipitants (salting out), centrifugation, osmotic shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0130] The antibodies of the present invention may be used alone, or may be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase)-modifying moiety, or any combination thereof.

[0131] Detectable markers used for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes that produce a detectable product.

[0132] Therapeutic agents that can be conjugated or complexed to the antibodies of the present invention include, but are not limited to, 1. radionuclides, 2. biotoxins, 3. cytokines, such as IL-2, 4. gold nanoparticles / nanorods, 5. viral particles, 6. liposomes, 7. magnetic nanoparticles, 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), and the like.

[0133] Thymic stromal lymphopoietin (TSLP) Thymic stromal lymphopoietin (TSLP) is a short-chain cytokine with a four-helix bundle folding structure and belongs to the IL-2 cytokine family. As a novel cytokine, TSLP is normally expressed in epithelial cells on the surface of the lung, skin, and intestinal barrier. Animal experiments have shown that TSLP is highly expressed in the lungs of allergen-induced model mice, and that TSLP receptor-deficient mice exhibit significantly reduced asthma symptoms, while lung-specific TSLP transgenic mice exhibit Th2-type inflammation and tracheal inflammation with enhanced IgE expression and are hyperresponsive. Further studies have shown that TSLP is expressed in bone marrow-derived TSLP. It has been suggested that TSLP activates dendritic cells and upregulates costimulatory molecules, resulting in the production of the Th2 chemokine CCL17. Therefore, TSLP is a prerequisite for the development of airway allergic inflammation. It is an upstream regulator of many inflammatory pathways in various diseases, including asthma, and is crucial for the development and maintenance of airway inflammation. Furthermore, studies have shown that TSLP is also a cytokine involved in the pathogenesis of atopic dermatitis (AD). More importantly, researchers have found that TSLP levels are elevated in many different tumors, and that tumors can be induced to express another protein called BCL-2, which protects tumors from death. Therefore, TSLP is crucial for tumor survival.

[0134] Thymic stromal lymphopoietin receptor (TSLPR) The TSLPR receptor is a type I transmembrane protein and a member of the hematopoietic cytokine receptor family. The functional TSLPR complex consists of TSLPR and IL-7Ra. TSLPR is also known as cytokine receptor-like molecule 2 or type I cytokine receptor σ1. TSLP initiates intracellular type 2 signaling by binding to its high-affinity heterodimeric receptor complex, which consists of its specific receptor, TSLPR, which shares 24% homology with the common receptor γ chain of IL-2, IL-4, IL-9, and IL-15, but does not contain the δ-common chain of the IL-2 family or the IL-7Rα subunit (CD127) in cells coexpressing TSLPR and IL-7Rα. TSLP first binds to TSLPR and then recruits the IL-7Rα chain.

[0135] Drug Composition The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the above-mentioned antibody, its active fragment, or a fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier, and the pH value is usually about 5 to 8, preferably about 6 to 8, depending on the properties of the formulated substances and the disease to be treated. The formulated pharmaceutical composition can be administered by any conventional route, including, but not limited to, intraperitoneal, intravenous, or topical administration.

[0136] The pharmaceutical compositions of the present invention can be used to directly bind to the TSLP protein molecule, and are therefore useful for treating TSLP-related diseases or conditions (including immune system diseases and tumor diseases), and may be used in combination with other therapeutic agents.

[0137] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described nanobody (or complex thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. The pharmaceutical formulation corresponds to the dosage form. The pharmaceutical composition of the present invention may also be an injection, which can be prepared by conventional methods using, for example, physiological saline or an aqueous solution containing glucose and other excipients. In the case of an injection or solution, the pharmaceutical composition is prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight daily. The polypeptide of the present invention can also be used in combination with other therapeutic agents.

[0138] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, and this safe and effective amount is usually at least about 10 μg / kg body weight and in many cases less than about 50 mg / kg body weight, preferably about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage should be determined taking into account factors such as the mode of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0139] Anti-TSLP nanoantibody In the present invention, the amino acid sequence of the VHH chain of the anti-TSLP Nanobody is selected from one or more of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:34 or SEQ ID NO:38.

[0140] In one preferred embodiment of the present invention, the anti-TSLP Nanobody comprises a monomer, a dimer (bivalent antibody), a tetramer (tetravalent antibody), and / or a multimer (multivalent antibody).

[0141] Typically, the anti-TSLP Nanobody comprises two VHH chains having the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:34 or SEQ ID NO:38.

[0142] In another preferred embodiment, the VHH chains are linked via a linking peptide.

[0143] In another preferred embodiment, the connecting peptide is selected from the following sequence: (GaSb)x, where a, b, and x=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a=4 and b=1, and x=4).

[0144] In another preferred embodiment, the sequence of the connecting peptide is GGGGSGGGGSGGGGSGGGGS.

[0145] Labeled nanoantibodies In one preferred embodiment of the present invention, the Nanobody carries a detectable marker, more preferably selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker. Colloidal gold markers may be prepared by methods known to those skilled in the art. In one preferred embodiment of the present invention, TSLP nanoantibodies are labeled with colloidal gold to obtain colloidal gold-labeled nanoantibodies.

[0146] Detection Method The present invention also relates to methods for detecting TSLP protein, which essentially involve obtaining a cell and / or tissue sample, lysing the sample in a medium, and detecting the level of TSLP protein in the lysed sample. In the detection method of the present invention, the sample used is not particularly limited, and a typical example is a sample containing cells present in a cell preservation solution.

[0147] kit The present invention also provides a kit containing the antibody (or fragment thereof) or cassette of the present invention, and in one preferred embodiment of the present invention, the kit further comprises a container, instructions for use, buffer solution, etc.

[0148] The present invention also provides a detection kit for detecting TSLP levels, which includes an antibody that recognizes the TSLP protein, a lysis medium for dissolving the sample, and general-purpose reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit may also be an in vitro diagnostic device.

[0149] application As described above, the Nanobody of the present invention has broad biological and clinical application value, and its applications cover many fields, including the diagnosis and treatment of TSLP-related diseases or conditions, basic medical research, biological research, etc. One preferred application is for use in clinical diagnosis and targeted therapy of TSLP.

[0150] The main advantages of the present invention are as follows: (a) The Nanobodies of the invention can effectively block the interaction between TSLP and TSLPR. (b) The Nanobodies of the invention have stronger blocking activity than the control antibody tezepelumab. (c) The nanobodies of the present invention can be expressed in Pichia pastoris, with high expression yields in shake culture and fermentation tank yields reaching 17-23 g / L. (d) The Nanobodies of the invention have significantly better inhibitory effects on the pSTAT5 signaling pathway in BaF3 / TSLPR-IL7R cells than the control antibody tezepelumab.

[0151] The present invention will be further described below with reference to specific examples. These examples are used only to illustrate the present invention and should not be construed as limiting the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples generally follow conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts are by weight. Unless otherwise stated, all materials or reagents used in the examples of the present invention are commercially available products.

[0152] Example 1 Screening for TSLP-specific nanobodies The amino acid sequence of human TSLP with a furin cleavage site mutation was optimized for human codons, then cloned into the pFUSE vector and transfected into HEK293F cells. Human TSLP protein was obtained by purifying the purified protein. After mixing with an adjuvant, four Xinjiang Bactrian camels were immunized weekly for a total of seven times. Peripheral blood samples were collected from the camels, RNA was isolated, and VHH gene fragments were amplified. The fragments were then cloned into the pMECS vector and electrotransformed into TG1-responsive cells to construct high-quality phage-display nanoantibody libraries. All four libraries achieved capacities of over 1 x 109 CFU and fragment insertion rates of over 80%.

[0153] Phage display technology was used to screen for TSLP-specific nanoantibodies. After three cycles of adsorption-washing-assembly, each library yielded a collection of specific nanoantibody phages. 1,200 clones were selected for ELISA detection and sequencing, resulting in 312 antibody strains with different sequences.

[0154] Example 2: Screening of TSLP Nanobodies by Flow Cytometry The above nanobodies with different sequences were cloned and inoculated into TB medium. Cultured and induced with IPTG overnight, the cells were lysed, and the supernatants were collected and used to detect blocking activity. Cultured CHOZEN / TSLPR stably transfected cells were divided into 96-well plates, with 3E5 cells per well. The plates were centrifuged at 3000 rpm for 3 minutes, the supernatant discarded, and then lysates of each antibody and TSLP-biotin protein were added and incubated for 20 minutes. After centrifugation and the supernatant discarded, diluted SA-PE antibodies were added and incubated at 4°C for 20 minutes. After further centrifugation and the supernatant discarded, 200 μL of PBS-resuspended cells were added to each well, and the PE signals of the samples were detected using a flow cytometer. Results indicated that a total of 64 candidate antibodies had the function of blocking the interaction between TSLP and TSLPR. Following sequencing, 31 different antibody families were selected and expressed and purified. For the purification method, please refer to Example 4 in Patent CN110144011A.

[0155] The blocking activity of these 31 purified antibodies was further assayed by flow cytometry. Cultured HEK293F / TSLPR transiently transfected cells were divided into 96-well plates, each containing 3E5 cells. The plates were centrifuged at 3000 rpm for 3 minutes, the supernatant discarded, and a gradient dilution of each antibody (2-fold dilution starting from 20 μg / mL) and TSLP-biotin protein were added and incubated for 20 minutes. Tezepelumab was used as the control antibody. The plates were then centrifuged, the supernatant discarded, and the diluted SA-PE antibody was added and incubated for 20 minutes at 4°C. The plates were then centrifuged, the supernatant discarded, and 200 μL of PBS-resuspended cells were added to each well. The PE signal of the samples was detected by flow cytometry. The results are shown in Figures 1A and 1B. The blocking activity of 14 antibodies was significantly better than that of the control antibody tezepelumab. The antibody numbers of these 14 antibodies are Nb1-41, Nb1-51, Nb1-59, Nb3-18, Nb3-43, Nb5-31, Nb6-29, Nb7-54, Nb10-55, Nb10-63, Nb10-87, Nb11-6, Nb11-72, and Nb11-75, respectively.

[0156] Example 3 Measurement of antibody affinity Bio-layer interferometry (BLI) was used to detect the binding kinetics of 14 blocking TSLP nanoantibodies. For kinetic measurements, candidate antibodies were diluted to 5 μg / mL in PBST buffer, and TSLP-Fc antigen was diluted in PBST buffer at six concentrations (2-fold gradient, starting from 20 nM). The instrument was operated at 30°C with a shaker speed of 1000 rpm. The antibody was captured with a protein A-coated probe for 60 s, then bound to the gradient-diluted antigen. The binding time was 240 s, and the dissociation time was 300 s. The antibody was regenerated twice with 10 mM glycine (pH 1.7), each time for 5 s. Analysis was performed using ForteBio Analysis version 9.0, and the binding rate (Kon), dissociation rate (Kdis), and dissociation constant (KD) were calculated using fitting in global mode. The results are shown in Figure 2.

[0157] Example 4 Expression of a humanized bivalent antibody in Pichia pastoris The candidate antibody was humanized, leaving the variable region intact, and the four framework region sequences were humanized. The modification method was based on the method described in Example 4 of Patent CN2018101517526. The amino acid sequence of the humanized antibody is shown in Table 1.

[0158] [Table 1]

[0159] The above humanized antibodies were constructed in a bivalent form and linked with a (G4S)4 linker. The linked sequences are shown in SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42. They were then expressed in Pichia pastoris. The expression method was outlined as follows: (1) The above TSLP nanoantibody dimer sequences were constructed in the pPICZaA vector. (2) They were linearized with Sac I restriction enzyme and then electrotransfected into X-33-sensitive cells. (3) The electrotransfected samples were plated onto bleomycin-resistant YPD plates at different concentrations and cultured in an incubator at 30°C for 3-4 days. (4) After single clones were formed on the plates, single clones from the plates at different concentrations were selected and placed in BMGY medium. When the OD value of the BMGY medium reached approximately 20, the cells were harvested, transferred to BMMY medium, and cultured at 28°C and 250 rpm. (5) Sampling was then performed every 24 hours, and methanol was added to a final volume of 1%. The samples were centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected and stored at -20°C. Induction was continued for 5 consecutive days, and the culture was terminated. (6) The resulting supernatant samples were subjected to SDS-PAGE analysis. The expression levels of the bivalent single-domain antibodies in the shaking culture are shown in Figure 3.

[0160] Example 5 Detection of blocking activity of humanized bivalent antibodies by ELISA The blocking activity of these purified bivalent humanized antibodies was detected by ELISA. Human TSLP antigen protein was aliquoted into a 96-well plate and incubated overnight at 4°C. After washing five times with PBST, 300 μL of 1% BSA blocking solution was added and incubated at 37°C for 2 hours. After washing five times with PBST, 50 μL of gradient-diluted antibody sample was added, followed by 50 μL of 0.02 μg / mL biotinylated TSLPR protein per well and incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of SA-HRP (diluted 1:100,000) was added and incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of TMB staining solution was added and incubated at 37°C for 10 minutes. The reaction was stopped by adding 50 μL of 2M H2SO4 per well, and the absorbance was measured at 450 nm using a microplate reader. As shown in Figure 4, the blocking activity of the three humanized bivalent antibodies was significantly better than that of the control antibody tezepelumab.

[0161] Example 6 Inhibitory effect of humanized bivalent antibodies on the pSTAT5 signal pathway in BaF3 / TSLPR-IL7R cells Well-grown BaF3 / TSLPR-IL7R cells were centrifuged at 1000 rpm for 5 minutes, resuspended in PBS, and plated at 1x105 cells / well in a 96-well plate. 25μL of diluted human TSLP factor was mixed with gradient-diluted bivalent humanized antibodies or tezepelumab and incubated at 37°C for 30 minutes. The mixture was then added to the cells and incubated at 37°C, 5% CO2 for 20 minutes. After washing with PBS, the cells were fixed with formaldehyde and further incubated with pre-chilled formaldehyde for 10 minutes. Finally, the cells were incubated with anti-phosphorylated STAT5 PE-labeled antibody for 30 minutes. After washing, the cells were loaded onto a flow cytometer and the PE signal was detected. As shown in Figure 5, the inhibitory effects of two of the humanized antibodies on the pSTAT5 signaling pathway in BaF3 / TSLPR-IL7R cells were significantly greater than those of the control antibody tezepelumab.

[0162] Example 7: Evaluation of the yield of a humanized bivalent antibody in a 7 L fermentation tank Glycerol stocks of the above three antibody-expressing strains were amplified at a ratio of 1:100 to obtain primary seeds, which were then transferred to fresh medium for secondary seed culture. Once the secondary seed culture passed, they were inoculated into a 7L fermentation tank for fermentation. Ammonia water was automatically added and the fermentation pH was adjusted to 6.0. During the fermentation process, the pH, wet cell weight, and OD of the fermentation liquid were monitored periodically. The agitation speed, tank pressure, and oxygen introduction rate were adjusted according to changes in the dissolved oxygen level. Glycerol-supplemented medium and methanol-supplemented medium were added at different stages of fermentation according to changes in the wet cell weight and dissolved oxygen level. Fermentation was terminated after induction with methanol for 160–200 h. The yields of the above three humanized bivalent antibodies were measured and found to be 17 g / L, 19 g / L, and 23 g / L, respectively. These antibody yields far exceeded all previously reported fermentation expression yields of antibodies, reaching the industry's highest level.

[0163] Sequence information: SEQ ID NO:1: GFTLDDSDMG SEQ ID NO:2: ISSLGGT SEQ ID NO:3: APGTDRYSDCPNEYSV SEQ ID NO:4: QVQLQESGGGSVQAGGSLRLSCTAS SEQ ID NO:5: WYRQAPGDECELVST SEQ ID NO:6: YYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYC SEQ ID NO:7: WGQGTQVTVSS SEQ ID NO:8: QVQLQESGGGSVQAGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTDRYSDCPNEYSVWGQGTQVTVSS SEQ ID NO:9: CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTTGGATGATTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGGATGAGTGCGAGTTGGTCTCAACTATTAGTAGTTTGGGTGGCACATACTATGCAGAC TCCGTGAAGGGCCGATTCACCATCTCCCATGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTCACGACACGGCCGTGTATTACTGTGCGCGGGGACAGACCGTTATAGCGACTGCCCTAATGAGTATAGCGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO:10: EVQLLESGGGLVQPGGSLRLSCTAS SEQ ID NO:11: WGQGTLVTVSS SEQ ID NO:12: EVQLLESGGGLVQPGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTDRYSDCPNEYSVWGQGTLVTVSS SEQ ID NO: 13: GAGGTTCAATTGTTGGAATCTGGTGGTGGTTTGGTTCAACCAGGTGGTTCTTTGAGATTGTCTTGTACTGCTTCTGGTTTCACCTTGGACGATTCTGATATGGGATGGTACAGACAAGCTCCAGGAGATGAGTGTGAGTTGGTTTCTACTATCTCTTCCTTGGGTGGAACCTACTACGCTGAT TCTGTCAAGGGTCGTTTCACTATTTCTCACGATAATGCTAAGAACACCGTTTACTTGCAAATGAACTCTTTAAAGCCACATGATACTGCCGTTTACTACTGTGCTCCTGGTACTGATAGATACTCTGACTGTCCAAACGAATACTCCGTTTGGGGTCAGGGTACTTTGGTTACTGTCTCTTCC SEQ ID NO: 14: GFTFDDSDMG SEQ ID NO: 15: ISSDGMT SEQ ID NO: 16: AATKYSSDYDVAEDWRRGVCGDMDY SEQ ID NO:17: QVQLQESGGGSVQAGETLRLSCTAS SEQ ID NO: 18: WYRQAPGNECELVSI SEQ ID NO: 19: YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYC SEQ ID NO:20: WGKGTQVTVSS SEQ ID NO:21: QVQLQESGGGSVQAGETLRLSCTASGTFDDDSDMGWYRQAPGNECELVSIISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTQVTVSS SEQ ID NO:22: CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGAGACTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTTTGATGATTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGAATGAGTGCGAGTTGGTCTCAATTATTAGTAGTGATGGTATGACACTACTATGCAGACTCCGTGAAGGGCC GATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTGTATTACTGTGCGGCGACGAAGTACTCCAGCGACTATGACGTAGCTGAGGATTGGAGACGCGGGGTCTGTGGAGACATGGACTACTGGGGCAAAGGAACCCAGGTCACGTCTCCTCA sequence number 23: EVQLLESGGGLVQPGETLRLSCTAS sequence number 24: WGKGTLVTVSS sequence number 25: EVQLLESSGGGLVQPGETLRLSCTASGFTFDDSMGWYRQAPGNECELVSIISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTLVTVSS sequence number 26: GAAGTCCAATTGCTTGAATCCGGTGGTGGATTAGTTCAACCAGGTGAGACCTTGAGACTGTCCTGTACCGCTTCCGGTTTCACTTTCGACGACTCCGACATGGGTTGGTACAGACAGGCTCCAGGTAATGAGTGTGAGTTGGTTTCTATTATTTCCTCTGATGGTATGACTTACTACGCTGATTCTGTTAAGGGTA GATTCACTATCTCTCAAGACAATGCTAAGAACACTGTTTACTTGCAAATGAACTCTTTGAAGCCTGAAGATACCGCCGTCTACTACTGTGCTGCCACCAAGTACTCCTCCGATTATGATGTCGCTGAAGATTGGAGAAGAGGAGTTTGTGGAGATATGGATTACTGGGGTAAAGGTACTTTGGTTACCGTTTCTTCT SEQ ID NO:27: GFTSGGSDMG SEQ ID NO:28: ISSDGST SEQ ID NO:29: AATDYGLGPPPSSTGQCYGMDY SEQ ID NO:30: QVQLQESGGGSVQAGGSLRLSCTAS SEQ ID NO:31: WYRQAPGNECDLVSY SEQ ID NO:32: YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYC SEQ ID NO:33: WGKGTQVTVSS SEQ ID NO:34: QVQLQESGGGSVQAGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISSDGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATDYGLGPPPSSTGQCYGMDYWGKGTQVTVSS SEQ ID NO:35: CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTCTGGCGGTTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGAATGAGTGCGACTTGGTTCCATATATTAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAG GGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACAGGGCCGTGTATTACTGTGCGGCCACCGACTATGGGTTGGGGCCGCCCCCTTCTTCGACGGGCCAATGTTACGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCCA sequence number 36: EVQLLESGGGLVQPGGSLRLSCTAS sequence number 37: WGKGTLVTVSS sequence number 38: EVQLLESSGGGLVQPGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISSDGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYCAATDYGLGPPSSTGQCYGMDYWGKGTLVTVSS sequence number 39: GAAGTTCAGTTGTTGGAATCAGGTGGTGGTTTGGTTCAACCAGGAGGTTCTCTGAGATTGTCTTGTACTGCTTCCGGTTTCACTTCCGGAGGTTCTGACATGGGATGGTACCGTCAAGCTCCTGGTAACGAGTGCGACTTGGTTTCTTACATCTCTTCCGACGGTTCCACTTACTACGCTGATTCTGTTAAG GGTAGATTCACTATTTCTCAAGACAACGCTAAGAATACTGTTTACTTGCAGATGAACTCTTTGAAGCCAGAGGATACCGCTGTTTACTATTGTGCCGCCACTGATTACGGTTTGGGTCCTCCACCATCTTCTACTGGACAATGTTACGGTATGGATTACTGGGGTAAAGGTACCCTGGTCACCGTTTCCTCT sequence number 40: EVQLLESSGGGLVQPGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTDRYSDCPNEYSVWGQGTLVTVSSGGGGGSGGGSGGGGSGGGGSEVQLLESSGGGLVQPGGSLRLSCTASGFTLDDSDMGWYRQAPGDECELVSTISSLGGTYYADSVKGRFTISHDNAKNTVYLQMNSLKPHDTAVYYCAPGTRYSDCPNEYSVWGQGTLVTVSS sequence number 41: EVQLLESSGGGLVQPGETLRLSCTASGFTFDDSMGWYRQAPGNECELVSIISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTLVTVSSGGGGSGGGGS GGGGSGGGGSEVQLLESSGGGLVQPGETLRLSCTASGFTFDDSMGWYRQAPGNECELVSIISSDGMTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYCAATKYSSDYDVAEDWRRGVCGDMDYWGKGTLVTVSS SEQ ID NO:42: EVQLLESGGGLVQPGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISSDGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATDYGLGPPPSSTGQCYGMDYWGKGTLVTVSSGGGGSGGGGS GGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCTASGFTSGGSDMGWYRQAPGNECDLVSYISSDGSTYYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYCAATDYGLGPPPSSTGQCYGMDYWGKGTLVTVSS

[0164] All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. After reading the above content of the present invention, it should be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalents thereof are within the scope of the claims of the present invention.

Claims

1. An anti-TSLP nanobody, characterized in that the CDR region, which is the complementarity determining region of the VHH chain in said nanobody, consists of CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO:

3.

2. The anti-TSLP Nanobody according to claim 1, characterized in that the VHH chain of said anti-TSLP Nanobody further comprises a framework region FR, said framework region FR being one or more selected from the group consisting of: (1) FR1 represented by SEQ ID NO: 4: FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO: 7; (2) FR1 represented by SEQ ID NO: 10, FR2 represented by SEQ ID NO: 5, FR3 represented by SEQ ID NO: 6, and FR4 represented by SEQ ID NO:

11.

3. The anti-TSLP nanobody according to claim 1, characterized in that the amino acid sequence of the VHH chain of said anti-TSLP nanobody is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, or a combination thereof.

4. An anti-TSLP antibody, characterized by comprising the anti-TSLP nanobody according to claim 1, which is an antibody against a TSLP epitope.

5. An anti-TSLP antibody, characterized in that it comprises one or more anti-TSLP nanobodies according to claim 1.

6. The antibody of claim 4 or 5, wherein the antibody comprises a monomeric, bivalent, and / or multivalent antibody.

7. A polynucleotide encoding a protein selected from the group consisting of the anti-TSLP nanobody of claim 1, the anti-TSLP antibody of claim 4 or the anti-TSLP antibody of claim 5.

8. An expression vector comprising the polynucleotide of claim 7.

9. A host cell comprising the expression vector of claim 8.

10. 1. A method for producing an anti-TSLP nanobody, characterized in that the method comprises the steps of: (a) culturing the host cell of claim 9 under conditions suitable for the production of Nanobodies. to obtain a culture comprising said anti-TSLP nanobody; (b) isolating or recovering said anti-TSLP Nanobody from said culture; and (c) purifying and / or modifying the anti-TSLP nanobody obtained in step (b).

11. An immunoconjugate characterized in that it comprises: (a) an anti-TSLP nanobody according to claim 1, an anti-TSLP antibody according to claim 4 or an anti-TSLP antibody according to claim 5; and (b) a conjugate moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a magnetic nanoparticle, a viral coat protein or a VLP, or a combination thereof.

12. A multispecific antibody, characterized in that it comprises an anti-TSLP nanobody according to claim 1, an anti-TSLP antibody according to claim 4 or an anti-TSLP antibody according to claim 5.

13. A recombinant protein characterized in that it comprises: (i) an anti-TSLP nanobody according to claim 1, an anti-TSLP antibody according to claim 4 or an anti-TSLP antibody according to claim 5; and (ii) A tag sequence to aid in expression and / or purification.

14. A pharmaceutical composition comprising: (i) an anti-TSLP nanobody according to claim 1, an anti-TSLP antibody according to claim 4 or an anti-TSLP antibody according to claim 5; and (ii) A pharmaceutically acceptable carrier.

15. Use of the anti-TSLP antibody of claim 1, the anti-TSLP antibody of claim 4 or the anti-TSLP antibody of claim 5, characterized in that the use is for (a) the manufacture of a drug for preventing and / or treating a disease associated with TSLP, and / or (b) the manufacture of a reagent, detection plate or kit for detecting TSLP.

16. 1. A method for detecting TSLP protein in a sample, comprising the steps of: (1) contacting a sample with the anti-TSLP nanobody of claim 1, the anti-TSLP antibody of claim 4 or the anti-TSLP antibody of claim 5; (2) detecting the formation of an antigen-antibody complex, where the formation of the complex indicates the presence of TSLP protein in the sample;

17. 1. A detection reagent for TSLP protein, characterized in that it comprises: (i) an anti-TSLP nanobody according to claim 1, an anti-TSLP antibody according to claim 4 or an anti-TSLP antibody according to claim 5; and (ii) A vector that is permissive for detection.

18. A kit for detecting TSLP protein, comprising the immune complex of claim 7 and an instruction manual.

19. Use of the immunoconjugate according to claim 11 for the manufacture of an imaging agent for detecting TSLP protein in the body.

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