A shark nano-antibody fusion protein against TSLP and its production method, detection kit and application

Abstract

The present invention relates to the field of biological detection. The present invention provides an anti-TSLP shark nano-antibody fusion protein and its production method, detection kit and application. The present invention also provides a CBA kit and a detection method comprising the shark nano-antibody fusion protein. Specifically, the CBA kit of the present application includes a pair of antibody fusion proteins, which are used as coating antibodies and detection antibodies in the CBA method, respectively. The kit provided in the present application can complete the quantitative detection of TSLP protein, has high specificity and detection sensitivity, and has good development and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an anti-TSLP shark nano-antibody fusion protein and a production method, a detection kit and application thereof. Background Art

[0002] Thymic Stromal Lymphopoietin (TSLP) is a type I cytokine similar to IL-7. It is composed of a short chain structure of four helical bundles and is mainly secreted by thymic stromal cells and epithelial cells, and a small amount is secreted by mast cells and macrophages. TSLP plays a key role in balancing innate immunity and adaptive immunity, especially in initiating and promoting allergic inflammation mediated by Th2 cells. In addition, TSLP also shows an important role in the pathological development of various malignant tumors such as breast cancer, pancreatic cancer, cervical cancer, colorectal cancer, etc. In view of its role in various diseases, TSLP is gradually becoming a new target for the treatment of allergic asthma and other allergic diseases.

[0003] The biological mechanism of TSLP protein is mainly through binding to its receptor complex, a heterodimer composed of TSLP receptor (TSLPR) and IL-7 receptor α (IL-7Rα), activating the downstream STAT5 signaling pathway, thereby inducing the maturation of dendritic cells (DC cells) and the Th2 differentiation of CD4+T cells, and promoting the release of cytokines such as IL-4, IL-5, and IL-13. These cytokines are not only closely related to allergic diseases such as asthma, but also participate in pathological changes in tissues, such as airway remodeling and fibrosis. Through this pathway, TSLP promotes and aggravates the pathological processes of diseases such as asthma, allergic dermatitis, and allergic rhinitis.

[0004] TSLP protein plays a key role in the early upstream activities of the inflammatory response, especially in airway inflammation. Studies have shown that the expression of TSLP in the airways of asthma patients is significantly increased, and its level is closely related to the expression of Th2 cytokines and chemokines, and is directly related to the severity of the disease. TSLP can not only effectively activate mast cells and natural killer T cells (NKT), trigger the release of Th2 cytokines such as IL-13, further aggravating airway inflammation, but also promote DC cells to secrete chemokines TARC and MDC, attract Th2 cells and eosinophils to infiltrate, and initiate and maintain allergic reactions.

[0005] In addition, upregulation of TSLP protein is closely related to a variety of allergic diseases (such as eczema, food allergy and allergic rhinitis). Studies have shown that upregulation of TSLP triggers the activation of the JAK / STAT signaling pathway through the binding of TSLPR to DC cells, ultimately leading to overactivation of Th2 cells and overexpression of corresponding factors (such as IL-4, IL-13 and IL-5), thereby inducing Th2-type inflammatory responses. This series of pathological changes not only promotes the progression of allergic diseases, but is also closely related to the aggravation of the course of chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). In summary, TSLP protein plays a vital role in the pathological development of a variety of immune-related diseases. Its expression in asthma, allergic dermatitis, allergic rhinitis and a variety of malignant tumors is closely related to the progression of the disease. No development technology for kits for TSLP detection has been found in the prior art, so there is an urgent need to develop clinical detection methods for TSLP protein, which will not only help to diagnose these diseases early, but also provide a key reference for the formulation of targeted therapeutic strategies and provide a scientific basis for precision medicine.

[0006] Shark single-domain antibodies (VNARs, Variable New Antigen Receptors) exhibit high thermal stability and high acid stability due to their unique structure and physiological properties, making them potential for application in multiple special application scenarios.

[0007] Therefore, there is a need in the art to develop shark nanobody fusion proteins for detecting TSLP. Summary of the invention

[0008] The purpose of the present invention is to provide a method for detecting TSLP protein with high sensitivity, wide detection range and high specificity.

[0009] The present invention provides an anti-TSLP shark nano-antibody fusion protein and a fusion protein combination.

[0010] The present invention provides a detection kit comprising an anti-TSLP shark nano-antibody fusion protein combination and application thereof.

[0011] In the first aspect of the present invention, an anti-TSLP shark nanobody fusion protein is provided, wherein the fusion protein comprises a shark nanobody variable region (VNAR) portion located at the N-terminus and an IgG1 Fc fragment located at the C-terminus, wherein the VNAR comprises a complementary determining region CDR and a hypervariable loop HV selected from the following group:

[0012] (1) CDR1 shown in SEQ ID NO: 4; CDR3 shown in SEQ ID NO: 7; HV2 shown in SEQ ID NO: 5; and HV4 shown in SEQ ID NO: 6; and

[0013] (2) CDR1 shown in SEQ ID NO: 8; CDR3 shown in SEQ ID NO: 10; HV2 shown in SEQ ID NO: 9; and HV4 shown in SEQ ID NO: 6.

[0014] In another preferred embodiment, the positions of CDR1, HV2, HV4 and CDR3 in the VNAR are determined based on the IMGT encoding rules.

[0015] In another preferred embodiment, the variable region includes a framework region (FR).

[0016] In another preferred embodiment, the amino acid sequence of the VNAR is as shown in any one of SEQ ID NO: 1 or 2, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99% thereto.

[0017] In another preferred embodiment, the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 3.

[0018] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain the ability to specifically bind to TSLP.

[0019] In another preferred embodiment, the derivative sequence that has been added, deleted, modified and / or substituted at least one amino acid and can retain the ability to specifically bind to TSLP is an amino acid sequence with a homology or sequence identity of at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0020] In another preferred embodiment, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 11 or 12.

[0021] In the second aspect of the present invention, a polynucleotide is provided, which encodes the shark nanobody fusion protein as described in the first aspect of the present invention.

[0022] In another preferred embodiment, the polynucleotide may be RNA, DNA or cDNA.

[0023] In the third aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide as described in the second aspect of the present invention.

[0024] 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. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or combinations thereof.

[0025] In another preferred embodiment, the expression vector further includes a member selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.

[0026] In the fourth aspect of the present invention, a host cell is provided. The host cell contains the expression vector as described in the third aspect of the present invention, or the polynucleotide as described in the second aspect of the present invention is integrated into its genome.

[0027] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0028] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0029] In a fifth aspect of the present invention, an anti-TSLP shark nanobody fusion protein combination is provided, the combination comprising fusion proteins S1 and S2, the fusion protein comprising a shark nanobody variable region (VNAR) portion at the N-terminus and an IgG1 Fc fragment at the C-terminus,

[0030] Wherein, the VNAR of the fusion protein S1 comprises:

[0031] CDR1 shown in SEQ ID NO: 4; CDR3 shown in SEQ ID NO: 7; HV2 shown in SEQ ID NO: 5; and HV4 shown in SEQ ID NO: 6;

[0032] The VNAR of the fusion protein S2 comprises:

[0033] CDR1 shown in SEQ ID NO: 8; CDR3 shown in SEQ ID NO: 10; HV2 shown in SEQ ID NO: 9; and HV4 shown in SEQ ID NO: 6.

[0034] In another preferred embodiment, the amino acid sequence of VNAR of the fusion protein S1 is shown in SEQ ID NO:1.

[0035] In another preferred embodiment, the amino acid sequence of VNAR of the fusion protein S2 is shown in SEQ ID NO:2.

[0036] In another preferred embodiment, the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 3.

[0037] In another preferred embodiment, the amino acid sequence of the fusion protein S1 is shown in SEQ ID NO: 11.

[0038] In another preferred embodiment, the amino acid sequence of the fusion protein S2 is shown in SEQ ID NO: 12.

[0039] In a sixth aspect of the present invention, a method for producing a shark nanobody fusion protein is provided, comprising the steps of:

[0040] (a) culturing the host cell according to the fourth aspect of the present invention under conditions suitable for producing shark nanobody fusion protein, thereby obtaining a culture containing shark nanobody fusion protein;

[0041] (b) isolating and / or recovering the shark nanobody fusion protein from the culture; and

[0042] (c) Optionally, purifying and / or modifying the shark nanobody fusion protein obtained in step (b).

[0043] In the seventh aspect of the present invention, there is provided a use of an active ingredient, wherein the active ingredient is selected from the following group: the shark nanobody fusion protein as described in the first aspect of the present invention, the polynucleotide as described in the second aspect of the present invention, the expression vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, or the shark nanobody fusion protein combination as described in the fifth aspect of the present invention, or a combination thereof, wherein the active ingredient combination is used to prepare:

[0044] (a) drugs for preventing and / or treating diseases with high TSLP expression; and / or

[0045] (b) Reagents for detecting diseases with high TSLP expression.

[0046] In another preferred embodiment, the reagent is a diagnostic reagent, preferably, the diagnostic reagent is a detection sheet or a detection plate.

[0047] In another preferred embodiment, the diagnostic reagent is used to detect TSLP protein or a fragment thereof in a sample.

[0048] In another preferred embodiment, the TSLP high expression disease is selected from: inflammatory diseases, autoimmune diseases, tumors, or a combination thereof.

[0049] In another preferred embodiment, the TSLP high expression disease is an allergic disease.

[0050] In another preferred embodiment, the allergic disease is selected from the group consisting of asthma, atopic dermatitis, allergic conjunctivitis, or a combination thereof.

[0051] In the eighth aspect of the present invention, a TSLP protein detection kit is provided, the kit comprising the shark nanobody fusion protein as described in the first aspect of the present invention, or the shark nanobody fusion protein combination as described in the fifth aspect of the present invention.

[0052] In another preferred embodiment, the detection kit is used for double antibody sandwich detection.

[0053] In another preferred embodiment, the detection kit is used for CBA detection, ELISA detection and / or colloidal gold detection.

[0054] In another preferred embodiment, the kit is a CBA kit.

[0055] In another preferred embodiment, the detection kit comprises the fusion protein combination as described in the fifth aspect of the present invention, wherein the fusion protein S1 is fixed to a carrier and used to capture the TSLP protein, and the fusion protein S2 is coupled to a detection marker.

[0056] In another preferred embodiment, the detection kit comprises microspheres (such as fluorescent microspheres), and the microspheres are coated with the fusion protein S1.

[0057] In another preferred embodiment, the detection kit includes fusion protein S2 with biotin.

[0058] In another preferred embodiment, the detection kit further includes a TSLP protein standard.

[0059] In another preferred embodiment, the detection kit further comprises a component selected from the following group: fluorescent protein-labeled streptavidin, freeze-dried TSLP standard, washing solution, sample diluent, matrix buffer, or a combination thereof.

[0060] In another preferred embodiment, the fluorescent protein is phycoerythrin.

[0061] In the ninth aspect of the present invention, a method for detecting TSLP in a sample is provided, using the shark nanoantibody fusion protein as described in the first aspect of the present invention, or the shark nanoantibody fusion protein combination as described in the fifth aspect of the present invention, or the detection kit as described in the eighth aspect of the present invention for detection.

[0062] In another preferred embodiment, the method comprises the following steps:

[0063] 1) Immobilize the fusion protein S1 on the carrier;

[0064] 2) labeling the fusion protein S2 with a detectable marker;

[0065] 3) incubating the antibodies S1 and S2 with the sample to be tested, so that the antibodies S1 and S2 bind to TSLP in the sample;

[0066] 4) washing away the reagent not bound to the carrier, detecting the detectable marker, and thus detecting TSLP.

[0067] In another preferred embodiment, the detection is quantitative detection.

[0068] In another preferred embodiment, the carrier is a microsphere, preferably a fluorescent microsphere.

[0069] In another preferred embodiment, the detectable marker is biotin.

[0070] In another preferred embodiment, step 4) comprises the steps of:

[0071] Avidin labeled with fluorescent protein is added and incubated to connect the biotin on the fusion protein S2, and the fluorescent protein is detected.

[0072] In another preferred embodiment, the avidin is streptavidin.

[0073] In another preferred embodiment, the fluorescent protein is phycoerythrin.

[0074] In another preferred embodiment, the method further comprises the steps of: detecting a TSLP standard to obtain a standard curve, and calculating the content of TSLP in the sample to be tested according to the standard curve.

[0075] In another preferred embodiment, the sample to be tested is selected from the following group: cell culture fluid, whole blood, serum, plasma, body fluid, or a combination thereof.

[0076] In the tenth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0077] (i) the shark nanobody fusion protein as described in the first aspect of the present invention, or the shark nanobody fusion protein combination as described in the fifth aspect of the present invention, as an active ingredient; and

[0078] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0079] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized agent.

[0080] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the shark nanobody fusion protein as described in the first aspect of the present invention, or the shark nanobody fusion protein combination as described in the fifth aspect of the present invention and 0.01 to 99.99% of a pharmaceutically acceptable carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0081] The technical effect of the present invention is: providing antibodies and antibody pairs that specifically target TSLP protein, which have high sensitivity, high accuracy and high specificity when used in CBA kits to detect TSLP protein.

[0082] It should be 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 (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The following drawings are used to illustrate specific embodiments of the present invention, but are not used to limit the scope of the present invention.

[0084] Figure 1 Shown is the result of SDS-PAGE identification of TSLP-VNAR-Fc nanobody protein purity.

[0085] Figure 2 The binding results of shark nanoantibody conjugates 1# and 2# with antigens human IL-7, IL-2, TSLPR, IL-4, IL-5, and IL-13 are shown.

[0086] Figure 3 The standard curve of human TSLP concentration detection is shown. The horizontal axis is the LOG10 value of the calibrator concentration, and the vertical axis is the LOG10 value of the fluorescence value. DETAILED DESCRIPTION

[0087] After extensive and in-depth research, the inventors have developed for the first time a shark nano-antibody fusion protein against TSLP and its production method, detection kit and application. Specifically, the present invention provides a TSLP shark nano-antibody conjugate and a TSLP protein CBA detection kit comprising the antibody conjugate. The kit of the present invention has higher sensitivity and a wider detection range. The kit also has extremely high specificity, can avoid interference from human IL-7, IL-2, TSLPR, IL-4, IL-5, and IL-13, makes the detection more accurate, and has good development and application prospects. On this basis, the present invention was completed.

[0088] the term

[0089] In order to more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive.

[0090] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0091] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0092] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic response), ie, has a reasonable benefit / risk ratio.

[0093] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. It should be understood by those skilled in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs.

[0094] TSLP

[0095] In the present specification, the term "thymic stromal lymphopoietin (TSLP)" refers to a type I cytokine similar to IL-7, which consists of a short chain structure composed of four helical bundles, is mainly secreted by thymic stromal cells and epithelial cells, and is secreted in small amounts by mast cells and macrophages, with a molecular weight of about 15-20 kD. The natural full-length or long-form human TSLP protein is easily enzymatically cleaved, for example, by the PCSK3 enzyme. The PCSK3 enzyme can specifically target positions 127 to 130 of the long-form TSLP protein and truncate the protein, wherein the amino acid numbering is based on the sequence of the full-length human TSLP protein. In order to prevent such enzymatic cleavage and stabilize TSLP in its long form, certain mutations can be introduced into the enzymatic cleavage site (such as R127 and R130). Therefore, in the present invention, TSLP with R127A and R130A mutations (e.g., SEQ ID NO: 15) is used, and the amino acid sequence is shown in SEQ ID NO: 15.

[0096] SEQ ID No:15:

[0097] YDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKARKAKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ

[0098] The present invention provides an antibody, a detection kit and a detection method for detecting TSLP (especially TSLP protein).

[0099] Antibody

[0100] In 1995, Flajnik discovered a natural heavy chain antibody in cartilaginous fishes, the antibody new antigen receptor (IgNAR). IgNAR is a unique antibody that has been found in several different types of sharks, including nurse sharks, wobbegong sharks, bamboo sharks, striped hound sharks, and dogfish sharks. IgNAR is different from traditional antibodies in that it is a dimer composed of two heavy chains, each of which consists of five constant regions, a hinge region, and a variable region.

[0101] In this application, the term "shark nanobody" or "shark antibody" refers to an antibody new antigen receptor, i.e., IgNAR. The variable region of IgNAR, or VNAR, contains only two complementary determining regions (CDRs), i.e., CDR1 and CDR3. Due to the absence of CDR2, VNARs are the smallest naturally occurring immunoglobulins to date, with a molecular weight of approximately 12 kDa. VNAR may also contain two hypervariable loops (HV) HV2 and HV4. Through the grooves on the hypervariable loops and the smaller size, VNAR can enter the grooves that general antibodies cannot enter, bind to antigens, and have a strong antigen affinity. VNAR has the advantages of strong tissue penetration, higher stability, recognition of antigen hidden epitopes, and the ability to develop new targets when treating tumors. Therefore, based on the above advantages, the combination of VNAR with antibodies, cytokines, CAR-NK, etc. has great prospects for drug development.

[0102] In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The intensity or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0103] In the present application, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0104] In the present application, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. Conservative amino acid substitutions include substitutions that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). These conservative substitutions are preferably generated by amino acid replacement according to Table 1 below.

[0105] Table 1 Amino acid residue substitution

[0106]

[0107] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0108] As used herein, the term "vector", "expression system" or "expression vector" refers to a nucleic acid sequence containing a desired coding sequence and a control sequence in an operable connection so that a host transformed with these sequences can produce the encoded protein. To achieve transformation, the expression system can be included on a vector; however, the relevant nucleic acid molecule can also be subsequently integrated into the host chromosome.

[0109] As used herein, the term "host cell" is a cell that can support the replication or expression of an expression vector. The host cell can be a prokaryotic cell such as E. coli, or a eukaryotic cell such as a yeast cell, an insect cell, an amphibian cell, or a mammalian cell.

[0110] As used herein, the terms "transfection", "stable transfection" or "transient transfection" refer to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed. A variety of transfection methods are known to those skilled in the art. For example, transfection is accomplished in the presence of an expression vector and a high concentration of calcium phosphate, by electroporation, by using a bacteriophage or viral expression vector to insert into a host cell, by mechanical insertion of nucleic acids, or even by culturing host cells in the presence of unpackaged nucleic acid fragments. Successful transfection is usually confirmed when any indication of manipulation of the vector of interest appears in the host cell.

[0111] Shark Nanobody Fusion Protein

[0112] As used in the present invention, the terms "shark nanobody fusion protein", "shark nanobody conjugate" and "fusion protein" are used interchangeably and all refer to the isolated fusion protein containing shark nanobody VNAR and IgG Fc fragment provided in the first aspect of the present invention.

[0113] The VNAR region in the anti-TSLP shark nanobody fusion protein of the present invention comprises a complementary determining region CDR and a hypervariable loop HV selected from the following group:

[0114] (1) CDR1 shown in SEQ ID NO: 4; CDR3 shown in SEQ ID NO: 7; HV2 shown in SEQ ID NO: 5; and HV4 shown in SEQ ID NO: 6;

[0115] (2) CDR1 shown in SEQ ID NO: 8; CDR3 shown in SEQ ID NO: 10; HV2 shown in SEQ ID NO: 9; and HV4 shown in SEQ ID NO: 6.

[0116] In another preferred embodiment, the amino acid sequence of the VNAR is as shown in any one of SEQ ID NO: 1 or 2.

[0117] In another preferred embodiment, the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 3.

[0118] In another preferred embodiment, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 11 or 12.

[0119] The term "CDR3" refers to the TCR region that directly contacts the antigen. The CDR3 sequence of shark single-domain antibodies is longer than that of IgG, usually 8-18 amino acids.

[0120] In this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art.

[0121] The shark nanobody conjugate of the present application does not bind to proteins that are unrelated to the target. Here, "unrelated proteins" refer to proteins other than the TSLP protein as the target; here, "no binding" means: when the binding ability of the shark nanobody conjugate of the present invention to the TSLP protein as its target is taken as 100%, the binding ability of the shark nanobody conjugate of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0122] The shark nanobody conjugate of the present application does not bind to TSLP of other animal species. Here, "other animal species" refers to animal species other than humans, such as rhesus monkeys, crab-eating monkeys, rats, mice, etc.; here, "no binding" means: when the binding ability of the shark nanobody conjugate of the present invention to the TSLP protein as its target is taken as 100%, the binding ability of the shark nanobody conjugate of the present invention to TSLP of other animal species is less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0123] CBA

[0124] Cytometric Bead Array (CBA), also known as cytokine microsphere detection technology, is a multiplex protein quantitative detection method based on a flow cytometric detection system, which can simultaneously detect multiple indicators in a single sample. BD uses the characteristic of flow cytometers to amplify fluorescent signals in series, attaches the soluble factors to be tested to some microparticles with a diameter similar to that of cells, and can detect various soluble factors in the tested samples. The basic principle of CBA is similar to that of ELISA, that is, using tiny, dispersed particles to capture liquid analytes, and using flow cytometry to detect the fluorescence emitted by the "sandwich"-like particles-the analyte complex, thereby determining the amount of the analyte.

[0125] Each CBA microsphere is of uniform size, has a specific fluorescence intensity, and is coated with a specific capture antibody (Capture Antibody) suitable for specific analysis (such as other antibodies or soluble proteins), providing a capture surface similar to an ELISA plate. When the microspheres and the sample solution to be tested are mixed, the specific antibodies on the microspheres bind to the corresponding antigens or proteins in the sample (serum, plasma or cell culture fluid), and then the fluorescently labeled detection antibodies are added to form a "sandwich" complex. Finally, the specific target protein is detected by flow cytometry. Each CBA microsphere carries red fluorescence of different intensities. The target protein is qualitatively determined by detecting the difference in the fluorescence intensity of the microspheres in the FL3 channel of the flow cytometer; the detection antibody is labeled with PE fluorescein and detected in the FL2 channel of the flow cytometer, and the target protein is quantified by the PE fluorescence intensity of the detection antibody.

[0126] In this specification, "avidin" is a glycoprotein, each molecule of which is composed of 4 subunits and can bind closely to 4 biotin molecules. Streptavidin extracted from Streptomyces is more commonly used.

[0127] In this specification, biotin is a small molecule growth factor widely distributed in animals and plants, also known as coenzyme R or vitamin H. Avidin is an alkaline glycoprotein extracted from ovalbumin, which has a very high affinity for biotin. The avidin molecule combined with the enzyme reacts with the biotin molecule combined with the specific antibody, which not only plays a multi-stage amplification role, but also develops color due to the catalytic action of the enzyme when encountering the corresponding substrate, thereby achieving the effect of measuring the amount of antibody bound to it. Therefore, combining avidin and biotin with CBA can greatly improve the sensitivity of CBA. The application of biotin-avidin system in CBA has various forms, which can be used for indirect coating and can also be used for final reaction amplification. The enzyme-labeled antibody in conventional CBA can also be replaced by a biotinylated antibody, and then connected to an avidin-enzyme conjugate to amplify the reaction signal.

[0128] Detection Kits

[0129] The present invention provides a detection kit for detecting TSLP protein, which comprises the shark nanobody fusion protein or shark nanobody fusion protein combination of the present invention.

[0130] In one embodiment, the detection kit uses a double antibody sandwich method (sandwich method) for detection. The detection method of the present invention can use CBA detection.

[0131] In a specific embodiment, the kit of the present application includes a first antibody and a second antibody, wherein the first antibody is a coated antibody, which has been fixed to a solid phase carrier (such as a microsphere); the second antibody is a detection antibody, which is labeled with biotin. Preferably, the microsphere particle size is a fluorescent microsphere. Further preferably, the particle size is 5 μm. In a specific embodiment, polystyrene microspheres of China Nano can be used.

[0132] In a specific embodiment, the first antibody and the second antibody are shark nanobody conjugates of the present application.

[0133] Furthermore, the CBA kit also includes devices or reagents necessary for detecting TSLP.

[0134] Specifically, the CBA kit also includes peroxidase or fluorescent protein labeled streptavidin, TSLP standard, substrate, coating antibody diluent, washing solution, blocking solution / sample diluent and stop solution.

[0135] In a specific embodiment, the fluorescent protein is preferably phycoerythrin (PE), and the streptavidin labeled therewith is a streptavidin-phycoerythrin conjugate (SA-PE).

[0136] In a specific embodiment, the TSLP standard in the kit is human TSLP protein; the coating antibody diluent is phosphate buffered saline (PBS) pH 7.4; the washing solution is phosphate buffered saline (PBS) containing 0.05% Tween20; the blocking solution / sample diluent is phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300.

[0137] Preferably, the CBA kit comprises the following components:

[0138] 1) Microspheres pre-coated with antibodies: shark nanoantibody conjugate 1# (i.e., first antibody), 2.5 mL / tube, 1 tube;

[0139] 2) Detection antibody: shark nanoantibody conjugate 2# (i.e., second antibody) (labeled with biotin), 2.5 mL / tube, 1 tube;

[0140] 3) Standard: lyophilized TSLP protein, 10 ng / tube, 1 tube;

[0141] 4) Streptavidin-phycoerythrin conjugate (SA-PE), 2.5 mL / tube, 1 tube;

[0142] 5) Matrix buffer: 2.5 ml / tube, 1 tube;

[0143] 6) Washing buffer: phosphate buffer (10XPBST), pH 7.4, 5 ml / bottle, 1 bottle;

[0144] Detection Methods

[0145] The present invention also provides a method for detecting TSLP using the antibody or kit of the present invention. The detection method of the present invention can be a double antibody sandwich method. The detection method of the present invention can use CBA detection.

[0146] The present invention forms an antibody-biotin-avidin system (ABAS) by biotin-labeling the shark nano-antibody conjugate with phycoerythrin-labeled streptavidin. The strong binding with high affinity can achieve a multi-stage amplification effect, so that the kit of the present application has higher sensitivity and a wider detection range.

[0147] A specific method is to bind a known amount of a first antibody to the surface of a solid support. The sample to be tested is then applied to the surface so that any cells and / or TSLP substances in the serum present therein are captured by the fixed first antibody. Unbound substances are preferably removed by one or more washing steps. A second antibody labeled with biotin, i.e., a detection antibody, is then added and allowed to bind to any cells and / or TSLP substances in the serum captured by the first antibody, so that each unit of TSLP simultaneously binds to two antibodies to form a "sandwich". The amount of the bound second antibody is then determined in a direct or indirect detection method. Specifically, the label or enzyme can be directly or indirectly linked to the second antibody via a link such as a biotin-streptavidin or biotin-avidin link.

[0148] In one embodiment, the detection method of the present invention comprises the following steps:

[0149] Fluorescent microspheres were coated with shark nanoantibody conjugate 1# (i.e., the first antibody);

[0150] Labeling biotin on shark nanobody conjugate 2# (i.e., the second antibody);

[0151] The microspheres coated with the first antibody, the sample to be tested, and the biotin-labeled second antibody are sequentially added to the flow tube and incubated;

[0152] Add phycoerythrin-labeled streptavidin to the flow tube and incubate;

[0153] The standard curve was fitted using the MFI value of the TSLP standard, and the MFI value of the sample was substituted into the equation to calculate the TSLP content in the sample.

[0154] In a specific embodiment, the method for quantitatively detecting the TSLP protein content in a sample using the CBA kit of the present application comprises the following steps:

[0155] (1) Coating: The coating antibody shark nanobody conjugate 1# (i.e., the first antibody) was prepared into a coating antibody working solution with a concentration of 1 g / L using a coating antibody diluent (phosphate buffered saline (PBS), pH 7.4), and then mixed in clean EP at a ratio of 10 μg / 1E7 microspheres and incubated at room temperature for 2 hours;

[0156] (2) Blocking: Discard the coating antibody working solution in the EP tube by centrifugation, wash twice with washing buffer (phosphate buffered saline (PBS) containing 0.05% Tween20), then add blocking solution (phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300) at a volume of 100 μL / well, and place on a shaker (120 rpm) at room temperature for 2 hours for blocking;

[0157] (3) Preparation of protein standards: Take 7 EP tubes and number them in sequence. Add 150 μL of sample diluent to each tube 1-6 and place them on the EP tube rack. Use the sample diluent to prepare the standard protein (human TSLP protein) into a liquid with a concentration of 10 μg / L. Pipet 50 μL and add it to the first EP tube. Then, pipette 50 μL of liquid from the first EP tube and add it to the second EP tube for four-fold dilution. Repeat this process until the sixth EP tube. The concentrations are 10000 pg / mL, 2500 pg / mL, 625 pg / mL, 156.3 pg / mL, 39.6 pg / mL, 9.7 pg / mL, and 2.4 pg / mL, respectively.

[0158] (4) Sample addition: Add the microspheres coated with the first antibody, protein standard solution, and biotin-labeled second antibody in order at a volume of 25 μL / tube, and incubate at room temperature for 2 hours on a shaker (500 rpm);

[0159] (5) Adding SA-PE: Add 25 μL / tube to EP and incubate on a shaker (500 rpm) for 0.5 h;

[0160] (6) Washing: Discard the reaction solution in the EP tube by centrifugation, wash twice with washing solution, and resuspend the microspheres with sample diluent;

[0161] (7) Detection: The MFI value of each EP tube was measured at a wavelength of 585 nm using a flow cytometer, and a standard curve was fitted based on the MFI value of the standard.

[0162] The antibody, detection kit and detection method of the present application can be used to detect TSLP in cell culture fluid or human serum.

[0163] Pharmaceutical composition

[0164] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0165] The pharmaceutical composition of the present invention may contain any anti-tumor drug (such as an anti-tumor antibody) connected to the shark nanobody fusion protein of the present invention against TSLP protein, and thus can be used to treat tumors. In addition, other therapeutic agents may also be used simultaneously.

[0166] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned nano antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0167] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 10 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0168] The main advantages of the present invention include:

[0169] 1) The present invention provides antibodies and antibody pairs that specifically target TSLP protein, which can be used in CBA kits to detect TSLP protein and can greatly expand the detection range and improve the detection sensitivity. Multiple repeated tests show that it has high accuracy.

[0170] 2) The antibodies and combinations of the present invention do not cross-react with the interference of human IL-7, IL-2, TSLPR, IL-4, IL-5, and IL-13, avoiding the interference of similar cytokines and improving the detection specificity.

[0171] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0172] Example 1 Preparation method of TSLP shark antibody-Fc fusion protein

[0173] This example uses the TSLP shark nanoantibody G12 and H1 sequences obtained in the previous screening (see application number CN202411058132X) to prepare TSLP-VNAR-Fc fusion protein using mammalian cells. Primers HF (ccttaagggcgtgcagtgccaggtgcagctgcaggagtc; SEQ ID NO. 13) and HR (gatttgggctcgctagctgaggagacggtgacctggg; SEQ ID NO. 14) were used to amplify the positive single-chain variable region gene, and the amplification reaction system is as follows:

[0174] Sterilized phagemid template: 0.5 μL

[0175] HF primer (10μM): 1μL

[0176] HR primer (10 μM): 1 μL

[0177] 2 xPhanta Max Master Mix: 15μL

[0178] Deionized water: 12.5 μL

[0179] Total: 30 μL.

[0180] The reaction procedure is as follows:

[0181] 95℃ 3 min

[0182] 95℃ 15s 30 cycles

[0183] 62℃ 15s

[0184] 72℃ 20s

[0185] 72℃ 5min

[0186] Maintain at 4°C.

[0187] The TSLP shark antibody G12 and H1 genes were cloned into the pcDNA3.1(+) vector with human Fc (SEQ ID NO.3) by homologous recombination. The reaction system was as follows, and the reaction conditions were 37°C for 30 min:

[0188] Vector pABG1-Fc (0.02 pmol): 0.4 μL

[0189] PD-L1 target fragment (0.06 pmol): 0.3~3μL

[0190] Exnase II: 1 μL

[0191] 5xCE II buffer: 2 μL

[0192] Sterile deionized water: 3.6~6.3μL

[0193] Total: 10 μL.

[0194] Antibody G12:

[0195] QWVEQTPTTTTKEAGESLTINCVLRDSSCALANTYWYFTKKGAPNKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYNPLLRYACRYEGGGTILTVK (SEQ ID NO: 1)

[0196] Antibody H1:

[0197] ARVEQTPTTTTKEAGESLTINCVLSESSCALDSTSWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYNPLLRYNCRYEGGGTILTVK (SEQ ID NO: 2)

[0198] Amino acid sequence of Fc:

[0199] VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3)

[0200] The plasmid was extracted using endotoxin-free plasmid extraction reagent, quantified by spectrophotometer and sent for sequencing. FreeStyleTM293-F cells were collected by centrifugation one day before transfection and the cell density was adjusted to 1×10 6 cells / mL, transfection ratio: (cell: culture medium = 20:10); Polyethylenimine Lineae (PEI) 2μL / mL; plasmid 1 μg / mL; that is, aspirate 40μg of plasmid and dissolve it in 2 mL of Opti-MEM culture medium, mix gently, then add 80 μL of PEI transfection reagent, vortex for 10s, mix thoroughly, and stand at 25℃ for 13 min; add 2 mL of the mixed solution to 40 mL of FreeStyleTM293-F cells in good condition, mix gently, and culture in a horizontal shaker at 37℃, 120 rpm, and 5% CO2 for 4 days. Collect the supernatant for SDS-PAGE electrophoresis analysis. Based on the protein purification system, ProteinA affinity purification was used, and the sample was eluted and collected with 0.1 M citric acid-sodium citrate buffer. The antibody concentration was quantified by spectrophotometer, and the antibody purity was identified by SDS-PAGE. Figure 1 As shown, obvious bands appeared at the relative molecular mass of 37-50KD, which was consistent with the predicted molecular weight.

[0201] Example 2 Specificity identification of shark nanobody conjugates

[0202] 1. Antibody recognition antigen specificity experiment:

[0203] Dilute human TSLP, IL-7, IL-2, TSLPR, IL-4, IL-5, and IL-13 to 10 μg / mL with coating solution, coat the ELISA plate at 2-8°C overnight, and block at room temperature for 2 hours the next day. After washing the plate, add 100 μL of diluted cell culture supernatant and incubate at room temperature for 1 hour. After washing the plate, add diluted enzyme-labeled secondary antibody and incubate at room temperature for 1 hour. Finally, wash the plate for color development and read the absorbance at OD450nm. The results are as follows: Figure 2 As shown, antibodies 1# and 2# did not bind to human IL-7, IL-2, TSLPR, IL-4, IL-5, and IL-13 antigens, indicating that antibodies 1# and 2# have good specificity for TSLP protein. These antibodies can be paired for experiments. The test results of antibodies 1# and 2# are shown in Table 2.

[0204] Table 2 Antibody specificity detection

[0205]

[0206] 2. Anti-human TSLP antibody pairing experiment

[0207] The two antibodies were biotin-labeled using the Sulfo-NHS-LC- Biotin kit, and the specific steps were strictly followed in accordance with the kit instructions. After labeling, the free biotin was dialyzed using a dialysis bag, and the resulting solution was the biotin-labeled antibody.

[0208] Dilute the two antibodies to 10 μg / mL with coating solution, coat the fluorescent microspheres at 2-8℃, and incubate at room temperature for 2 hours. After centrifugation, add blocking solution and block at room temperature for 2 hours. After washing, add preservation solution and count for later use.

[0209] Add matrix buffer, human TSLP protein, antibody-coated microspheres, and biotin-labeled antibodies in sequence and incubate at room temperature for 2 hours. Then add diluted phycoerythrin-labeled streptavidin and incubate at room temperature for 0.5 hours. Finally, after washing, use a flow cytometer to read the fluorescence value. The results are shown in Table 3 below. Antibody 1# and antibody 2# can be paired for assembling a quantitative detection kit for detecting human TSLPCBA.

[0210] Table 3 Antibody pairing test results

[0211]

[0212] Example 3 Composition of Human TSLP CBA Quantitative Detection Kit

[0213] The antibody 1# was coated with fluorescent microspheres as the capture antibody, and the antibody 2# was biotin-labeled as the detection antibody to prepare a CBA kit. The reagent composition, specifications, sources, storage conditions, etc. in the kit are shown in Table 4.

[0214] 1) Microspheres pre-coated with antibodies: shark nanoantibody conjugate 1# (i.e., first antibody), 2.5 mL / tube, 1 tube;

[0215] 2) Detection antibody: shark nanoantibody conjugate 2# (i.e., second antibody) (labeled with biotin), 2.5 mL / tube, 1 tube;

[0216] 3) Standard: lyophilized human TSLP protein, 10 ng / tube, 1 tube;

[0217] 4) Streptavidin-phycoerythrin conjugate (SA-PE), 2.5 mL / tube, 1 tube;

[0218] 5) Matrix buffer: 5 mL / tube, 1 tube;

[0219] 6) Washing buffer: phosphate buffer (10XPBST), pH 7.4, 5 mL / bottle, 1 bottle;

[0220] The components are shown in Table 4 below:

[0221] Table 4 Composition of CBA kit

[0222]

[0223] Example 4 Evaluation of performance indicators of human TSLP CBA kit

[0224] The human TSLP CBA kit in Example 3 was used to quantitatively detect human TSLP in the quality control sample.

[0225] 1. Linear range:

[0226] (1) Dilute the human TSLP protein with sample diluent to prepare a protein standard, and dilute the human TSLP protein with cell culture medium to prepare a quality control sample as the sample to be tested. Fit a standard curve based on the MFI value of the protein standard. Substitute the MFI value of the sample to be tested into the standard curve to obtain the theoretical concentration value of human TSLP in the sample to be tested, and then calculate the recovery rate of the sample to be tested (recovery rate = theoretical concentration value / actual concentration value * 100%), so as to analyze the quantitative detection effect of the CBA kit in Example 3 for human TSLP in cell culture medium.

[0227] (2) Quantitative detection steps:

[0228] 1) Preparation of protein standards: Take 8 EP tubes and number them in sequence. Add 300 μL of sample diluent to each tube starting from the second tube and place them on the EP tube rack. Use sample diluent to prepare the standard protein to a concentration of 10 ng / mL in the first tube, draw 100 μL and add it to the second EP tube for four-fold dilution, and so on to the seventh EP tube. The concentrations are 10000 pg / mL, 2500 pg / mL, 625 pg / mL, 156.3 pg / mL, 39.6 pg / mL, 9.7 pg / mL, and 2.4 pg / mL.

[0229] 2) Preparation of quality control samples: Take 6 EP tubes, take samples from 10000 pg / mL respectively, and prepare quality control samples of 6250 pg / mL, 1250 pg / mL, 250 pg / mL, 50 pg / mL, 10 pg / mL, and 2 pg / mL;

[0230] 3) Sample addition: Add matrix buffer, protein standard solution, microspheres coated with the first antibody, and biotin-labeled second antibody in order at a volume of 25 μL / tube, and incubate at room temperature for 2 hours on a shaker (500 rpm);

[0231] 4) Add SA-PE: add 25 μL / tube to EP and incubate the reaction on a shaker (500 rpm) for 0.5 h;

[0232] 5) Washing: Discard the reaction solution in the EP tube by centrifugation, wash twice with washing solution, and resuspend the microspheres with sample diluent;

[0233] 6) Detection: Use flow cytometry to measure the MFI value of each EP tube at a wavelength of 585 nm, fit a standard curve based on the MFI value of the standard, and substitute the MFI value of the quality control sample into the equation to calculate the concentration of the quality control sample.

[0234] (3) Results and discussion

[0235] 1) The linear range test data of human TSLP standard is shown in Table 5:

[0236] Table 5 Detection data of human TSLP standard

[0237]

[0238] The detection curve of human TSLP was recovered within the concentration range of 2.4~10000pg / mL.

[0239] The curve equation is Figure 3 Shown: y = 0.0849x2 + 0.485x - 1.0834

[0240] (y: Log of Con; x: Log of MFI), R² = 0.9996.

[0241] 2. Specificity

[0242] (1) Take different cytokines and dilute them to a concentration of 10 ng / mL. Detect their concentrations according to the kit procedure. The specificity of the kit is expressed by the cross-reaction rate (%). The calculation method is: cross-reaction rate (%) = detection concentration / theoretical concentration × 100%.

[0243] (2) The specific detection data of the human TSLP kit are shown in Table 6.

[0244] Table 6 Specificity of human TSLP kit detection

[0245]

[0246] (3) Results and discussion:

[0247] The results in Table 6 show that the cross-reaction rate of related cytokines is ≤0.03%. The above results show that when the human TSLP CBA kit provided by the present application is used to quantitatively detect human TSLP in a sample, it is not interfered by related cytokines in the sample, indicating that the detection method provided by the present application has good specificity.

[0248] 3. Accuracy:

[0249] (1) In 3 different batches of experiments, each analysis batch contained 3 sets of samples to be tested, and each set of samples to be tested contained 6 concentrations (2, 10, 50, 250, 1250, 6250 pg / mL). The precision of the method is expressed by the coefficient of variation CV%, CV% = SD / mean value × 100. The accuracy is expressed by the relative error RE%, RE% = (mean detection concentration - theoretical concentration) / theoretical concentration × 100. The accuracy of the kit was analyzed by the above parameters. The analysis results are shown in Table 7.

[0250] Table 7 Test data of samples to be tested

[0251]

[0252] (2) Results and discussion

[0253] The results in Table 7 show that the intra-batch coefficient of variation CV≤5% at the six concentration levels. The accuracy (RE) ≤5%. The above results show that the quantitative detection of human TSLP in samples using the human TSLP CBA kit provided by the present application can meet the intra-batch coefficient of variation CV≤5% and accuracy (RE) ≤5%, indicating that the detection method provided by the present application has good accuracy.

[0254] 4. Precision

[0255] (1) In 3 different batches of experiments, each analysis batch contained 3 sets of samples to be tested, and each set of samples to be tested contained 6 concentrations (2, 10, 50, 250, 1250, 6250 pg / ml). The precision of the method was expressed by the inter-batch coefficient of variation CV%, CV% = SD / mean value × 100%. The reproducibility of the test kit was analyzed by the above parameters. The analysis results are shown in Table 8.

[0256] Table 8 Experimental data on the quantitative detection precision of human TSLP

[0257]

[0258] (2) Results and discussion

[0259] The results in Table 8 show that the inter-batch coefficient of variation CV% of 6 concentration levels (including 2 concentrations each of low, medium and high) is ≤5%. The above results show that the inter-batch coefficient of variation CV% of the human TSLP in the sample quantitatively detected by using the human TSLP CBA kit provided by the application can meet ≤5%, indicating that the detection method provided by the application has good reproducibility.

[0260] 5. Sensitivity

[0261] (1) In the experiment of 3 batches of different kits, each analysis batch was tested 20 times of sample dilution. The sensitivity calculation method is: calculate the mean value M and standard deviation SD of the measurement results, and bring the value of the mean value M plus 2 times the standard deviation SD into the standard curve in Example 4. The calculated result is the sensitivity. The analysis results are shown in Table 9.

[0262] Table 9 Human TSLP CBA Detection Kit Sensitivity Experimental Data

[0263]

[0264] (2) Results and discussion

[0265] The results in Table 9 show that the sensitivity of the three batches of kits is ≤0.5 pg / mL. The above results show that the kit provided by the present application has good sensitivity.

[0266] sequence:

[0267] Antibody CDR sequences:

[0268] Antibody G12:

[0269] CDR1: DSSCALAN (SEQ ID NO: 4)

[0270] HV2:NKESLSN (SEQ ID NO: 5)

[0271] HV4: KASKS (SEQ ID NO: 6)

[0272] CDR3: KAYNPLLRYACR (SEQ ID NO: 7)

[0273] Antibody H1:

[0274] CDR1:ESSCALDS (SEQ ID NO: 8)

[0275] HV2: KKESLSN (SEQ ID NO: 9)

[0276] HV4: KASKS (SEQ ID NO: 6)

[0277] CDR3: KAYNPLLRYNCR (SEQ ID NO: 10)

[0278] G12 Shark Nanobody Fusion Protein (#1) Sequence:

[0279] QWVEQTPTTTTKEAGESLTINCVLRDSSCALANTYWYFTKKGAPNKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYNPLLRYACRYEGGGTILTVKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

[0280] H1 Shark Nanobody Fusion Protein (#2) Sequence:

[0281] ARVEQTPTTTTKEAGESLTINCVLSESSCALDSTSWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYNPLLRYNCRYEGGGTILTVKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12)

[0282] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the present application.

Claims

1. An anti-TSLP shark nanobody fusion protein, characterized in that: The fusion protein comprises a shark nanobody variable region (VNAR) portion located at the N-terminus and an IgG1 Fc fragment located at the C-terminus, wherein the VNAR comprises the following complementary determining region CDR and hypervariable loop HV: (1) CDR1 shown in SEQ ID NO:4; CDR3 shown in SEQ ID NO:7; HV2 shown in SEQ ID NO:5; and HV4 shown in SEQ ID NO:6; or (2) CDR1 shown in SEQ ID NO:8; CDR3 shown in SEQ ID NO:10; HV2 shown in SEQ ID NO:9; and HV4 shown in SEQ ID NO:

6.

2. The anti-TSLP shark nanobody fusion protein according to claim 1, characterized in that: The amino acid sequence of the VNAR is shown in SEQ ID NO: 1 or 2.

3. The anti-TSLP shark nanobody fusion protein according to claim 1, characterized in that: The amino acid sequence of the Fc fragment is shown in SEQ ID NO:

3.

4. The anti-TSLP shark nanobody fusion protein according to claim 1, characterized in that: The amino acid sequence of the anti-TSLP shark nanobody fusion protein is shown in SEQ ID NO: 11 or 12.

5. A polynucleotide, characterized in that The polynucleotide encodes the anti-TSLP shark nanobody fusion protein according to claim 1.

6. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 5.

7. A host cell, comprising the expression vector according to claim 6, or the polynucleotide according to claim 5 integrated into its genome.

8. An anti-TSLP shark nanobody fusion protein combination, characterized in that: The combination includes fusion proteins S1 and S2, wherein the fusion protein comprises a shark nanobody variable region (VNAR) portion located at the N-terminus and an IgG1 Fc fragment located at the C-terminus, Wherein, the VNAR of the fusion protein S1 comprises: CDR1 shown in SEQ ID NO:4; CDR3 shown in SEQ ID NO:7; HV2 shown in SEQ ID NO:5; and HV4 shown in SEQ ID NO:6; The VNAR of the fusion protein S2 comprises: CDR1 shown in SEQ ID NO:8; CDR3 shown in SEQ ID NO:10; HV2 shown in SEQ ID NO:9; and HV4 shown in SEQ ID NO:

6.

9. The anti-TSLP shark nanobody fusion protein combination according to claim 8, characterized in that: The amino acid sequence of the fusion protein S1 is shown in SEQ ID NO:11; and the amino acid sequence of the fusion protein S2 is shown in SEQ ID NO:

12.

10. A method for producing shark nanobody fusion protein, characterized in that: The method comprises the steps of: (a) culturing the host cell according to claim 7 under conditions suitable for producing shark nanobody fusion protein, thereby obtaining a culture containing shark nanobody fusion protein; (b) isolating and / or recovering the shark nanobody fusion protein from the culture.

11. A use of an active ingredient, characterized in that: The active ingredient is selected from the following group: the anti-TSLP shark nanobody fusion protein according to claim 1, or the shark nanobody fusion protein combination according to claim 8, and the active ingredient combination is used to prepare a reagent for detecting TSLP.

12. A TSLP protein detection kit, characterized in that: The kit comprises the anti-TSLP shark nanobody fusion protein according to claim 1, or the shark nanobody fusion protein combination according to claim 8.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) the anti-TSLP shark nanobody fusion protein according to claim 1, or the shark nanobody fusion protein combination according to claim 8, as an active ingredient; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

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