Anti-CD70 single-domain antibody and application thereof

Abstract

The invention discloses an anti-CD70 single domain antibody and application thereof. The single-domain antibody comprises a CDR1 of which the amino acid sequence is as shown in SEQ ID No. 1, a CDR2 of which the amino acid sequence is as shown in SEQ ID No. 2 and a CDR3 of which the amino acid sequence is as shown in SEQ ID No. 3. The anti-CD70 single-domain antibody disclosed by the invention can be specifically bound with epitopes existing on CD70, has good affinity, and has an obvious inhibition effect on CD70 positive tumor cells by taking the anti-CD70 single-domain antibody as a chimeric antigen receptor and CAR-T cells formed by an antigen binding structural domain.

Description

Technical Field

[0001] The present application relates to the field of biomedical technologies, and particularly to an anti-CD70 single-domain antibody and its uses. Background Art

[0002] CD70, namely tumor necrosis factor receptor superfamily member 7 (TNFSF7), has a chain length of 193 amino acids (a.a.) and a molecular weight of 21.1 kDa. It is a type II transmembrane glycoprotein, with 155 amino acids at the C-terminus located outside the membrane. Disulfide bonds are formed between the first and third, and the second and fourth cysteines in the extracellular part to maintain the spatial structure of the extracellular portion. Usually, it exists as a homotrimer on the membrane.

[0003] CD70 is mainly expressed on activated immune cells such as T cells and B cells, and is expressed after various stimuli such as antigen recognition or cytokines. In addition, its interaction with the ligand CD27 not only plays a role in the second signal for T cell activation, but also the interaction between the two can regulate the proliferation and differentiation of immune cells such as B cells and NK cells.

[0004] Under pathological conditions, CD70 is highly expressed in various tumor tissues. For example, a large number of studies have shown that CD70 is highly expressed in various tumor tissues such as lymphoma, renal cell carcinoma, glioblastoma, breast cancer, and hematogenous malignancies (such as Hodgkin lymphoma, multiple myeloma, leukemia, etc.). At the same time, it has been observed that the expression of CD27 in these tissues increases or decreases synchronously with CD70. It is possible that as a ligand / receptor pair, they form a feedback loop that promotes the proliferation and differentiation of lymphocytes, and instead plays a role in mediating the occurrence of diseases. After the interaction between CD70 and CD27, CD27 is activated, and TRAF2 / 5 binds to its intracellular segment, further activating the NF-κB and c-Jun kinase signaling pathways, leading to cell proliferation, survival, and differentiation, etc. In addition, some people also believe that after the binding of CD70 and CD27, it may also cause CD27 intracellular segment to bind Siva, thereby inducing caspase-mediated apoptosis.

[0005] In view of the above phenomena, currently, the preparation of specific antibodies targeting CD70, CAR-T therapy, antibody drugs, and antibody-drug conjugates are all under intense development.

[0006] Single-domain antibodies are special antibodies derived from camelids or cartilaginous fish, which naturally lack light chains and contain only heavy chains. Cloning the variable region of a single-domain antibody can produce a single-domain antibody consisting of only one heavy chain variable region, called a VHH antibody. The crystal diameter of a VHH antibody is only 2.5nm and the length is 4nm, so it is also called a nanobody. The size of a nanobody is only one-tenth of that of a traditional IgG antibody, and it is the smallest naturally occurring fragment that can bind to an antigen. In addition, nanobodies have good tolerance to the environment, high conformational stability, and better clinical therapeutic effects. At the same time, these small protein molecules are easier to synthesize and are cheaper. The unique properties of nanobodies have given them a broader application prospect in the precise diagnosis of diseases and immune targeted therapy. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide an anti-CD70 single-domain antibody and its use to solve the problems in the prior art.

[0008] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0009] The first aspect of the present application protects an anti-CD70 single-domain antibody, which comprises a CDR1 with an amino acid sequence as shown in SEQ ID No.1, a CDR2 with an amino acid sequence as shown in SEQ ID No.2, and a CDR3 with an amino acid sequence as shown in SEQ ID No.3.

[0010] The anti-CD70 single-domain antibody of the present invention is produced by immunostimulating alpacas to produce high-titer antibodies through the recombinant protein of the CD70 extracellular segment, and then constructing a library with a capacity of more than 10 9 The phage antibody library was obtained through panning and amplification. Its reduced molecular weight is less than 40Kda, which is 1 / 10 of that of ordinary antibodies. Through ELISA testing, it was found that the candidate antibodies are relatively safe; through flow cytometry, it was found that the candidate antibodies can recognize the CD70 antigen on the cell surface.

[0011] The anti-CD70 single-domain antibody of the present invention has high affinity and can be used as the antigen domain of a chimeric antigen receptor to prepare CAR-T cells.

[0012] GFTYSTYC (SEQ ID No. 1)

[0013] ISSDGTT (SEQ ID No. 2)

[0014] AADRRCGWYPDEYNY (SEQ ID No. 3)

[0015] Preferably, the framework region FR of the single-domain antibody comprises FR1 with the amino acid sequence shown in SEQ ID No. 5, FR2 with the amino acid sequence shown in SEQ ID No. 6, FR3 with the amino acid sequence shown in SEQ ID No. 7, and FR4 with the amino acid sequence shown in SEQ ID No. 8.

[0016] QVKLVQSGGGSVQAGGSLRLSCAAS(SEQ ID No.5)

[0017] MGWFRQAPGKQREGVAV(SEQ ID No.6)

[0018] KYADSVKGRFTISISKDNAKNTLYLQMNSLKPEDTAMYYC(SEQ ID No.7)

[0019] WGQGTQVTVSS(SEQ ID No.8)

[0020] Preferably, the heavy chain variable region of the single-domain antibody comprises the sequence shown in SEQ ID NO.4.

[0021] SEQ ID No.4 (the framework region is in bold, Underline is the CDR region):

[0022]

[0023] In the present invention, the anti-CD70 single-domain antibody can be derived from alpaca, dromedary camel, Bactrian camel, llama or camel single-domain antibody.

[0024] In the present invention, the preparation method of the anti-CD70 single-domain antibody is as follows: inserting the coding gene of the anti-CD70 single-domain antibody into an expression vector to obtain a recombinant expression vector, introducing the recombinant expression vector into cells and culturing them, and then separating and purifying to obtain the anti-CD70 single-domain antibody.

[0025] The second aspect of the present application protects a chimeric antigen receptor, which comprises an antigen-binding domain, and the antigen-binding domain comprises the single-domain antibody as described above.

[0026] The present invention constructs a chimeric antigen receptor using the anti-CD70 single-domain antibody, which can specifically recognize CD70-positive tumor cells and kill them efficiently. At the same time, it can also release the cytokine IFN-γ to play a role in cell killing, and has good application prospects in the field of diseases related to CD70 expression.

[0027] Preferably, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain. The signal peptide comprises a CD8α signal peptide, the hinge region includes a CD8α hinge region, the transmembrane region includes any one or a combination of at least two of a CD8α transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region, and the signal transduction domain includes an immunoreceptor tyrosine-based activation motif.

[0028] More preferably, the signal transduction domain further comprises a costimulatory molecule, and the costimulatory molecule includes any one or a combination of at least two of 4-1BB, the intracellular region of CD28, OX40, ICOS, or the intracellular region of DAP10.

[0029] Further preferably, the chimeric antigen receptor sequentially comprises a CD8α signal peptide, a single-domain anti-CD70 antibody, a CD8α hinge region, a CD8α transmembrane region, the costimulatory molecule 4-1BB, and an immunoreceptor tyrosine-based activation motif.

[0030] In the present invention, the method for preparing the chimeric antigen receptor comprises the following steps:

[0031] (a) Transforming the aforementioned nucleic acid or a vector containing the same into a host cell, or directly culturing the aforementioned cell;

[0032] (b) Expressing the nucleic acid molecule;

[0033] (c) Recovering the chimeric antigen receptor described above from the culture.

[0034] The third aspect of the present application protects a biological material, which comprises at least one of the following:

[0035] B1) A nucleic acid molecule encoding the chimeric antigen receptor or the single-domain antibody described above;

[0036] B2) A vector containing the nucleic acid molecule described in B1);

[0037] B3) A cell containing the nucleic acid molecule described in B1) or the vector described in B2).

[0038] B4) A recombinant virus containing the vector described in B2).

[0039] Preferably, the vector is selected from any one of a lentiviral vector, a retroviral vector, and an adeno-associated viral vector.

[0040] More preferably, it is a lentiviral vector.

[0041] Preferably, the recombinant virus is obtained by transfecting a cell with the vector described in B2) and an auxiliary plasmid. The cell is a T cell.

[0042] Preferably, the cell is a chimeric antigen receptor immune cell, and the chimeric antigen receptor immune cell expresses the chimeric antigen receptor as described above.

[0043] Preferably, the chimeric antigen receptor immune cell comprises any one or more of T cells, B cells, and NK cells.

[0044] In the present invention, the method for preparing the cell comprises the following steps: introducing the single-domain antibody, the chimeric antigen receptor, or the nucleic acid or vector as described above into a target cell.

[0045] The fourth aspect of the present application protects the use of the single-domain antibody, the chimeric antigen receptor, and the biomaterial as described above in at least one of the following:

[0046] C1) Preparing a product for in vitro detection of CD70;

[0047] C2) Preparing a pharmaceutical composition for preventing or treating a disease related to CD70 expression.

[0048] The fifth aspect of the present application protects a product for in vitro detection of CD70, and the product comprises the single-domain antibody or the chimeric antigen receptor as described above.

[0049] Preferably, the product further comprises a diagnostic agent conjugated or coupled to the single-domain antibody or the chimeric antigen receptor. The conjugation or coupling is achieved by, but not limited to, chemical conjugation, gene fusion, non-covalent binding, or other means.

[0050] Preferably, the diagnostic agent includes a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, a photosensitizing diagnostic agent. Paramagnetic ions that can be used in the present invention include: chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and erbium(III). Fluorescent agents that can be used in the present invention include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine. Chemiluminescent agents that can be used in the present invention include luminol, isoluminol, aromatic acridinium esters, imidazole, acridinium salts, and oxalate esters. Bioluminescent agents that can be used in the present invention include luciferin, luciferase, and aequorin. Enzymes that can be used in the present invention include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0051] Preferably, specific examples of the product include a kit. The kit further includes a solid-phase carrier, and the aforementioned antibody is immobilized on the solid-phase carrier (such as a microplate, a coverslip, a microbead) or exists freely.

[0052] In a sixth aspect of the present application, there is provided a pharmaceutical composition, which comprises a chimeric antigen receptor as described above; or, a single-domain antibody as described above; or, chimeric antigen receptor immune cells in the biomaterial as described above.

[0053] Preferably, the pharmaceutical composition further includes a therapeutic agent conjugated or coupled to the single-domain antibody or the chimeric antigen receptor.

[0054] More preferably, the therapeutic agent includes cytotoxins, antimetabolites, and radionuclides. Cytotoxins include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, didehydroxymethotrexate, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or homologs. Antimetabolites include methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracycline antibiotics (such as daunorubicin (previously known as daunomycin) and doxorubicin), antibiotics (such as actinomycin D (previously known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents (such as vincristine and vinblastine) duocarmycin, calicheamicin, maytansine, and auristatin and their derivatives. Cytotoxins can be conjugated to the antibodies of the present invention using existing linker technologies in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. The antibodies of the present invention can also be conjugated to radioisotopes to generate cytotoxic radiopharmaceuticals. Examples of radioisotopes that can be conjugated to antibodies for treatment include, but are not limited to, iodine 131, indium 111, yttrium 90, and lutetium 177.

[0055] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0056] More preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, surfactants, disintegrants, coating materials, excipients, solubilizers, diluents, pH regulators, binders, wetting agents, colorants, emulsifiers, bacteriostatic agents, cosolvents, osmotic pressure regulators, fillers, antioxidants or buffers.

[0057] The seventh aspect of the present application protects the use of the pharmaceutical composition as described above in the preparation of a drug for preventing or treating a disease related to CD70 expression.

[0058] Preferably, the related diseases are tumors, chronic inflammatory diseases, immune diseases and infectious diseases related to CD70 expression

[0059] More preferably, the tumors include lymphoma, acute myeloid leukemia, multiple myeloma, renal cell carcinoma, esophageal cancer, mesothelioma, gastric cancer, adenocystic carcinoma, ovarian cancer, endometrial cancer, breast cancer, head and neck squamous cell carcinoma, glioma, lung cancer, osteosarcoma, thyroid cancer, melanoma, pancreatic cancer, Hodgkin's disease, non-Hodgkin lymphoma, glioblastoma, renal cell carcinoma, nasopharyngeal carcinoma, glioma, myeloma and lymphoma.

[0060] More preferably, the immune diseases include rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis.

[0061] The present application also protects a method for non-diagnostic and non-therapeutic detection of CD70 protein or nucleic acid molecules encoding the same in a test sample, the method comprising the following steps: contacting the test sample with the single-domain antibody, the chimeric antibody, the biological material or the product as described above; detecting the formation of a complex of the CD70 protein or nucleic acid molecules encoding the same and the single-domain antibody and the chimeric antibody as described above. Further, the method for detecting CD70 protein or nucleic acid molecules encoding the same in a test sample is applied in vitro for non-diagnostic and non-therapeutic purposes.

[0062] The present application also protects a method for preventing or treating a disease related to CD70 expression, by administering an effective dose of the above-mentioned pharmaceutical composition to a subject.

[0063] Wherein, the subject is usually a mammal, such as a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. Primates such as monkeys, apes or Homo sapiens.

[0064] Among them, the effective dose refers to the dose at which the drug can exhibit its pharmacological effect. Because a certain dose of the drug must be absorbed by the body to reach a certain drug concentration, and only when a certain drug concentration is reached can the drug effect occur. If the dose is too small, an effective concentration cannot be obtained in the body, and the drug cannot exert its effective effect. However, if the dose is too large and exceeds a certain limit, the effect of the drug may undergo a qualitative change and may produce varying degrees of toxicity to the body. Therefore, to exert the effective effect of the drug while avoiding its adverse reactions, it is necessary to strictly control the dosage range of medication.

[0065] In the context of the present invention, the terms "framework region", "framework" or "FR" refer to the amino acid sequences inserted between the CDRs. The terms "single-domain antibody", "VHH", "VHH antibody fragment" and "single-domain antibody" are used interchangeably and represent the variable domain of a single heavy chain of those types of antibodies found in camelids, which are naturally devoid of light chains. In the absence of light chains, each single-domain antibody has three CDRs, denoted as CDR1, CDR2, and CDR3, respectively. The term "affinity" refers to the binding ability between a macromolecule and the antigen to which it binds, particularly the binding ability between a single-domain antibody and the antigen to which it binds.

[0066] Compared with the prior art, the beneficial effects of the present application are as follows:

[0067] 1) In the present invention, non-immunized alpacas are immunized with CD70 recombinant protein to construct a phage display single-domain antibody library. Anti-CD70 antibodies are screened according to this phage display antibody library. The obtained anti-CD70 single-domain antibodies can specifically bind to the CD70 antigen and have good affinity. Through the determination of the antibody half-maximal effective concentration (EC 50 ), it can be known that its EC 50 (unit: ng / mL) is 158.6.

[0068] 2) The anti-CD70 single-domain antibody provided by the present invention has good affinity. Using it as an antigen-binding domain to construct a chimeric antigen receptor, and preparing T cells (CAR-T cells) using this chimeric antigen receptor. The CAR-T cells have killing activity against tumor cells with medium or high expression of CD70, and after co-culture with cells with medium or high expression of CD70, they secrete the cytokine IFN-γ efficiently. Therefore, the anti-CD70 single-domain antibody of the present invention can be effectively applied to immunotherapy and is of great significance for the development of tumor therapeutic drugs. Brief Description of the Drawings

[0069] Figure 1 Shown is a graph of the antibody half-maximal effective concentration of the anti-CD70 single-domain antibody (CD 70-A4) detected by flow cytometry in Example 2 of the present application.

[0070] Figure 2A Shown is the FACS detection result graph of the anti-CD70 single-domain antibody recognizing 293T cells in Example 3 of the present application.

[0071] Figure 2B Shown is the FACS detection result graph of the anti-CD70 single-domain antibody recognizing 293T-CD70 cells in Example 3 of the present application.

[0072] Figure 3 Shown is the plasmid map of the chimeric antigen receptor lentiviral vector targeting CD70 in Example 4 of the present application.

[0073] Figure 4 Shown is the schematic diagram of the chimeric antigen receptor structure expressing CD70 in Example 4 of the present application.

[0074] Figure 5 Shown is the FACS detection result graph of the chimeric antigen receptor expression rate of T lymphocytes in Example 6 of the present application.

[0075] Figure 6 Shown is the killing effect of CAR-T cells on 293T cells in Example 7 of the present application.

[0076] Figure 7 Shown is the killing effect of CAR-T cells on renal cell carcinoma cells 786-O in Example 7 of the present application.

[0077] Figure 8 Shown is the killing effect of CAR-T cells on renal cell carcinoma cells ACHN in Example 7 of the present application.

[0078] Figure 9 Shown is the bar graph of IFNγ secreted by CAR-T cells in Example 8 of the present application.

[0079] Figure 10 Shown is the HD SIN03 CD19 CAR map in Example 4 of the present application. Detailed implementation manners

[0080] In order to make the invention purpose, technical solutions and beneficial effects of the present application clearer, the present application will be further described below in conjunction with embodiments. It should be understood that the embodiments are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following embodiments are all conventional methods unless otherwise specified. Those familiar with this technology can easily understand other advantages and effects of the present application from the content disclosed in this description.

[0081] The following describes the embodiments of the present invention through specific specific examples. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0082] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are used to describe specific embodiments, rather than to limit the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include the plural forms.

[0083] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar to or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0084] Example 1 Obtaining an anti-CD70 single-domain antibody

[0085] In this example, the phage display technology was used to screen the alpaca VHH immune library immunized with the recombinant protein of the extracellular domain of CD70. Through cell screening, a high-affinity anti-CD70 single-domain antibody was obtained. The steps are as follows:

[0086] 1.1 Construction of the phage antibody library

[0087] Alpacas were immunized with the recombinant protein of the extracellular domain of CD70 (Acro, catalog number: CD7-H52H7). After detecting the serum titer by ELISA, peripheral blood was drawn; lymphocytes were separated to obtain a precipitate of peripheral blood mononuclear lymphocytes, total RNA was extracted, and then reverse transcribed into the first-strand cDNA using RNA as a template. Then, the VHH gene was amplified by nested PCR; the VHH gene fragment was cloned into the pShort phagemid, and the reaction product was electrotransformed into competent cells, and the phage was separated and purified by the PEG8000 / NaCl precipitation method to obtain an antibody library; the concentration was adjusted, and it was aliquoted and stored frozen at -80 °C in the refrigerator for later use.

[0088] 1.2 Screening of the phage antibody library

[0089] 1) First, incubate 293T cells with the antibody library for negative screening. Then, take the supernatant and incubate it with 293T-CD70-GFP cells (CD70 positive, see Example 5 for the construction process) and 293T cells respectively;

[0090] 2) Wash 4 times with PT buffer pre-cooled at 4°C; infect NEB alpha 5F’ cells, add helper phage M13K07, and culture overnight; plate by the Drop method and count the enrichment degree the next day; separate and purify the phage by the PEG8000 / NaCl precipitation method and enter the next round of screening;

[0091] 3) After enrichment appears, use the obtained phage as a template, amplify the VHH region, perform next-generation sequencing, and obtain the candidate antibody VHH-A4.

[0092] The amino acid sequence of the candidate antibody VHH-A4 is the sequence shown in SEQ ID NO.4.

[0093] Example 2

[0094] In this example, the candidate antibody obtained in Example 1 was subjected to expression, purification of the VHH Fc single-domain antibody, and determination of antibody affinity. The steps are as follows:

[0095] 2.1 Expression and purification of the VHH Fc single-domain antibody

[0096] To further identify these antibodies, it is necessary to express the antibodies in mammalian cells. Therefore, a recombinant plasmid of the recombinant VHH-A4 antibody (i.e., VHH-CD70-A4 Fc) with a mouse Fc tag was constructed, and then expression and purification were carried out. Expression and purification can be achieved by existing technical means, specifically as follows:

[0097] The method for constructing the recombinant plasmid can be conventional. For example, the coding sequence of VHH-CD70-A4 Fc was synthesized and cloned into the eukaryotic expression plasmid pcDNA3.4. After large-scale plasmid extraction, CHO cells were electrotransformed, cultured for 3 - 7 days, the supernatant was collected, and purified by Protein A affinity chromatography column.

[0098] The absorbance value of OD280 was detected by an enzyme-linked immunosorbent assay (ELISA) reader and the concentration was calculated, and the purity and molecular weight were detected by SDS-PAGE gel.

[0099] The quality test results of the CD70 VHH antibody with a mouse Fc tag are shown in Table 1.

[0100] Table 1

[0101]

[0102] 2.2 Antibody Affinity Assay

[0103] The anti-CD70 VHH antibody with a murine Fc tag obtained in step 2.1 was assayed for the half-maximal effective concentration of the antibody by flow cytometry.

[0104] The half-maximal effective concentration of the antibody (EC 50 ) is a measurement method for evaluating the strength of the interaction between an antibody and its target. During the assay in Example 2, the antibody was serially diluted to different concentrations (starting from 4 μg / mL, serially diluted 4-fold, a total of 10 gradients), combined with 293T-CD70 cells overexpressing CD70 (293T-CD70-GFP, for the preparation process, see Example 5 in detail), and then incubated with APC goat anti-mouse IgG (Biolegend, catalog number: 405308). The fluorescence intensity was measured by flow cytometry to characterize the degree of antibody binding to the cells. EC 50 can be used to compare the affinities of different antibodies for the same target, or to compare the binding strengths of the same antibody under different experimental conditions, and is a commonly used antibody performance evaluation index.

[0105] Based on the measurement results, the half-maximal effective concentration can be calculated, that is, the antibody concentration required for the antibody to bind to 293T cells overexpressing CD70, such that the binding of the target cells reaches half. The measurement results are as shown in Figure 1 the figure.

[0106] As can be seen from Figure 1 , the EC 50 (unit: ng / mL) of the VHH-A4 antibody was 158.6 respectively.

[0107] Example 3

[0108] In this Example 3, flow cytometry was performed on the anti-CD70 single-domain antibody.

[0109] Wild-type 293T (CD70 negative, stored in our company) was incubated with the purified anti-CD70 VHH antibody A4 with a murine Fc tag obtained in step 2.1 of Example 2 in an ice bath for 30 min, and then incubated with APC-labeled goat anti-mouse IgG antibody for 30 min. Flow cytometry (FACS) was used for detection, and the results are shown in Figure 2A . Meanwhile, wild-type 293T without adding the anti-CD70 VHH antibody and APC-labeled goat anti-mouse IgG antibody was used as the blank control group (Ctrl); wild-type 293T without adding the anti-CD70 VHH antibody but adding APC-labeled goat anti-mouse IgG antibody was used as the control group (secAb).

[0110] The 293T-CD70 cells (293T-CD70-GFP) that stably overexpress the CD70 protein (the preparation process is detailed in Example 5) were incubated with the purified anti-CD70 VHH antibody A4 with a mouse Fc tag obtained in Step 2.1 of Example 2 in an ice bath for 30 min, and then incubated with an APC-labeled goat anti-mouse IgG antibody for 30 min. Detection was performed using a flow cytometer (FACS), and the results are shown in Figure 2B . Meanwhile, 293T-CD70-GFP without the addition of the anti-CD70 VHH antibody and the APC-labeled goat anti-mouse IgG antibody was used as the blank control group (Ctrl); 293T-CD70-GFP without the addition of the anti-CD70 VHH antibody but with the addition of the APC-labeled goat anti-mouse IgG antibody was used as the control group (secAb).

[0111] From Figure 2A and Figure 2B it can be seen that the anti-CD70 single-domain antibody of the present application can specifically recognize the CD70 antigen on the cell surface.

[0112] Example 4

[0113] In this Example 4, a lentiviral vector expressing a chimeric antigen receptor of the anti-CD70 VHH single-domain antibody was prepared.

[0114] The schematic diagram of the chimeric antigen receptor is as shown in Figure 4 and includes a CD8α signal peptide, an anti-CD70 single-domain antibody (anti-CD70VHH), a CD8α hinge region, a transmembrane region, and an immunoreceptor tyrosine-based activation motif (CD3ζ).

[0115] Among them, the amino acid sequence (SEQ ID NO.9) of the CD8α signal peptide is:

[0116] MALPVTALLLPLALLLHAARP.

[0117] The amino acid sequence of anti-CD70 VHH is as shown in SEQ ID NO.4.

[0118] The amino acid sequence (SEQ ID NO.10) of the CD8α hinge region and the transmembrane region is:

[0119] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0120] The amino acid sequence of the 4-1BB intracellular region (SEQ ID NO.11) is:

[0121] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

[0122] The amino acid sequence of CD3ζ (SEQ ID NO.12) is as follows:

[0123] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0124] 4.1 Construction of lentiviral vector

[0125] The anti-CD70 VHH Fc antibody fragment containing the CD8α signal peptide (abbreviated as CD8αsingal CD70 VHH fragment) was synthesized from the antibody obtained in Example 2, and then the CD8αsingal CD70 VHH fragment was respectively recombined and ligated with the CD8ahinge-TM-41BB-CD3Z fragment and the digested HD SIN03 CD19 CAR plasmid to obtain a lentiviral vector. The lentiviral vector map is as Figure 3 shown.

[0126] The specific construction method is as follows:

[0127] 4.1.1 Preparation of CD8αsingal CD70 VHH fragment

[0128] Prepare the PCR reaction system according to Table 3 (the reagents in the table are from TOYOBO Inc.), amplify each anti-CD70 single-domain antibody fragment, and perform the reaction according to the PCR program shown in Table 4. The primer sequences are:

[0129] CD8a leader-F (SEQ ID NO.13):

[0130] CTGCAGGTCGACTCTAGAGGATCCCACCATGGCCTTACCAGTGA

[0131] CD8H-R (SEQ ID NO.14):

[0132] GTCGCGGCGCTGGCGTCGTGGT

[0133] Table 3

[0134]

[0135]

[0136] Table 4

[0137]

[0138] After the reaction ended, the PCR products were subjected to 1% agarose gel electrophoresis, and the PCR amplification fragments were recovered. The length of each PCR amplification fragment was about 480 bp, that is, the anti-CD70 VHH antibody fragment containing the CD8α signal peptide, and the quantification was performed by the ultraviolet absorption method.

[0139] In the case of knowing the amino acid sequence, the above-mentioned CD8α singal CD70 VHH fragment can also be obtained by synthesizing its coding nucleotides.

[0140] 4.1.2 Preparation of CD8a hinge-TM-41BB-CD3Z fragment

[0141] Using two primers, CD8H2-F and Vector-R, with the HD CD19 CAR plasmid as the template, PCR amplification was carried out. The PCR reaction system was prepared according to Table 5, and the PCR reaction was carried out according to the procedure in Table 6.

[0142] 1% agarose electrophoresis was performed, and the fragment with a length of 700 bp was recovered using the ZymocleanTM Gel DNA Recovery kit to obtain the CD8a hinge-TM-41BB-CD3Z fragment.

[0143] CD8H2-F (SEQ ID NO.15): CGACGCCAGCGCCGCGACCACC

[0144] Vector-R (SEQ ID NO.16): TCGATAAGCTTGATATCG

[0145] Table 5

[0146]

[0147]

[0148] 4.1.3 Preparation of digested vector

[0149] 5 μg of the HD SIN03 CD19 CAR plasmid constructed in the laboratory (the plasmid map is shown in Figure 10 ) was digested with BamHI and EcoRI double enzymes. After a 1-hour water bath reaction at 37 °C, the vector was recovered, and the length was about 7710 bp.

[0150] 4.1.4 Ligation

[0151] Link the CD8αsingal CD70 VHH fragment in step 4.1.1 of this example, the CD8αhinge-TM-41BB-CD3Z fragment in step 4.1.2 of this example, and the double-digested vector in step 4.1.3 of this example according to the recombination reaction system in Table 6. The reaction conditions are: 37 °C, 30 min, to obtain a recombinant product. The recombination reaction system is shown in Table 6.

[0152] Table 6

[0153] Reagent Dosage HD SIN03 CD19 CAR backbone in Step 4.1.3 154.2 ng CD8α singal CD70VHH fragment in Step 4.1.1 10 ng CD8α hinge-TM-41BB-CD3Z fragment in Step 4.1.2 14 ng 5×CE buffer 2 μL <![CDATA[Exnase TM II]]> 1.2 μL Sterile deionized water (PCR grade water) Make up to 12 μL

[0154] 4.2 Transformation

[0155] Take 12 μL of the recombinant product obtained in step 4.1.4 of this example and heat-transform it into Escherichia coli stbl3 competent cells (100 μL) at 42 °C. Use a kana-resistant plate to select positive monoclonal strains for PCR identification.

[0156] Using LV-F2 and LV-R as primers, prepare according to the reaction system in Table 7, and then perform a PCR reaction according to the program in Table 8 to obtain a PCR product. After the PCR is completed, select positive clones for further sequencing identification, and the sequencing results are as expected.

[0157] LV-F2 (SEQ ID NO.17): TCTTGGTTCATTCTCAAGCCTC

[0158] LV-R (SEQ ID NO.18): GCAACATAGTTAAGAATACC

[0159] Table 7

[0160] Reagent Volume (μL) Taq PCR Master Mix 10 10 μM F LV-F2 1 10 μM R LV-R 1 Template DNA bacterial solution 1 Sterile deionized water (PCR grade water) 7

[0161] Table 8

[0162]

[0163]

[0164] After the PCR is completed, select the correctly identified clones and send them to the company for sequencing (the sequencing primers are LV-F2 and LV-R, and the primers are shown in Table 12). Finally, select the correct clones to shake the bacteria and extract the plasmid using a plasmid large extraction kit. The positive clone is the constructed lentiviral vector HDSIN03-CD70 CAR, and the map is shown in Figure 3 .

[0165] Example 5

[0166] In this example, the lentiviral vector obtained in Example 4 was subjected to lentiviral packaging, concentration, and titer detection, including the following:

[0167] 5.1 Lentivirus packaging

[0168] 1) Seed 293T cells at a density of 6.0×10 6 cells in a 10-cm culture dish and incubate overnight at 37°C in 5% CO 2 2 for virus packaging. The culture medium is DMEM containing 10% fetal bovine serum (FBS).

[0169] 2) Dissolve 5.4 μg of the lentiviral vector obtained in Example 4, 6.2 μg of the helper plasmid pMDlg-RRE, 6.2 μg of the helper plasmid pRSV-REV, and 2.4 μg of the envelope plasmid VSVg in 0.8 mL of serum-free DMEM medium and mix well.

[0170] 3) Dissolve 60.6 μg of PEI (1 μg / μL) in 0.8 mL of serum-free DMEM medium, vortex at 1000 rpm for 5 seconds, and incubate at 25°C for 5 min to obtain a PEI mixture.

[0171] 4) Formation of transfection complexes: Add the PEI mixture obtained in step 3) to the DNA mixture, mix gently immediately after addition, and incubate at 25°C for 20 min to obtain transfection complexes.

[0172] 5) Drop 1.6 mL of the transfection complexes obtained in step 4) into 10 mL of DMEM medium containing 293T cells. After 6 h, replace with fresh medium (DMEM containing 10% fetal bovine serum) and incubate for 48 h, then collect the supernatant of the virus solution.

[0173] 5.2 Lentivirus concentration

[0174] Filter the supernatant of the virus solution in step 5.1 of this example through a 0.45-μm filter membrane and collect it in a 50-mL centrifuge tube. Add 1 / 4 of the PEG-NaCl virus concentrate, mix well by inverting up and down, and place at 4°C overnight. Centrifuge at 3500 rpm for 30 min at 4°C. Discard the supernatant, add RPMI 1640 medium (containing 10% FBS), dissolve and resuspend the virus precipitate. Aliquot the concentrated lentivirus suspension into 50-μL portions and store in a finished product tube at -80°C.

[0175] 5.3 Lentivirus titer detection

[0176] 1) Seed 500 μL of Jurkat cells (2×10 5 cells) in a 24-well culture plate to form a cell suspension.

[0177] 2) Add the concentrated lentivirus in step 5.2 of this example to the cell suspension in volumes of 1 μL, 0.2 μL, and 0.04 μL respectively, and add polybrene to a final concentration of 8 μg / mL. Then, incubate overnight at 37 °C in 5% CO 2 After overnight incubation, replace the fresh medium;

[0178] 3) After 72 h of infection, centrifuge at 500×g for 5 min, discard the supernatant, collect the cells, and resuspend the cells with 100 μL of PBS + 2% FBS; add the MonoRab TM Rabbit Anti-Camelid VHH Cocktail [iFluor 488] antibody at a dilution ratio of 1:100 and incubate on ice for 30 min; then wash once with flow buffer (PBS containing 2% FBS), add 300 μL of flow buffer to resuspend the cells, and detect the infection efficiency using a flow cytometer; take cell samples with a positive rate of 5 - 20% and calculate the titer.

[0179] The titer calculation formula is as follows: Titer (TU / mL) = number of cells (10 5 ) × positive rate / volume of virus (mL).

[0180] Meanwhile, 293T-CD70 cells (293T-CD70-GFP) overexpressing CD70 protein were constructed using the method of this example as follows:

[0181] The CD70-GFP lentivirus was obtained by co-transfecting the CD70-GFP plasmid (purchased from Yunzhou Biotech), pMGlg-RRE, pRSV-REV, and VSVg plasmid. The steps were the same as step 2) in step 5.1 of this example to obtain the CD70-GFP lentivirus;

[0182] Inoculate 1×106 293T cells (preserved by our company) into a 6-well plate, and add 1 mL of the obtained CD70-GFP lentivirus to obtain 293T-CD70 cells (293T-CD70-GFP) overexpressing CD70 protein.

[0183] Example 6

[0184] In this example, the lentivirus prepared in Example 5 was used to transduce T lymphocytes, including the following steps:

[0185] 6.1. Activation of T lymphocytes

[0186] Adjust the density of human PBMC with T cell medium (X-VIVO + 10% FBS + 300 U / mL IL-2) to 1×10 6cells / mL, add 1 / 100 volume of T Cell Transact (commercially available magnetic beads conjugated with CD3 and CD28), inoculate into a culture container and activate for 24 h to obtain activated T cells.

[0187] 6.2. T cell infection

[0188] Collect the activated T cells obtained in step 6.1, adjust the cell density to 3×10 5 cells / mL, add the concentrated lentivirus in step 5.2 of Example 5 according to a multiplicity of infection MOI = 10, and add polybrene to a final concentration of 8 μg / mL; culture overnight at 37 °C and 5% CO 2 environment and then replace with fresh medium. Passage every 2 - 3 days.

[0189] 6.3. Chimeric antigen receptor expression

[0190] Five days after the infection in step 6.2 of this example, take 3×10 5 T cells, centrifuge at 4 °C and 400 g for 5 min, discard the supernatant, and wash once with flow buffer (PBS + 2% bovine serum); resuspend the cells with 50 μL of buffer, add Rabbit Anti-Camelid VHH Antibody (iFluor488) antibody at a ratio of 1:1000, and incubate on ice for 30 min; after washing once with buffer, add 300 μL of buffer to resuspend the cells, and use a flow cytometer to detect the chimeric antigen receptor infection efficiency of T lymphocytes. The results are shown in Figure 5 .

[0191] Meanwhile, use simultaneously processed T cells (T) and CAR-T not incubated with the antibody (Unstained Ctrl) as controls.

[0192] From Figure 5 it can be seen that there is an obvious positive cell population in the infected CAR-T cells, indicating that CAR-T cells expressing the chimeric antigen receptor have been successfully constructed, and the antigen-binding domain of the chimeric antigen receptor contains an anti-CD70 single-domain antibody. The infection efficiency of the CAR-T cells corresponding to the anti-CD70 single-domain antibody is 60.05%.

[0193] Example 7

[0194] In this example, the toxicity of the CAR-T cells prepared in Example 6 to target cells was detected in real time and dynamically using real-time cell analysis technology (RTCA), including the following steps:

[0195] 1) Prepare the RTCA instrument and E-plate: Turn on the RTCA instrument, start the software and log in. Select the required experimental type, place the E-plate into the RTCA instrument and check whether the electrode array is clean and undamaged.

[0196] 2) Stabilize the baseline: Add 50 μL of 1640 medium containing 10% FBS to the E-plate and wait for a few minutes to stabilize the baseline.

[0197] 3) Digest and collect wild-type 293T, 786-O (Cell Bank of the Chinese Academy of Sciences), and ACHN (Cell Bank of the Chinese Academy of Sciences) cells as target cells respectively. Wash them with PBS buffer and suspend the cells in 1640 medium containing 10% FBS so that the number of each type of cell is 15,000 per well.

[0198] 4) Examine and count the cells: Examine the cell morphology and health status and count the cells using a hemocytometer.

[0199] 5) Inoculate the cells: Take 100 μL of the counted cells and inoculate them into the E-plate.

[0200] 6) Real-time monitoring: Place the E-plate into the RTCA instrument, start the real-time monitoring program, and record the impedance or capacitance data.

[0201] 7) CAR-T treatment: During the real-time monitoring process, add effector cells to the 3 types of target cells in step 3) of this example at an effector-to-target ratio (E:T) of 2:1 and 0.5:1 respectively. The effector cells are 50 μL of CAR-T cells obtained in Example 6.

[0202] 8) Monitor the tumor cell proliferation, calculate the killing activity of CAR-T through Cell index, lysis% = (1 - relative Cell index treatment / relative Cell index target ) × 100%, where relative Cell index treatment is the ratio of the Cell index at a certain time point in the co-culture group to the initial Cell index, and relative Cell index target is the ratio of the Cell index at a certain time point in the co-culture group of tumor cells alone to the initial Cell index.

[0203] The results are as Figures 6 - 8 shown. The CAR-T cells constructed in the present invention have no obvious killing effect on CD70-negative 293T cells, but have obvious killing activity on CD70-positive tumor cells 786-O and CD70-positive ACHN cells, indicating that the CAR-T cells constructed in the present invention not only have high tumor killing ability but also have high specificity.

[0204] Example 8

[0205] In this example, the detection of the secretion of the CAR-T cell factor IFN-γ includes the following:

[0206] 8.1 Cell culture supernatant

[0207] The experiment was divided into three groups.

[0208] The first group (spontaneous or MOCK) was: the effector cells in Example 6 were cultured alone at 37°C for 24 h.

[0209] The second group (293T) was: the effector cells in Example 6 and 293T cells negative for CD70 were co-cultured at 37°C for 18 h.

[0210] The second group (293T-CD70) was: the effector cells in Example 6 and target cells positive for CD70 (293T-CD70) were co-cultured at 37°C for 24 h with an effector-to-target ratio of 1:1.

[0211] Then, the cell cultures of each group were centrifuged at 400×g for 10 min to remove the precipitate, and the supernatant was taken and stored at -80°C for later detection.

[0212] Meanwhile, a Ctl T group (not infected with lentivirus) was established.

[0213] 8.2 Detection of IFN-γ

[0214] 8.2.1 Reagents: Human IFN-γ ELISA Kit (Linkage Biotechnology, catalog number: EK180-96) was used for detection. Before detection, all reagents and samples were restored to 25°C, and 1× wash buffer, 1× detection buffer, and detection antibodies were prepared according to the instructions.

[0215] 8.2.2 Preparation of standards and samples Standards: The standard stock solution was serially diluted 2-fold with 5% 1640 medium, with a total of 8 dilution gradients, including zero concentration.

[0216] Samples: The samples were diluted according to the ratio with 5% 1640 medium.

[0217] 8.2.3 Detection steps

[0218] (1) Immerse the ELISA plate: Add 300 μL of 1× wash buffer and let it stand for 30 s. After discarding the wash buffer, pat the microplate dry on absorbent paper.

[0219] (2) Add standards: Add 100 μL of the 2-fold serially diluted standards to the standard wells, and add 100 μL of 5% 1640 medium to the blank well.

[0220] (3) Adding sample: Add 100 μL of the cell culture supernatant obtained in step 8.1 of this example to the sample well;

[0221] (4) Adding detection antibody: Add 50 μL of the diluted detection antibody (diluted 1:100) to each well;

[0222] (5) Incubation: Seal the plate with a sealing film, shake at 300 rpm, and incubate at 25 °C for 2 h;

[0223] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well to wash the plate, wash 6 times, and pat dry on absorbent paper after each wash;

[0224] (7) Adding enzyme and incubating: Add 100 μL of the diluted streptavidin labeled with horseradish peroxidase (diluted 1:100) to each well;

[0225] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25 °C for 45 min;

[0226] (9) Washing: Repeat step (6);

[0227] (10) Adding substrate for color development: Add 100 μL of the color development substrate TMB to each well, protect from light, and incubate at 25 °C for 15 min;

[0228] (11) Adding stop solution: Add 100 μL of stop solution to each well and mix thoroughly;

[0229] (12) Detection and reading: Use an enzyme-linked immunosorbent assay (ELISA) reader for dual-wavelength detection, measure the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and the calibrated OD value is the measured value at 450 nm minus the measured value at the reference wavelength. The results are shown in Figure 9 .

[0230] It can be seen from Figure 9 that the spontaneous MOCK group is a separate CAR-T cell group, and almost no release of the cytokine IFN-γ can be detected. Similarly, almost no IFN-γ factor can be detected in the co-culture of CAR-T cells and 293T cells negative for CD70, while a relatively high content of IFN-γ factor can be detected in the co-culture of CAR-T cells and 293T-CD70 cells (CD70+) positive for CD70.

[0231] Comparative Example 1

[0232] Example 1 simultaneously screened for candidate antibodies, named VHH-17 antibody. The VHH-17 antibody was constructed to obtain the corresponding CAR-T cells using the same method as the VHH-A4 antibody, that is, the same steps in Examples 4-6. The antigen-binding domain of the chimeric antigen receptor contains VHH-17. Then, the RTCA method of Example 7 was used for target cell cytotoxicity detection. The results are shown in Figures 6 - 8 。

[0233] The sequence of the VHH-17 antibody is:

[0234] EVQLVQSGGGSVQAGGSLRLSCAASGYTYSSNYMGWFRQASGKEREGVAAIARDGST SYADSVKGRFTISKDNANNTLYLQMNSLKPEDTAMYYCAADQLGPAHFAVVVGYGYWGQ GTQVTVSS(SEQ ID NO.19)

[0235] Comparative Example 2

[0236] Example 1 simultaneously screened for candidate antibodies, named VHH-28 antibody. The VHH-28 antibody was constructed to obtain the corresponding CAR-T cells using the same method as the VHH-A4 antibody, that is, the same steps in Examples 4-6. The antigen-binding domain of the chimeric antigen receptor contains VHH-28. Then, the RTCA method of Example 7 was used for target cell cytotoxicity detection. The results are shown in Figures 6 - 8 。

[0237] The sequence of the VHH-28 antibody is:

[0238] QVQLVESGGGSVQAGGSLRLSCAASGYTYGSYYMGWFRQAPGKEREGVAAIDSAGR TSYTDSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYCAADQLGPAHFVVVVGFGYRG QGTQVTVSS(SEQ ID NO.20)

[0239] From Figures 6 to 8 it can be seen that the CAR-T cells constructed with VHH-A4 of the present invention have no obvious killing activity against 293T cells; at different effector-to-target ratios, the killing activity against CD70-positive 786-O cells can reach more than 80%; at an effector-to-target ratio of 2:1, the killing activity against CD70-positive ACHN cells reaches more than 80%, which is much higher than the CAR-T cells constructed with VHH-17 and VHH-28. Taken together, the CAR-T cells constructed with VHH-A4 of the present invention not only have high tumor killing ability but also high specificity.

[0240] In summary, the present invention screens and prepares anti-CD70 single-domain antibodies with high affinity, which can efficiently and specifically bind to CD70. Using it as an antigen-binding domain to construct chimeric antigen receptors and CAR-T cells, the obtained CAR-T cells have obvious killing activity and specificity against CD70-positive tumor cells, and can secrete high levels of cytokines. The above results indicate that the single-domain antibodies of the present invention can be effectively applied to immunotherapy, which is of great significance for the development of tumor therapeutic drugs.

[0241] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit this application. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of this application.

Claims

1. A single-domain antibody against CD70, characterized in that, the single-domain antibody comprises CDR1 with the amino acid sequence shown in SEQ ID No.1, CDR2 with the amino acid sequence shown in SEQ ID No.2, and CDR3 with the amino acid sequence shown in SEQ ID No.

3.

2. The single-domain antibody according to claim 1, characterized in that, the variable region of the heavy chain of the single-domain antibody comprises the sequence shown in SEQ ID No.

4.

3. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor comprises an antigen-binding domain, and the antigen-binding domain comprises the single-domain antibody according to claim 1 or 2.

4. The chimeric antigen receptor according to claim 3, characterized in that, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane region and a signal transduction domain. The signal peptide comprises the CD8α signal peptide, the hinge region comprises the CD8α hinge region, the transmembrane region includes any one or a combination of at least two of the CD8α transmembrane region, the CD28 transmembrane region or the DAP10 transmembrane region, and the signal transduction domain includes an immunoreceptor tyrosine-based activation motif.

5. A biological material, characterized in that, the biological material comprises at least one of the following: B1) A nucleic acid molecule encoding the single-domain antibody according to claim 1 or 2 or the chimeric antigen receptor according to claim 3 or 4; B2) A vector containing the nucleic acid molecule as described in B1); B3) A cell containing the nucleic acid molecule as described in B1) or the vector as described in B2); B4) A recombinant virus containing the vector as described in B2).

6. The biological material according to claim 5, characterized in that, the vector is selected from any one of a lentiviral vector, a retroviral vector and an adeno-associated viral vector; and / or, the cell is a chimeric antigen receptor immune cell, and the chimeric antigen receptor immune cell expresses the chimeric antigen receptor according to claim 3 or 4; Preferably, the chimeric antigen receptor immune cell comprises any one or more of T cells, B cells and NK cells.

7. Use of the single-domain antibody according to claim 1 or 2 or the chimeric antigen receptor according to claim 3 or 4 or the biological material according to claim 5 in at least one of the following: C1) Preparing a product for in vitro detection of CD70; C2) Preparing a drug for preventing or treating a disease related to CD70 expression.

8. A product for in vitro detection of CD70, characterized in that, the product comprises the single-domain antibody according to claim 1 or 2 or the chimeric antigen receptor according to claim 3 or 4.

9. The product according to claim 8, characterized in that, the product further comprises a diagnostic agent conjugated or coupled to the single-domain antibody or the chimeric antigen receptor.

10. The product according to claim 9, characterized in that, the diagnostic agent includes any one or more of a radionuclide, a contrast agent, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme and a photosensitizing diagnostic agent.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the single-domain antibody as described in claim 1 or 2; or, the chimeric antigen receptor as described in claim 3 or 4; or, the biological material as described in claim 5.

12. The pharmaceutical composition according to claim 11, wherein, the pharmaceutical composition further comprises a therapeutic agent conjugated or coupled to the single-domain antibody or the chimeric antigen receptor.

13. Use of the pharmaceutical composition according to claim 11 or 12 in the preparation of a medicament for preventing or treating a disease related to CD70 expression; preferably, the related disease is a tumor, a chronic inflammatory disease, an immune disease, and an infectious disease related to CD70 expression.

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