Specific antibodies targeting tumor necrosis factor receptor superfamily factor 7

Abstract

The application discloses specific antibodies targeting tumor necrosis factor receptor superfamily factor 7. The specific antibodies comprise a heavy chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6. The specific antibodies provided by the application have high affinity and specificity. CAR-T cells constructed by using the specific antibodies can specifically recognize tumor cells, secrete cytokine IFN-gamma, and efficiently kill tumor cells.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to specific antibodies targeting tumor necrosis factor receptor superfamily 7 factor. Background Technology

[0002] Whether it's cancer or neurological and inflammatory diseases, tumor necrosis factor (TNF) is always present. CD70, as a member of the TNF superfamily, is a type II transmembrane protein that has the ability to regulate the activation, proliferation, and differentiation of T cells and B cells, playing an important role in regulating the immune response.

[0003] Tumor necrosis factor receptor superfamily 7 (TNFSF7), also known as CD27 Ligand or CD70, is a type II transmembrane glycoprotein with a molecular weight of approximately 50 kDa. It is mainly composed of an extracellular binding domain, a transmembrane domain, and a cytoplasmic domain, and exists in a trimer form. The TNFSF7 receptor is CD27. TNFSF7 promotes the activation, proliferation, and differentiation of T cells and B cells, and regulates the immune response. Under normal conditions, TNFSF7 is mainly expressed in activated lymphocytes. Under pathological conditions, TNFSF7 is highly expressed in various tumor tissues; therefore, TNFSF7 can serve as a potential target for tumor therapy. Furthermore, TNFSF7 is closely related to tumor development and patient prognosis, and can serve as a novel biomarker for early cancer diagnosis and a new target for monitoring disease prognosis.

[0004] Current research on TNFSF7 mainly focuses on monoclonal antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor T-cell therapy (CAR-T). However, some literature reports that ADCs can cause significant adverse events such as thrombocytopenia, and there are currently very few drugs targeting CD70 approved for marketing globally. Therefore, there is an urgent need for an antibody that can specifically bind to tumor necrosis factor receptor superfamily 7 factor (TNFSF7). Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this application is to provide a specific antibody targeting tumor necrosis factor receptor superfamily factor 7 to solve the problems in the prior art.

[0006] This invention uses recombinant CD70 extracellular protein to stimulate alpacas to produce high-titer antibodies, and employs phage display technology to construct a library with a capacity greater than 10... 9Phage display of a camel VHH immune library was used, and specific antibodies (VHH-A5 or CD-A5) targeting tumor necrosis factor receptor superfamily 7 were obtained through panning and amplification. The specific antibodies of this invention can recognize the CD70 antigen on the cell surface with high affinity. Simultaneously, a chimeric antigen receptor and CAR-T cells containing the chimeric antigen receptor were constructed using the specific antibodies of this invention, and it was found that they have cytotoxic activity against CD70-positive tumor cells and can efficiently secrete the cytokine IFN-γ. This invention is based on these findings.

[0007] To achieve the above objectives, the present invention specifically adopts the following technical solution.

[0008] The first aspect of this application protects a specific antibody targeting tumor necrosis factor receptor superfamily 7 factor, said specific antibody comprising a heavy chain variable region, said heavy chain variable region comprising complementarity-determining regions CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6.

[0009]

[0010] In some embodiments, the heavy chain variable region further includes frame regions FR1, FR2, FR3, and FR4 of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7.

[0011] Serial Number sequence FR1 1 EVQLVQSGGGSVQAGGSLRLSCKAS FR2 3 MGWFRQAPGKEREGVAA FR3 5 HYADSVKGRFTISRDNAENTVTLQMNSLKPEDTAMYYC FR4 7 WGQGTQVTVSS

[0012] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID No. 8.

[0013] In the following sequences, bold text represents framed areas. underline For CDR area:

[0014]

[0015] In some embodiments, the specific antibody is a nanobody, also called a single-domain antibody (sdAb), which refers to an antibody containing only the heavy chain variable region. Nanobodies are a class of naturally occurring single-heavy-chain antibodies found in animals such as alpacas and camels. The molecular weight of their antigen-binding site is only 10% of that of immunoglobulin G (IgG) antibodies. Due to the simple structure of nanobodies, and the fact that the relatively long CDR3 region can form a large protrusion that inserts into certain conserved "cavitation structures" to form a stable antigen-antibody complex,

[0016] In some embodiments, the specific antibody includes an antigen-binding fragment, which refers to any antibody fragment capable of binding to the target antigen.

[0017] In this invention, the frame region or FR refers to the amino acid sequence inserted between CDRs; specific antibodies, VHH, VHH antibody fragments, nanobodies, single-domain antibodies, and antigen-binding fragments are used indiscriminately; affinity refers to the binding ability between an antibody and the antigen it binds to, especially the binding ability between a specific antibody and the antigen it binds to.

[0018] A second aspect of this application protects a chimeric antigen receptor comprising an antigen-binding domain, a hinge region, and a transmembrane domain, wherein the antigen-binding domain comprises a specific antibody as described above.

[0019] In some embodiments, the hinge region is selected from the CD8α hinge region.

[0020] In some embodiments, the transmembrane domain is selected from one or more of CD8α, CD28, or DAP10.

[0021] In some embodiments, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the aforementioned specific antibody, hinge region, and transmembrane domain.

[0022] The amino acid sequences of the CD8α hinge region and transmembrane region are as follows:

[0023] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID No. 10).

[0024] In some embodiments, the chimeric antigen receptor further comprises a signal peptide and / or a signal transduction domain.

[0025] In some embodiments, the signal peptide comprises a CD8α signal peptide. The amino acid sequence of the signal peptide comprises the sequence shown in SEQ ID NO. 9.

[0026] MALPVTALLLPLALLLHAARP (SEQ ID No. 9).

[0027] In some specific embodiments, the signal transduction domain comprises an immune receptor tyrosine activation motif or a co-stimulatory molecule. The co-stimulatory molecule comprises any one or a combination of at least two of the following: 4-1BB, the CD28 intracellular region, OX40, ICOS, or the DAP10 intracellular region. The amino acid sequence of the immune receptor tyrosine activation motif comprises the sequence shown in SEQ ID NO. 12. The amino acid sequence of the 4-1BB intracellular region comprises the sequence shown in SEQ ID No. 11.

[0028] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.11)

[0029] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No. 12).

[0030] In one embodiment, the chimeric antigen receptor comprises a CD8α signal peptide, the specific antibody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecule 4-1BB, and an immunoreceptor tyrosine activation motif.

[0031] A third aspect of this application protects a substance selected from one or more of the following:

[0032] Furthermore, the substance is a polynucleotide that encodes a specific antibody as described in the first aspect, or a chimeric antigen receptor as described in the second aspect.

[0033] Furthermore, the substance is a carrier, and the carrier contains the polynucleotides as described above.

[0034] In this invention, the vector refers to a vector that can introduce polynucleotides into host cells by transforming the host and promoting the expression of the introduced polynucleotides, for example, transcription and translation.

[0035] In some embodiments, the vector is typically constructed by inserting the polynucleotide into a suitable plasmid, and those skilled in the art can select the appropriate plasmid. For example, the types of plasmids include, but are not limited to: viral vectors, such as lentiviral vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors; and non-viral vectors, such as plasmids and transposon vectors. In some preferred embodiments, a suitable plasmid is the HD SIN03 CD19 CAR plasmid, and the constructed vector is HD SIN03-CD70 CAR.

[0036] Furthermore, the substance is a modified cell, which contains the vector as described above, or has the polynucleotides as described above integrated into its genome.

[0037] In this invention, a modified cell refers to a host cell in which the coding sequence of a specific antibody or chimeric antigen receptor is introduced, so that the host cell expresses the introduced coding sequence to express the specific antibody or chimeric antigen receptor. The host cell that receives and expresses the introduced coding sequence is the modified cell.

[0038] In some embodiments, the host cell is a T lymphocyte, B lymphocyte, natural killer cell, dendritic cell, cytotoxic T cell, tumor-infiltrating T cell, or regulatory T cell. In some embodiments, it is a T lymphocyte.

[0039] In some embodiments, methods for introducing the vector into host cells should be known to those skilled in the art, such as microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, etc. In some embodiments, virus-mediated transformation is used.

[0040] Furthermore, the substance is a recombinant virus, which contains the vector described above.

[0041] In some embodiments, the recombinant virus is prepared by viral packaging of the vector described above. Preferably, it is a recombinant lentivirus. The recombinant lentivirus can be used to infect host cells to prepare modified cells (CAR-T cells).

[0042] Furthermore, the substance is a viral vector system, which includes the vector as described above, as well as helper plasmids or host cells.

[0043] In some embodiments, the auxiliary plasmid may include a packaging plasmid and an envelope plasmid. In some embodiments, the packaging plasmid is pMDlg-RRE / pRSV-REV, and the envelope plasmid is VSVg; all three plasmids are commercially available. In some embodiments, the viral vector system includes the lentiviral vector HD SIN03-CD70 CAR, pMDlg-RRE, pRSV-REV, and VSVg.

[0044] Furthermore, the substance is an antibody-drug conjugate, which comprises a specific antibody as in the first aspect, or a chimeric antigen receptor as in the second aspect, and a conjugate thereto.

[0045] Furthermore, the substance is a pharmaceutical composition comprising a therapeutically effective amount of, for example, a specific antibody as described in the first aspect, or a chimeric antigen receptor as described in the second aspect, or a polynucleotide as described above, or a carrier as described above, or a modified cell as described above, or an antibody-drug conjugate as described above.

[0046] In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier includes any one or a combination of at least two of the following: a carrier, a surfactant, a disintegrant, a coating material, an excipient, a solubilizer, a diluent, a pH adjuster, a binder, a wetting agent, a colorant, an emulsifier, an antibacterial agent, a cosolvent, an osmotic pressure regulator, a filler, an antioxidant, or a buffer.

[0047] Furthermore, the substance is a product, the product comprising, as in the first aspect, a specific antibody, as in the second aspect, a chimeric antigen receptor, or a polynucleotide as described above, or a vector as described above, or a modified cell as described above, or a recombinant virus as described above, or a viral vector system as described above, or an antibody-drug conjugate as described above.

[0048] In some embodiments, the product is selected from one or more of a kit, nucleic acid membrane strip, chip, system, or device.

[0049] The fourth aspect of this application protects an application selected from one or more of the following:

[0050] Furthermore, the use of specific antibodies, such as those described in the first aspect, or chimeric antigen receptors, such as those described in the second aspect, or polynucleotides, or vectors, or modified cells as described above, in the preparation of products for in vitro detection of CD70.

[0051] In some embodiments, the in vitro CD70 detection product is typically targeted at the CD70 antigen, using the CD70 antigen as a biomarker for diagnosis. The product may also include a marker, which is typically used to label specific antibodies, including fluorescent markers, chromogenic markers, reporter genes, localization signals, etc. The antibody conjugated with the marker can be detected using common detection methods such as ELISA (enzyme-linked immunosorbent assay) and immunofluorescence techniques, immunoradiotherapy, immunoenzyme techniques, immunogold immunoassay, immunohistochemical staining (tissue) samples, flow cytometry, etc.

[0052] Furthermore, the use of specific antibodies, such as those described in the first aspect, or chimeric antigen receptors, such as those described in the second aspect, or polynucleotides, or vectors, or modified cells, as described above, in the preparation of drugs for the prevention or treatment of diseases related to CD70 expression.

[0053] In some embodiments, the drug comprises a therapeutically effective amount of a specific antibody, a chimeric antigen receptor, or a modified cell.

[0054] In some specific embodiments, the target of the drug includes, but is not limited to, mammals. Mammals include, for example, humans, other primates, pigs, cattle, horses, cats, dogs, sheep, goats, and rodents, specifically mice and rats, rabbits, guinea pigs, and hamsters.

[0055] In some specific embodiments, the relevant disease is a tumor, which is a malignant tumor expressing CD70, including 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's lymphoma, glioblastoma, renal cell carcinoma, nasopharyngeal carcinoma, glioma, myeloma, and lymphoma.

[0056] In some specific embodiments, the relevant disease is an immune disease, specifically an immune disease expressing CD70, including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

[0057] Furthermore, the use of specific antibodies, such as those described in the first aspect, or chimeric antigen receptors, such as those described in the second aspect, or polynucleotides, or vectors, or modified cells, as described above, in the preparation of drugs that regulate the activity or level of CD70 protein.

[0058] The fifth aspect of this application protects a method selected from one or more of the following:

[0059] Furthermore, a method for preparing a specific antibody as described in the first aspect or a chimeric antigen receptor as described in the second aspect includes the following steps:

[0060] a) Transform the polynucleotides or vectors described above into host cells, or directly culture the modified cells as described above;

[0061] b) Express the aforementioned polynucleotide;

[0062] c) The specific antibody or the chimeric antigen receptor as described above is recovered from the culture.

[0063] Furthermore, a method for preparing the modified cells as described in the third aspect includes the following steps:

[0064] The specific antibody as described in the first aspect, or the chimeric antigen receptor as described in the first aspect, or the polynucleotide as described above, or the vector as described above, is introduced into the target cell.

[0065] Furthermore, a method for detecting CD70 protein or its encoding nucleic acid molecule in a sample for non-diagnostic and non-therapeutic purposes includes the following steps:

[0066] The sample to be tested is contacted with the specific antibody as described in the first aspect, the chimeric antigen receptor as described in the second aspect, or the pharmaceutical composition as described above.

[0067] The formation of a complex of CD70 protein or the nucleic acid molecule encoding it with the specific antibody and the chimeric antigen receptor is detected.

[0068] Compared with the prior art, the beneficial effects of this application are as follows:

[0069] 1) The specific antibody targeting tumor necrosis factor receptor superfamily 7 of the present invention can specifically bind to CD70 antigen with good affinity, as indicated by the antibody half-maximal effective concentration (EC50). 50 The determination of its EC indicates that... 50 The concentration was 169.7 ng / mL.

[0070] 2) The specific antibody targeting tumor necrosis factor receptor superfamily 7 of the present invention has good affinity. It is used as the antigen-binding domain to construct a chimeric antigen receptor, and T cells (CAR-T cells) are prepared using the chimeric antigen receptor. CAR-T cells have killing activity against tumor cells with medium or high expression of CD70, and after co-culturing with cells with medium or high expression of CD70, they efficiently secrete the cytokine IFN-γ. Therefore, the specific antibody of the present invention can be effectively applied to immunotherapy and is of great significance for the development of tumor therapeutic drugs. Attached Figure Description

[0071] Figure 1 The graph shown is a flow cytometry curve of the half-maximal effective concentration of the antibody used to detect the specific antibody in Example 2 of this application.

[0072] Figure 2A The image shown is an FACS detection result of the specific antibody recognizing wild-type 293T (CD70 negative) in Example 3 of this application.

[0073] Figure 2B The image shown is an FACS detection result of 293T-CD70 cells (CD70 positive) overexpressing CD70 protein, as recognized by the specific antibody in Example 3 of this application.

[0074] Figure 3AThe image shown is a spectrum of plasmid HD SIN03 CD19 in Example 4 of this application.

[0075] Figure 3B The image shown is a map of the lentiviral vector expressing the chimeric antigen receptor in Example 4 of this application.

[0076] Figure 3C The diagram shown is a schematic diagram of the structure of the chimeric antigen receptor in Example 4 of this application.

[0077] Figure 4 The graph shown is a flow cytometry result of the chimeric antigen receptor expression rate in Example 9 of this application.

[0078] Figure 5A The image shows the killing effect of CAR-T cells on 293T cells in Example 10 of this application.

[0079] Figure 5B This is shown as the killing effect of CAR-T cells on renal cell carcinoma cells 786-O in Example 10 of this application.

[0080] Figure 5C The image shows the killing effect of CAR-T cells on renal cell carcinoma cells ACHN in Example 10 of this application.

[0081] Figure 6 The chart shown is a bar graph of IFNγ secreted by CAR-T cells in Example 11 of this application. Detailed Implementation

[0082] To make the inventive purpose, technical solution, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0083] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand 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, and 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.

[0084] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0085] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0086] Example 1

[0087] In this embodiment, phage display technology was used to pan the CD70-immunized alpaca VHH immune library, and high-affinity specific antibodies were obtained through cell screening. The steps included are as follows:

[0088] 1.1 Construction of a phage-specific antibody library

[0089] Alpacas were immunized with recombinant CD70 extracellular protein (Acro, catalog number: CD7-H52H7). After serum titer was detected by ELISA, peripheral blood was collected. Lymphocytes were isolated, peripheral blood mononuclear lymphocytes were collected, total RNA was extracted, and then reverse transcribed into first-strand cDNA using RNA as a template. The VHH gene was then amplified using nested PCR. The VHH gene fragment was cloned into pShort phage particles, and the electroporation product was transferred to… Phages were isolated and purified from competent cells using the PEG8000 / NaCl precipitation method to obtain an antibody library; the concentration was adjusted, aliquoted, and stored at -80℃ for later use.

[0090] 1.2 Screening of phage-specific antibody libraries

[0091] 1) First, 293T cells were co-incubated with the antibody library for negative screening. Then, the supernatant was collected and incubated with 293T-CD70-GFP cells (for details of CD70 positive construction, please refer to Example 8) and 293T cells respectively.

[0092] 2) Wash 4 times with PT buffer pre-cooled at 4℃; infect NEB alpha 5F' cells, add helper phage M13K07, and incubate overnight; plate using the Drop method, and calculate the enrichment level the next day; isolate and purify phages using PEG8000 / NaCl precipitation method, and proceed to the next round of screening;

[0093] 3) After enrichment, the obtained phage was used as a template to amplify the VHH region, and gene sequencing was performed to obtain a candidate antibody, named VHH-A5.

[0094] The amino acid sequence of the candidate antibody VHH-A5 is shown in SEQ ID No. 8.

[0095] Example 2: Antibody Expression, Purification, and Affinity Analysis

[0096] In this embodiment, the candidate antibody obtained in Example 1 was subjected to VHH Fc-specific antibody expression, purification, and antibody affinity determination. The steps included are as follows:

[0097] 2.1 Expression and purification of VHH Fc-specific antibodies

[0098] To further identify these antibodies, they need to be expressed in mammalian cells. Therefore, a recombinant plasmid containing a mouse Fc tag for the VHH-A5 antibody (i.e., VHH-CD70-A5 Fc) was constructed, and then expressed and purified. Expression and purification can be achieved using existing techniques, as detailed below:

[0099] The recombinant plasmid can be constructed using conventional methods, such as synthesizing the coding sequence of VHH-CD70-A5 Fc and cloning it into the eukaryotic expression plasmid pcDNA3.4. After plasmid extraction, CHO cells are electroporated and cultured for 3-7 days. The supernatant is collected and purified by Protein A affinity chromatography to obtain the recombinant VHH-A1 antibody (i.e., VHH-CD70-A5 Fc) with a mouse Fc tag.

[0100] The absorbance of OD280 was measured using an ELISA reader and the concentration was calculated. The purity and molecular weight were determined using an SDS-PAGE gel.

[0101] The quality test results of CD70 VHH antibodies with mouse Fc tags are shown in Table 1.

[0102] Table 1

[0103]

[0104] 2.2 Antibody Affinity Assay

[0105] The half-maximal effective concentration (IC50) of the anti-CD70 VHH antibody with mouse Fc tag obtained in step 2.1 was determined by flow cytometry.

[0106] half-maximal effective concentration (EC50) of antibody 50 This is a method for measuring the strength of the interaction between an antibody and its target. Antibodies were diluted to different concentrations (starting from 4 μg / mL, with subsequent 4-fold dilutions, for a total of 10 gradients), bound to 293T-CD70 cells overexpressing CD70 (293T-CD70-GFP, preparation process detailed in Example 8), and then incubated with APC goat anti-mouse IgG (Biolegend, catalog number: 405308). Fluorescence intensity was measured by flow cytometry to characterize the degree of antibody binding to cells. 50 It can be used to compare the affinity between different antibodies and the same target, or to compare the binding strength of the same antibody under different experimental conditions. It is a commonly used indicator for evaluating antibody performance.

[0107] Based on the measurement results, the half-maximal effective concentration (MCI) can be calculated, which is the antibody concentration required for the antibody to bind to 293T cells overexpressing CD70, ensuring that the binding of the target cells reaches half of the required concentration. (The measurement results are as follows...) Figure 1 As shown.

[0108] from Figure 1 It can be seen that the EC of specific antibodies 50 The concentration was 169.7 ng / mL.

[0109] Example 3: Specificity analysis of antibodies

[0110] In Example 3, the specific antibody was measured by flow cytometry.

[0111] Wild-type 293T (CD70 negative, stored in our company) was incubated with purified anti-CD70 VHH antibody A5 with a mouse Fc tag obtained in step 2.1 of Example 2 for 30 min on ice, and then incubated with APC-labeled goat anti-mouse IgG antibody for 30 min. The results were detected by flow cytometry (FACS). See [Figure number missing in original text]. Figure 2A Meanwhile, wild-type 293T cells without anti-CD70 VHH antibody and APC-labeled goat anti-mouse IgG antibody served as the blank control group (Ctrl); wild-type 293T cells without anti-CD70 VHH antibody but with APC-labeled goat anti-mouse IgG antibody served as the control group (secAb).

[0112] Stable CD70 protein-overexpressing 293T-CD70 cells (293T-CD70-GFP, preparation process detailed in Example 8) were incubated with purified anti-CD70 VHH antibody A5 with mouse Fc tag obtained in step 2.1 of Example 2 on ice for 30 min, followed by incubation 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 8]. Figure 2B Meanwhile, 293T-CD70-GFP without anti-CD70 VHH antibody and APC-labeled goat anti-mouse IgG antibody served as the blank control group (Ctrl); 293T-CD70-GFP without anti-CD70 VHH antibody but with APC-labeled goat anti-mouse IgG antibody served as the control group (secAb).

[0113] from Figure 2A and Figure 2B It is known that specific antibodies can specifically recognize the CD70 antigen on the cell surface.

[0114] Example 4: Construction of a lentiviral vector expressing a chimeric antigen receptor

[0115] In Example 4, a lentiviral vector expressing a chimeric antigen receptor was prepared, the chimeric antigen receptor containing the specific antibody VHH-A5.

[0116] 4.1 Construction of Lentiviral Vectors

[0117] Anti-CD70 VHH Fc antibody fragments containing the CD8α signal peptide (referred to as CD8αsingal CD70 VHH fragments) were synthesized using the antibodies obtained in Example 2. Then, the CD8αsingal CD70 VHH fragments were recombinantly ligated with CD8ahinge-TM-41BB-CD3Z fragments and enzyme-digested HD SIN03 CD19 CAR plasmids to obtain chronic viral vectors.

[0118] The specific construction method is shown in the following steps:

[0119] 4.1.1 Preparation of CD8αsingal CD70 VHH fragment

[0120] Prepare the PCR reaction system according to Table 3 (reagents in the table are from TOYOBO Inc.), amplify the specific antibody fragments against CD70, and perform the PCR reaction according to the PCR procedure shown in Table 4. The primer sequences are as follows:

[0121] CD8a leader-F (SEQ ID No.13):

[0122] CTGCAGGTCGACTCTAGAGGATCCCACCATGGCCTTACCAGTGA

[0123] CD8H-R (SEQ ID No. 14):

[0124] GTCGCGGCGCTGGCGTCGTGGT

[0125] Table 3

[0126] reagents Volume (μL) 10×buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO4]]> 3 10μM CD8a leader-F 1 10μM CD8H-R 1 Template DNA 1 Sterile deionized water (PCR grade water) 33 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0127] Table 4

[0128]

[0129]

[0130] After the reaction, the PCR products were subjected to 1% agarose gel electrophoresis to recover the PCR amplified fragments. Each PCR amplified fragment was about 480 bp in length, which is the anti-CD70 VHH antibody fragment containing the CD8α signal peptide. The fragments were quantified by ultraviolet absorption method.

[0131] Knowing the amino acid sequence, the CD8αsingal CD70 VHH fragment mentioned above can also be obtained by synthesizing its encoding nucleotide.

[0132] 4.1.2 Preparation of CD8a hinge-TM-41BB-CD3Z fragment

[0133] Using two primers, CD8H2-F and Vector-R, and with the HD CD19 CAR plasmid as a template, PCR amplification was performed. The PCR reaction system was prepared according to Table 5, and the PCR reaction was carried out according to the procedure in Table 6.

[0134] The fragment was recovered by 1% agarose gel electrophoresis and was 700 bp in length using the Zymoclean™ Gel DNA Recovery kit, yielding the CD8a hinge-TM-41BB-CD3Z fragment.

[0135] CD8H2-F (SEQ ID No. 15): CGACGCCAGCGCCGCGACCACC

[0136] Vector-R(SEQ ID No.16):TCGATAAGCTTGATATCG

[0137] Table 5

[0138] reagents Volume (μL) 10×buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO4]]> 3 10μMCD8αH-F 1 10μM Vector-R 1 Template DNA (HD CD19CAR) 1 Sterile deionized water (PCR grade water) 33 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0139] 4.1.3 Preparation of enzyme digestion vector

[0140] 5 μg of the laboratory-constructed HD SIN03 CD19 CAR plasmid (plasmid image shown) was used. Figure 3A The vector was digested with BamHI and EcoRI, reacted in a water bath at 37°C for 1 hour, and then recovered. The length of the vector was approximately 7710 bp.

[0141] 4.1.4 Connection

[0142] The CD8αsingal CD70 VHH fragment from step 4.1.1 of this embodiment, the CD8αhinge-TM-41BB-CD3Z fragment from step 4.1.2 of this embodiment, and the double-digested vector from step 4.1.3 of this embodiment were ligated according to the recombination reaction system in Table 6. The reaction conditions were 37℃ for 30 min to obtain the recombinant product. The recombinant reaction system is shown in Table 6.

[0143] Table 6

[0144] reagents Usage Step 4.1.3 HD SIN03 CD19 CAR skeleton 154.2ng Step 4.1.1 CD8αsingal CD70VHH fragment 10ng Step 4.1.2 CD8αhinge-TM-41BB-CD3Z fragment 14ng 5×CE buffer 2μL <![CDATA[Exnase TM II]]> 1.2μL Sterile deionized water (PCR grade water) Make up to 12μL

[0145] 4.2 Transformation

[0146] 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 at 42℃ (100 μL). Use Kana resistance plates to select positive monoclonal strains for PCR identification.

[0147] Using LV-F2 and LV-R as primers, the reaction system was prepared according to Table 7, and then the PCR reaction was carried out according to the procedure in Table 8 to obtain PCR products. After PCR, positive clones were selected for further sequencing and identification, and the sequencing results were as expected.

[0148] LV-F2(SEQ ID No.17):TCTTGGTTCATTCTCAAGCCTC

[0149] LV-R(SEQ ID No.18):GCAACATAGTTAAGAATACC

[0150] Table 7

[0151] reagents Volume (μL) Taq PCR Master Mix 10 10μM F LV-F2 1 10μM R LV-R 1 Template DNA bacterial solution 1 Sterile deionized water (PCR grade water) 7

[0152] Table 8

[0153]

[0154] After PCR, correctly identified clones were selected and sent to the company for sequencing (sequencing primers were LV-F2 and LV-R, see Table 12 for primers). Finally, the correctly selected clones were cultured and plasmids were extracted using a plasmid extraction kit. The positive clones were the constructed lentiviral vector HDSIN03-CD70 CAR, as shown in the diagram. Figure 3B .

[0155] A schematic diagram of the expressed chimeric antigen receptor is shown below. Figure 3C As shown, it includes the CD8α signal peptide, a specific antibody against CD70 (anti-CD70 VHH), the CD8α hinge region, the transmembrane region, and the immune receptor tyrosine activation motif (CD3ζ).

[0156] The amino acid sequence (SEQ ID No. 9) of the CD8α signal peptide is: MALPVTALLLPLALLLHAARP.

[0157] The amino acid sequence of anti-CD70 VHH is shown in SEQ ID No. 8.

[0158] The amino acid sequences (SEQ ID No. 10) of the CD8α hinge region and transmembrane region are as follows:

[0159] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0160] The amino acid sequence of the intracellular region of 4-1BB (SEQ ID No. 11) is as follows:

[0161] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0162] The CD3ζ amino acid sequence (SEQ ID No. 12) is as follows:

[0163] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0164] Example 5 Lentiviral Packaging

[0165] In this embodiment, the lentivirus vector obtained in Example 4 is packaged into a lentivirus, including the following:

[0166] 1) With 6.0×10 6293T cells were seeded in 10cm culture dishes and cultured overnight at 37°C with 5% CO2 to prepare for virus packaging. The culture medium was DMEM containing 10% fetal bovine serum (FBS).

[0167] 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 culture medium and mix well.

[0168] 3) Dissolve 60.6 μg PEI (1 μg / μL) in 0.8 mL of serum-free DMEM culture medium, vortex at 1000 rpm for 5 seconds, and incubate at 25 °C for 5 min to obtain PEI mixture;

[0169] 4) Formation of transfection complex: 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 the transfection complex.

[0170] 5) Add 1.6 mL of the transfection complex obtained in step 4) to 10 mL of DMEM medium containing 293T cells. After 6 hours, replace with fresh medium (DMEM containing 10% fetal bovine serum). After culturing for 48 hours, collect the viral supernatant.

[0171] Example 6 Lentiviral Concentration

[0172] In this embodiment, the lentivirus obtained in Example 5 is concentrated, including the following:

[0173] The supernatant of the viral suspension from Example 5 was filtered through a 0.45 μm filter membrane and collected into a 50 mL centrifuge tube. 1 / 4 of the PEG-NaCl viral concentrate was added, and the mixture was inverted and mixed thoroughly. The mixture was then incubated overnight at 4 °C. The mixture was centrifuged at 3500 rpm for 30 min at 4 °C. The supernatant was removed, and RPMI 1640 medium (containing 10% FBS) was added to dissolve and resuspend the viral precipitate. The concentrated lentivirus suspension was aliquoted into 50 μL portions and stored in finished tubes at -80 °C.

[0174] Example 7 Lentiviral titer detection

[0175] In this embodiment, the titer of the lentivirus obtained in Example 6 is detected, including the following:

[0176] 1) Add 500 μL of Jurkat cells (2 × 10⁻⁶) 5 (10 cells) were seeded into a 24-well culture plate to form a cell suspension;

[0177] 2) The concentrated lentivirus from Example 6 was added to the cell suspension in volumes of 1 μL, 0.2 μL, and 0.04 μL, respectively, and polybrene was added to a final concentration of 8 μg / mL. The cells were then incubated overnight at 37°C with 5% CO2, and the culture medium was replaced with fresh medium.

[0178] 3) 72 h after infection, centrifuge at 500×g for 5 min, discard the supernatant and collect the cells. Resuspend the cells in 100 μL PBS + 2% FBS; add MonoRab at a 1:100 dilution. TM Rabbit Anti-Camelid VHH Cocktail [iFluor 488] antibody was incubated on ice for 30 min; then the cells were washed once with flow cytometry buffer (PBS containing 2% FBS), and 300 μL of flow cytometry buffer was added to resuspend the cells. The infection efficiency was detected by flow cytometry; cell samples with a positive rate of 5-20% were taken and the titer was calculated.

[0179] The formula for calculating the titer is as follows:

[0180] Titer (TU / mL) = Cell number (10) 5 ) × Positive rate / Viral volume (mL).

[0181] Example 8: Construction of 293T-CD70 cells overexpressing CD70 protein

[0182] In this embodiment, the construction of 293T-CD70 cells overexpressing CD70 protein (293T-CD70-GFP) includes the following steps:

[0183] 1) CD70-GFP lentivirus was obtained by co-transfection of CD70-GFP plasmid (purchased from Yunzhou Biotechnology), pMGlg-RRE, pRSV-REV and VSVg plasmid, and the steps were the same as in Example 5 and Example 6, to obtain concentrated CD70-GFP lentivirus.

[0184] 2) Take 1×10 6 293T cells (preserved by our company) were seeded into 6-well plates, and 1 mL of the CD70-GFP lentivirus obtained above was added to obtain 293T-CD70 cells (293T-CD70-GFP) overexpressing CD70 protein.

[0185] Example 9 Construction of CAR-T cells

[0186] In this embodiment, the lentivirus transduction of T lymphocytes using the method described in Example 6 includes the following steps:

[0187] 9.1 Activation of T lymphocytes

[0188] Human PBMCs were adjusted to a density of 1×10⁻⁶ cells / mL using T cell culture medium (X-VIVO + 10% FBS + 300 U / mL IL-2). 6 Add 1 / 100 volume of T Cell Transact (commercially available magnetic beads coupled with CD3 and CD28) to the culture container and activate for 24 hours to obtain activated T cells.

[0189] 9.2 T-cell infection

[0190] The activated T cells obtained in step 9.1 of this embodiment were collected, and the cell density was adjusted to 3 × 10⁻⁶. 5 Cells / mL were added to the concentrated lentivirus of Example 6 at a multiplicity of infection (MOI) of 10, and polybrene was added to a final concentration of 8 μg / mL. The culture medium was replaced with fresh medium after incubation overnight at 37°C and 5% CO2. Subculture was performed every 2-3 days.

[0191] 9.3 Chimeric antigen receptor expression

[0192] In this embodiment, step 9.2, 5 days after infection, 3×10⁻⁶ samples were collected. 5 T cells were centrifuged at 400g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with flow cytometry buffer (PBS + 2% bovine serum). The cells were resuspended in 50 μL of buffer, and RabbitAnti-Camelid VHH Antibody (iFluor488) antibody was added at a 1:1000 ratio. The cells were incubated on ice for 30 min. After washing once with buffer, the cells were resuspended in 300 μL of buffer. The chimeric antigen receptor infection efficiency of T lymphocytes was detected by flow cytometry. The results are shown in [Figure 1]. Figure 4 .

[0193] Meanwhile, synchronously treated T cells (T) and CAR-T cells (Unstained Ctrl) that were not incubated with antibodies served as controls.

[0194] from Figure 4 The presence of a significant positive cell population in the infected CAR-T cells indicates the successful construction of CAR-T cells expressing a chimeric antigen receptor. The antigen-binding domain of the chimeric antigen receptor contains the VHH-A5 antibody. The infection efficiency of the CAR-T cells corresponding to the VHH-A5 antibody was 58.65%.

[0195] Example 10: Study on the toxicity of CAR-T cells to target cells

[0196] In this embodiment, the toxicity of the CAR-T cells prepared in Example 9 to target cells was detected in real time using Real-Time Cell Analysis (RTCA) technology, including the following steps:

[0197] 1) Prepare the RTCA instrument and E-plate: Turn on the RTCA instrument, start the software and log in, select the required experiment type, put the E-plate into the RTCA instrument and check that the electrode array is clean and undamaged.

[0198] 2) Stabilize the baseline: Add 50 μL of 1640 medium containing 10% FBS to the E-plate and wait a few minutes to stabilize the baseline.

[0199] 3) Wild-type 293T, 786-O (Chinese Academy of Sciences Cell Bank), and ACHN (Chinese Academy of Sciences Cell Bank) cells were digested and collected as target cells. The cells were washed with PBS buffer and suspended in 1640 medium containing 10% FBS, so that the number of each type of cell was 15,000 / well.

[0200] 4) Cell examination and counting: Examine the morphology and health of the cells and count the cells using a cell counting chamber.

[0201] 5) Cell seeding: Take 100 μL of the counted cells and seed them into an E-plate.

[0202] 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.

[0203] 7) CAR-T treatment: During real-time monitoring, effector cells were added to the three types of target cells in step 3) of this embodiment at an effector-to-target ratio (E:T) of 2:1 and 0.5:1, respectively. The effector cells were 50 μL of CAR-T cells obtained in Example 9.

[0204] 8) Monitor tumor cell proliferation and calculate CAR-T killing activity using the cell index: lysis% = (1 - relative cell index) treatment / relative Cell index target )×100%, where relative Cell index treatment The ratio of the cell index at a certain time point in the co-culture group to the initial cell index, relative to the cell index. target The ratio of the cell index at a certain time point to the initial value in the co-tumor cell culture group is shown in the figure. Figure 5A , 5B And 5C.

[0205] from Figures 5A-5C It can be seen that the CAR-T cells constructed using VHH-A5 in this invention have no significant killing activity (Lysis) against CD70-negative 293T cells; under different effector-target ratios, their killing activity (Lysis) against CD70-positive tumor cells 786-O is above 70%; under different effector-target ratios, their killing activity (Lysis) against CD70-positive tumor cells ACHN is above 55%. This indicates that the CAR-T cells constructed in this invention have no significant killing effect on CD70-negative 293T cells, but have significant killing activity against CD70-positive tumor cells 786-O and CD70-positive ACHN cells, demonstrating that the CAR-T cells constructed in this invention not only possess highly efficient tumor-killing ability but also high specificity.

[0206] Example 11 Study on IFN-γ secretion by CAR-T cells

[0207] In this embodiment, the secretion of CAR-T cytokine IFN-γ was detected, including the following:

[0208] 11.1 Cell Culture Supernatant

[0209] The experiment was divided into three groups.

[0210] The first group (spontaneous or MOCK) consisted of CAR-T cells from Example 9 cultured alone at 37°C for 24 hours.

[0211] The second group (293T) consisted of CAR-T cells from Example 9 and CD70-negative 293T cells co-cultured at 37°C for 18 hours.

[0212] The second group (293T-CD70) consisted of CAR-T cells from Example 9 and CD70-positive target cells (293T-CD70) co-cultured at 37°C for 24 hours, with an effector-to-target ratio of 1:1.

[0213] Then, the cell cultures of each group were centrifuged at 400×g for 10 min to remove the precipitate, and the supernatant was stored at -80℃ for testing.

[0214] 11.2 Detection of IFN-γ

[0215] 11.2.1 Reagents: The Human IFN-γELISA Kit (Lianke Biotechnology, catalog number: EK180-96) was used for detection. Before the test, all reagents and samples were brought to 25°C. 1× wash buffer and 1× test buffer were prepared according to the instructions for use to detect antibodies.

[0216] 11.2.2 Preparation of Standards and Samples Standards: The stock solution of the standard was diluted twice using 5% 1640 culture medium, with a total of 8 dilution gradients, including zero concentration.

[0217] Samples: Dilute the samples using 5% 1640 medium.

[0218] 11.2.3 Testing Procedures

[0219] (1) Soaking the microplate: Add 300 μL of 1× washing solution and let it stand for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper.

[0220] (2) Add standard: Add 100 μL of 2-fold serially diluted standard to the standard wells and add 100 μL of 5% 1640 culture medium to the blank wells;

[0221] (3) Add sample: Add 100 μL of the cell culture supernatant obtained in step 11.1 of this embodiment to the sample well;

[0222] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0223] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0224] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times. After each wash, pat the plate dry on absorbent paper.

[0225] (7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0226] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25°C for 45 min;

[0227] (9) Washing: Repeat step (6);

[0228] (10) Adding substrate for color development: Add 100 μL of TMB substrate to each well, incubate in the dark at 25°C for 15 min;

[0229] (11) Add stop solution: Add 100 μL of stop solution to each well and mix thoroughly;

[0230] (12) Detection readings: Dual-wavelength detection was performed using an ELISA reader, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The calibrated OD value was the measured value at 450 nm minus the measured value at the reference wavelength. Results are shown below. Figure 6 .

[0231] from Figure 6It can be seen that the spontaneous MOCK group is a CAR-T cell group with almost no IFN-γ release. Similarly, IFN-γ was almost undetectable in the co-culture of CAR-T cells and CD70-negative 293T cells, while a higher level of IFN-γ was detected in the co-culture of CAR-T cells and CD70-positive 293T-CD70 cells (CD70+).

[0232] IFN-γ is an effective pro-inflammatory agent. In addition to stimulating pro-inflammatory macrophages and inducing apoptosis of Treg cells, it also has cell inhibition, anti-proliferation and pro-apoptotic effects on tumor cells, playing an important role in cellular immunity and anti-tumor response. It is beneficial for CAR-T cells to exert their killing effect on tumor cells.

[0233] Comparative Example

[0234] Example 1 simultaneously screened for two candidate antibodies, VHH-17 and VHH-28. CAR-T cells containing VHH-17 and VHH-28 antibodies, respectively, were constructed using the same methods as in Examples 4 to 9 of this application, and target cell toxicity studies were performed using the same methods as in Example 10. Results are shown below. Figures 5A-5C .

[0235] The sequence of the VHH-17 antibody is as follows:

[0236] EVQLVQSGGGSVQAGGSLRLSCAASGYTYSSNYMGWFRQASGKEREGVAAIARDGST SYADSVKGRFTISKDNANNTLYLQMNSLKPEDTAMYYCAADQLGPAHFAVVVGYGYWGQ GTQVTVSS(SEQ ID No.19)

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

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

[0239] from Figures 5A to 5CIt can be seen that, under different effector-to-target ratios, the killing activity of CAR-T cells constructed with VHH-17 antibody and VHH-28 antibody against CD70-positive 786-O cells is within 40%; under different effector-to-target ratios, the killing activity of CAR-T cells constructed with VHH-17 antibody and VHH-28 antibody against CD70-positive ACHN cells is within 60%.

[0240] In summary, the present invention provides a specific antibody that protects against tumor necrosis factor receptor superfamily 7 factor, exhibiting high affinity for CD70 antigen and EC50. 50 The concentration was 169.7 ng / mL. This invention also protects chimeric antigen receptors containing specific antibodies and CAR-T cells expressing chimeric antigen receptors. CAR-T cells exhibit high killing activity against CD70-positive tumor cells, reaching up to 100%. Furthermore, the cytokine IFN-γ secreted by CAR-T cells exceeds 2000 pg / mL. These results indicate that the specific antibodies, chimeric antigen receptors, CAR-T cells, and related products of this invention can be effectively applied to immunotherapy and tumor treatment, and are of great significance for the development of drugs for immunotherapy and tumor treatment.

[0241] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. A nanobody targeting tumor necrosis factor receptor superfamily factor 7, characterized in that, The heavy chain variable region of the nanobody includes complementarity-determining regions CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6, respectively.

2. The nanobody as described in claim 1, characterized in that, The heavy chain variable region also includes frame regions FR1, FR2, FR3, and FR4, respectively, representing the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No.

7.

3. The nanobody as described in claim 2, characterized in that, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO.

8.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antigen-binding domain, a hinge region, and a transmembrane domain. The antigen-binding domain comprises a nanobody as described in any one of claims 1-3. The transmembrane domain is selected from one or more of CD8α, CD28, or DAP10. The hinge region is selected from the CD8α hinge region. The chimeric antigen receptor comprises the nanobody, the hinge region, and the transmembrane domain sequentially from the N-terminus to the C-terminus.

5. The chimeric antigen receptor as described in claim 4, characterized in that, The chimeric antigen receptor also includes a signal peptide and / or a signal transduction domain.

6. The chimeric antigen receptor as described in claim 5, characterized in that, The signal peptide is selected from the CD8α signal peptide; And / or, the signal transduction domain is selected from immune receptor tyrosine activation motifs and / or co-stimulatory molecules.

7. A substance characterized in that, The substance is selected from one or more of the following: B1) A polynucleotide, wherein the polynucleotide encodes a nanobody as described in any one of claims 1-3, or a chimeric antigen receptor as described in any one of claims 4-6; B2) A vector comprising the polynucleotide as described in B1); B3) The modified cell, said modified cell comprising the vector as described in B2); or the genome having integrated polynucleotides as described in B1); B4) A recombinant virus, wherein the recombinant virus is obtained by viral packaging of a vector as described in B2); B5) A viral vector system comprising a vector as described in B2) and an auxiliary plasmid or host cell; B7) A pharmaceutical composition comprising a therapeutically effective amount of a nanobody as described in any one of claims 1-3, or a chimeric antigen receptor as described in any one of claims 4-6, or a polynucleotide as described in B1), or a carrier as described in B2), or a modified cell as described in B3).

8. An application characterized in that, The application is selected from one or more of the following: 1) The use of the nanobody as described in any one of claims 1-3, or the chimeric antigen receptor as described in any one of claims 4-6, or the polynucleotide as described in claim B1) of claim 7, or the carrier as described in claim B2), or the modified cell as described in claim B3) in the preparation of products for in vitro detection of CD70; 2) The use of the nanobody as described in any one of claims 1-3, or the chimeric antigen receptor as described in any one of claims 4-6, or the polynucleotide as described in claim 7 (B1), or the carrier as described in claim 7 (B2), or the modified cell as described in claim 7 (B3), or the pharmaceutical composition as described in claim 7 (B7) in the preparation of a medicament for the prevention or treatment of diseases related to CD70 expression, wherein the diseases related to CD70 expression are selected from one or more of acute myeloid leukemia, renal cell carcinoma, esophageal cancer, mesothelioma, gastric cancer, adenocystic carcinoma, ovarian cancer, endometrial cancer, breast cancer, squamous cell carcinoma of the head and neck, glioma, lung cancer, osteosarcoma, thyroid cancer, melanoma, pancreatic cancer, nasopharyngeal carcinoma, myeloma, and lymphoma.

9. The application as described in claim 8, characterized in that, The myeloma includes multiple myeloma; And / or, the lymphoma includes Hodgkin's lymphoma and non-Hodgkin's lymphoma; And / or, the glioma comprises glioblastoma.

10. A method, characterized in that, The method is selected from one or more of the following: 1) A method for preparing a nanobody as described in any one of claims 1-3 or a chimeric antigen receptor as described in any one of claims 4-6, comprising the following steps: a) transforming a polynucleotide as described in claim B1) or a vector as described in claim B2) into a host cell, or directly culturing cells modified as described in claim B3); b) expressing the polynucleotide; c) recovering the nanobody or the chimeric antigen receptor from the culture; 2) A method for preparing the modified cells as described in claim 7 (B3), comprising the following steps: introducing the nanobody as described in any one of claims 1-3, or the chimeric antigen receptor as described in any one of claims 4-6, or the polynucleotide as described in claim 7 (B1) or the carrier as described in claim 7 (B2) into the target cells.

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