Specific antibodies targeting tumor necrosis factor receptor superfamily factor 7

Abstract

The invention discloses a specific antibody targeting tumor necrosis factor receptor superfamily Factor 7. The specific antibody comprises a heavy chain variable region, and the heavy chain variable region comprises complementarity determining regions CDR1, CDR2 and CDR3 of amino acid sequences shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6. The invention further discloses a detection method of the heavy chain variable region. The specific antibody provided by the invention has high affinity and specificity. The CAR-T cell constructed by adopting the specific antibody can specifically recognize tumor cells, secrete cell factors IFN-gamma and efficiently kill the tumor cells.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to specific antibodies targeting tumor necrosis factor receptor superfamily member 7 factor. Background Art

[0002] Whether it is tumor and cancer diseases, or nervous system and inflammatory diseases, tumor necrosis factor (TNF) is always involved. As a member of the TNF superfamily, CD70 is a type II transmembrane protein that has the ability to regulate the activation, proliferation, and differentiation of T cells and B cells, and plays an important role in regulating the immune response process.

[0003] Tumor necrosis factor receptor superfamily member 7 factor (TNFSF7), also known as CD27 Ligand, CD70. TNFSF7 is a type II transmembrane glycoprotein with a molecular weight of approximately 50 kDa, mainly composed of an extracellular binding region, a transmembrane region, and a cytoplasmic region, and exists in a trimeric form. The receptor of TNFSF7 is CD27, and the function of TNFSF7 can promote the activation, proliferation, and differentiation of T cells and B cells, and regulate the immune response. Under normal circumstances, TNFSF7 is mainly expressed in activated lymphocytes, and under pathological conditions, TNFSF7 is highly expressed in various tumor tissues. Therefore, TNFSF7 can be used as a potential target for tumor treatment. In addition, TNFSF7 is closely related to the occurrence and development of tumors and the prognosis of patients, and can be used as a new biomarker for early cancer diagnosis and a new target for monitoring disease prognosis.

[0004] Currently, the research on TNFSF7 mainly focuses on monoclonal antibodies, antibody-drug conjugates (ADCs), chimeric antigen receptor T cell therapies (CAR-T), etc. However, according to some literature reports, ADCs have important adverse events such as thrombocytopenia, and currently, there are 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 member 7 factor (TNFSF7). Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of this application is to provide specific antibodies targeting tumor necrosis factor receptor superfamily member 7 factor to solve the problems in the prior art.

[0006] The present invention uses the extracellular domain recombinant protein of CD70 to immunize alpacas to produce high-titer antibodies, and constructs a library with a capacity greater than 10 9The phage display camel VHH immune library was panned and amplified to obtain specific antibodies (VHH-A5 or CD-A5) targeting tumor necrosis factor receptor superfamily 7 factor. The specific antibodies of the present invention can recognize the CD70 antigen on the cell surface with high affinity. At the same time, chimeric antigen receptors and CAR-T cells containing the chimeric antigen receptors were constructed using the specific antibodies of the present invention, and it was found that they have killing activity against CD70-positive tumor cells and can efficiently secrete the cytokine IFN-γ. The present invention was completed on this basis.

[0007] To achieve the above object, the present invention specifically adopts the following technical solutions.

[0008] In a first aspect of the present application, there is provided a specific antibody targeting tumor necrosis factor receptor superfamily 7 factor, the specific antibody comprising a heavy chain variable region, the heavy chain variable region comprising complementarity-determining regions CDR1, CDR2, CDR3 having the amino acid sequences shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6.

[0009]

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

[0011] Sequence number Sequence FR1 1 EVQLVQSGGGSVQAGGSLRLSCKAS FR2 3 MGWFRQAPGKEREGVAA FR3 5 HYADSVKGRFTISRDNAENTVTLQMNSLKPEDTAMYYC FR4 7 WGQGTQVTVSS

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

[0013] In the following sequences, the bold represents the framework region, Underline and the CDR regions are:

[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 in animals such as alpacas and camels, and 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, the relatively long CDR3 region will form a relatively large protruding structure that inserts into some conserved "cavity structures" to form a stable antigen-antibody complex.

[0016] In some embodiments, the specific antibody comprises an antigen-binding fragment thereof, and the antigen-binding fragment refers to any antibody fragment capable of binding to a target antigen.

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

[0018] The second aspect of the present application protects a chimeric antigen receptor, which comprises an antigen-binding domain, a hinge region, and a transmembrane domain, and the antigen-binding domain comprises the 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 sequentially comprises the specific antibody, the hinge region, and the transmembrane domain from the N-terminus to the C-terminus as described above.

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

[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 specific embodiments, the signal peptide comprises the 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 certain specific embodiments, the signal transduction domain comprises an immunoreceptor tyrosine-based activation motif or a co-stimulatory molecule. The co-stimulatory molecule comprises 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. The amino acid sequence of the immunoreceptor tyrosine-based activation motif comprises the sequence shown in SEQ ID NO.12. The amino acid sequence of the intracellular region of 4-1BB 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, the co-stimulatory molecule 4-1BB, and an immunoreceptor tyrosine-based activation motif.

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

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

[0033] Further, the substance is a vector comprising the polynucleotide as described above.

[0034] In the present invention, the vector refers to a vector that can introduce a polynucleotide into a host cell, and the method is to transform the host and promote the expression of the introduced polynucleotide, for example, transcription and translation.

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

[0036] Further, the substance is a modified cell, and the modified cell contains the vector as described above, or the polynucleotide as described above is integrated into the genome.

[0037] In the present invention, a modified cell refers to introducing a coding sequence of a specific antibody or a chimeric antigen receptor into a host cell so that the host cell expresses the introduced coding sequence to express a specific antibody or a chimeric antigen receptor. The host cell that receives and expresses the introduced coding sequence is a 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 examples, it is a T lymphocyte.

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

[0040] Further, the substance is a recombinant virus, and the recombinant virus contains the vector described above.

[0041] In some embodiments, the recombinant virus is packaged from 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] Further, the substance is a virus vector system, and the virus vector system contains the vector as described above and an auxiliary plasmid or a host cell.

[0043] In some embodiments, the auxiliary plasmid may include a packaging plasmid and an envelope plasmid. In some examples, the packaging plasmid is pMDlg-RRE / pRSV-REV, the envelope plasmid is VSVg, and all three plasmids can be obtained commercially. In some examples, the virus vector system contains the lentiviral vector HD SIN03-CD70 CAR, pMDlg-RRE, pRSV-REV and VSVg.

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

[0045] Further, the substance is a pharmaceutical composition, and the pharmaceutical composition comprises a therapeutically effective amount of a specific antibody as in the first aspect, or a chimeric antigen receptor as in the second aspect, or a polynucleotide as described above, or a vector as described above, or a modified cell as described above, or an antibody-drug conjugate as described above.

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

[0047] Further, the substance is a product, and the product comprises a specific antibody as in the first aspect, a chimeric antigen receptor as in the second aspect, 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 certain embodiments, the product is selected from one or more of a kit, a nucleic acid strip, a chip, a system or a device.

[0049] The fourth aspect of the present application protects an application, and the application is selected from one or more of the following:

[0050] Further, the application of a specific antibody as in the first aspect, or a chimeric antigen receptor as in the second aspect, or a polynucleotide as described above or a vector as described above or a modified cell as described above in the preparation of a product for in vitro detection of CD70.

[0051] In certain embodiments, the product for in vitro detection of CD70 can generally target the target CD70 antigen and be used for diagnosis with CD70 antigen as a biomarker. The product may further comprise a label, and the label can generally be used to label a specific antibody, including: a fluorescent label, a chromogenic label, a reporter gene, a localization signal, etc. The antibody after linking the label can be detected by common detection methods such as ELISA (enzyme-linked immunosorbent assay) and immunofluorescence techniques, immunoradiometric techniques, immunoenzymatic techniques and immunogold techniques, immunohistochemical staining (tissue) samples, flow cytometry, etc.

[0052] Further, the application of a specific antibody as in the first aspect, or a chimeric antigen receptor as in the second aspect, or a polynucleotide as described above or a vector as described above or a modified cell as described above in the preparation of a drug for preventing or treating a disease related to CD70 expression.

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

[0054] In certain specific embodiments, the targets of the medicament include, but are not limited to: mammals. Mammals such as humans, other primates, pigs, cows, horses, cats, dogs, sheep, goats, and rodents, specifically mice, rats, rabbits, guinea pigs, and hamsters.

[0055] In certain specific embodiments, the related disease is a tumor, and the tumor is a malignant tumor expressing CD70, including lymphoma, acute myeloid leukemia, multiple myeloma, renal cell carcinoma, esophageal cancer, mesothelioma, gastric cancer, adenocarcinoma, 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 certain specific embodiments, the related disease is an immune disease, and the immune disease is an immune disease expressing CD70, including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

[0057] Furthermore, the use of the specific antibody as in the first aspect, or the chimeric antigen receptor as in the second aspect, or the polynucleotide as described above, or the vector as described above, or the modified cell as described above in the preparation of a medicament for regulating the activity or level of CD70 protein.

[0058] The fifth aspect of the present application protects a method, which is selected from one or more of the following:

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

[0060] a) Transforming the polynucleotide as described above or the vector as described above into a host cell, or directly culturing the modified cell as described above;

[0061] b) Expressing the polynucleotide;

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

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

[0064] Introduce 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 into the target cells.

[0065] Furthermore, a method for non-diagnostic and non-therapeutic detection of CD70 protein or nucleic acid molecule encoding the same in a sample to be tested, comprising the following steps:

[0066] Contact the sample to be tested with the specific antibody as described in the first aspect, the chimeric antigen receptor as described in the second aspect, and the pharmaceutical composition as described above;

[0067] Detect the formation of a complex of the CD70 protein or nucleic acid molecule encoding the same and the specific antibody and the chimeric antigen receptor.

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

[0069] 1) The specific antibody targeting tumor necrosis factor receptor superfamily member 7 of the present invention can specifically bind to the CD70 antigen with good affinity. Through the determination of the antibody half-maximal effective concentration (EC 50 ), it can be known that its EC 50 is 169.7 ng / mL.

[0070] 2) The specific antibody targeting tumor necrosis factor receptor superfamily member 7 of the present invention has good affinity. Using it as an antigen-binding domain, a chimeric antigen receptor is constructed, and T cells (CAR-T cells) are prepared using the 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 specific antibody of the present invention can be effectively applied to immunotherapy, which is of great significance for the development of tumor therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Shown is a graph of the antibody half-maximal effective concentration of the specific antibody detected by flow cytometry in Example 2 of the present application.

[0072] Figure 2A Shown is a FACS detection result graph of the specific antibody recognizing wild-type 293T (CD70 negative) in Example 3 of the present application.

[0073] Figure 2B Shown is a FACS detection result graph of the specific antibody recognizing 293T-CD70 cells (CD70 positive) overexpressing CD70 protein in Example 3 of the present application.

[0074] Figure 3AIt shows the map of plasmid HD SIN03 CD19 in Example 4 of this application.

[0075] Figure 3B It shows the map of the lentiviral vector expressing chimeric antigen receptor in Example 4 of this application.

[0076] Figure 3C It shows the schematic diagram of the structure of the spectrum chimeric antigen receptor in Example 4 of this application.

[0077] Figure 4 It shows the flow cytometry detection result graph of the chimeric antigen receptor expression rate in Example 9 of this application.

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

[0079] Figure 5B It shows the killing effect of CAR-T cells on renal cell carcinoma cell line 786-O in Example 10 of this application.

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

[0081] Figure 6 It shows the bar graph of IFNγ secreted by CAR-T cells in Example 11 of this application. Detailed implementation manners

[0082] In order to make the invention purpose, technical solutions and beneficial effects of this application clearer, the following further illustrates this application with examples. It should be understood that the examples are only used to explain this application and are not used to limit the scope of the application. The test methods used in the following examples are all conventional methods unless otherwise specified. Those familiar with this technology can easily understand other advantages and effects of this application from the content disclosed in this specification.

[0083] The following illustrates the implementation manners of the present invention through specific specific examples. 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 implementation manners. 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 the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than limiting 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 plural forms.

[0085] When an embodiment gives 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 or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0086] Example 1

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

[0088] 1.1 Construction of phage specific antibody library

[0089] Alpacas were immunized with the extracellular domain recombinant protein of CD70 (Acro, product number: CD7-H52H7). After detecting the serum titer by ELISA, peripheral blood was drawn; lymphocytes were separated to obtain peripheral blood mononuclear lymphocyte precipitates, total RNA was extracted, and then reverse transcribed into the first strand of 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 phages were 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.

[0090] 1.2 Screening of phage specific antibody library

[0091] 1) First, 293T cells were co-incubated with the antibody library for negative screening, and then the supernatant was taken and incubated with 293T-CD70-GFP cells (the construction process of CD70 positive is detailed in Example 8) and 293T cells respectively;

[0092] 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 PEG8000 / NaCl precipitation method and enter the next round of screening;

[0093] 3) After enrichment appears, use the obtained phage as a template, amplify the VHH region, perform gene sequencing, obtain candidate antibodies, and name them VHH-A5.

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

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

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

[0097] 2.1 VHH Fc-specific Antibody Expression and Purification

[0098] To further identify these antibodies, it is necessary to express the antibodies in mammalian cells. Therefore, a recombinant plasmid of the recombinant VHH-A5 antibody (i.e., VHH-CD70-A5 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:

[0099] The method for constructing the recombinant plasmid can be conventional. For example, the coding sequence of VHH-CD70-A5 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 to obtain the recombinant VHH-A1 antibody (i.e., VHH-CD70-A5 Fc) with a mouse Fc tag.

[0100] Detect the absorbance value of OD280 by an enzyme-linked immunosorbent assay (ELISA) reader and calculate the concentration, and detect the purity and molecular weight by SDS-PAGE gel.

[0101] The quality detection results of the CD70 VHH antibody with a mouse Fc tag are shown in Table 1.

[0102] Table 1

[0103]

[0104] 2.2 Antibody Affinity Determination

[0105] The anti-CD70 VHH antibody with a murine Fc tag obtained in Step 2.1 was subjected to flow cytometry to determine the half-maximal effective concentration (EC50) of the antibody.

[0106] The half-maximal effective concentration (EC 50 ) is a method for measuring the strength of the interaction between an antibody and its target. The antibody was diluted to different concentrations (starting from 4 μg / mL, serially diluted 4-fold, for a total of 10 gradients), and after binding to 293T-CD70 cells overexpressing CD70 (293T-CD70-GFP, the preparation process is detailed in Example 8), it was 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.

[0107] 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 Figure.

[0108] From Figure 1 it can be seen that the EC 50 of the specific antibody is 169.7 ng / mL.

[0109] Example 3 Specificity Analysis of the Antibody

[0110] In this Example 3, flow cytometry was performed on the specific antibody.

[0111] Wild-type 293T cells (CD70-negative, stored in our company) were incubated with the purified anti-CD70 VHH antibody A5 with a murine 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. Flow cytometry (FACS) was used for detection, and the results are shown in Figure 2A . At the same time, wild-type 293T cells without the addition of the anti-CD70 VHH antibody and the APC-labeled goat anti-mouse IgG antibody were used as the blank control group (Ctrl); wild-type 293T cells without the addition of the anti-CD70 VHH antibody but with the addition of the APC-labeled goat anti-mouse IgG antibody were used as the control group (secAb).

[0112] The 293T-CD70 cells (293T-CD70-GFP) stably overexpressing the CD70 protein (the preparation process is detailed in Example 8) were incubated with the purified anti-CD70 VHH antibody A5 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 a 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 a control group (secAb).

[0113] From Figure 2A and Figure 2B , it can be seen that the specific antibody 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 this Example 4, a lentiviral vector expressing a chimeric antigen receptor was prepared, and the chimeric antigen receptor contains the specific antibody VHH-A5.

[0116] 4.1 Construction of the lentiviral vector

[0117] 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 recombinantly ligated with the CD8ahinge-TM-41BB-CD3Z fragment and the digested HD SIN03 CD19 CAR plasmid to obtain a lentiviral vector.

[0118] The specific construction method is as follows:

[0119] 4.1.1 Preparation of the CD8αsingal CD70 VHH fragment

[0120] The PCR reaction system was prepared according to Table 3 (the reagents in the table are from TOYOBO Inc.) to amplify each specific antibody fragment against CD70, and the reaction was carried out according to the PCR program shown in Table 4. The primer sequences are:

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

[0122] CTGCAGGTCGACTCTAGAGGATCCCACCATGGCCTTACCAGTGA

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

[0124] GTCGCGGCGCTGGCGTCGTGGT

[0125] Table 3

[0126] Reagent Volume (μL) 10×buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO 4 > 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, 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 quantified by the ultraviolet absorption method.

[0131] In the case of knowing the amino acid sequence, the above CD8α singal CD70 VHH fragment can also be obtained by synthesizing its coding nucleotides.

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

[0133] 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.

[0134] 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.

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

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

[0137] Table 5

[0138] Reagent Volume (μL) 10×buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO 4 > 3 10μMCD8αH-F 1 10μMVector-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 Digested Vector

[0140] Digest 5 μg of the HD SIN03 CD19 CAR plasmid constructed in the laboratory (plasmid map shown in Figure 3A ) with BamHI and EcoRI double enzymes. After a 1-hour water bath reaction at 37°C, recover the vector, with a length of approximately 7710 bp.

[0141] 4.1.4 Ligation

[0142] Ligate 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 digested vector in step 4.1.3 of this example according to the recombinant reaction system in Table 6. The reaction conditions are: 37°C, 30 min, to obtain the recombinant product. The recombinant reaction system is shown in Table 6.

[0143] Table 6

[0144] Reagent Dosage HD SIN03 CD19 CAR backbone in Step 4.1.3 154.2 ng CD8αsingal CD70VHH fragment in Step 4.1.1 10 ng CD8αhinge-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

[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 (100 μL) at 42°C. Select positive monoclonal strains with a kana-resistant plate for PCR identification.

[0147] Using LV-F2 and LV-R as primers, prepare according to the reaction system in Table 7, and then perform PCR reaction according to the program in Table 8 to obtain the PCR product. After PCR, select positive clones for further sequencing identification, and the sequencing results are as expected.

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

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

[0150] Table 7

[0151] Reagent 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, the correctly identified clones were selected and sent to the company for sequencing (the sequencing primers were LV-F2 and LV-R, and the primers are shown in Table 12). Finally, the correct clones were selected for shaking bacteria, and plasmids were extracted using a plasmid large extraction kit. The positive clone was the constructed lentiviral vector HDSIN03-CD70 CAR, and the map is shown in Figure 3B 。

[0155] The schematic diagram of the expressed chimeric antigen receptor is as shown in Figure 3C shown, including the CD8α signal peptide, the specific antibody against CD70 (anti-CD70 VHH), the CD8α hinge region, the transmembrane region, and the immunoreceptor tyrosine-based activation motif (CD3ζ).

[0156] Among them, 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 as shown in SEQ ID No.8.

[0158] The amino acid sequence (SEQ ID No.10) of the CD8α hinge region and transmembrane region is:

[0159] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

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

[0161] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

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

[0163] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0164] Example 5 Lentivirus packaging

[0165] In this example, the lentiviral vector obtained in Example 4 was packaged into lentivirus, including the following:

[0166] 1) At 6.0×10 6Inoculate 293T cells into a 10-cm culture dish and incubate overnight at 37°C with 5% CO 2 Prepare for virus packaging. The culture medium is 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 of 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 a PEI mixture.

[0169] 4) Formation of transfection complex: Add the PEI mixture obtained in step 3) to the DNA mixture, gently mix immediately after addition, and incubate at 25°C for 20 min to obtain a transfection complex.

[0170] 5) Drop 1.6 mL of the transfection complex 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 collect the virus supernatant after culturing for 48 h.

[0171] Example 6 Lentivirus concentration

[0172] In this example, the lentivirus obtained in Example 5 is concentrated as follows:

[0173] Filter the virus supernatant of Example 5 through a 0.45-μm filter membrane and collect it into a 50-mL centrifuge tube. Add 1 / 4 of the PEG-NaCl virus concentrate, invert and mix well, 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; dispense the concentrated lentivirus suspension into 50-μL aliquots, store in a finished product tube, and store at -80°C.

[0174] Example 7 Lentivirus titer detection

[0175] In this example, the lentivirus obtained in Example 6 is titer-detected as follows:

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

[0177] 2) Add the concentrated lentivirus from Example 6 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 at 37 °C in 5% CO 2 After culturing overnight, 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 in 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:

[0180] Titer (TU / mL) = number of cells (10 5 ) × positive rate / volume of virus (mL).

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

[0182] In this example, to construct 293T - CD70 cells (293T - CD70 - GFP) overexpressing CD70 protein, the following steps are included:

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

[0184] 2) Inoculate 1 × 10 6 293T cells (preserved by our company) into a 6 - well plate, and add 1 mL of the above - obtained CD70 - GFP lentivirus to obtain 293T - CD70 cells (293T - CD70 - GFP) overexpressing CD70 protein.

[0185] Construction of CAR - T cells in Example 9

[0186] In this example, transduce T lymphocytes with the lentivirus from Example 6, including the following steps:

[0187] 9.1. Activation of T lymphocytes

[0188] Adjust the density of human PBMC to 1×10 6 cells / mL with T cell medium (X-VIVO + 10% FBS + 300 U / mL IL-2), 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.

[0189] 9.2. T cell infection

[0190] Collect the activated T cells obtained in step 9.1 of this example, adjust the cell density to 3×10 5 cells / mL, add to the concentrated lentivirus of Example 6 according to a multiplicity of infection MOI = 10, and add polybrene to a final concentration of 8 μg / mL; culture overnight at 37°C in a 5% CO 2 environment and then replace with fresh medium. Passage every 2 - 3 days.

[0191] 9.3. Chimeric antigen receptor expression

[0192] Five days after the infection in step 9.2 of this example, take 3×10 5 T cells, centrifuge at 4°C and 400 g for 5 min, discard the supernatant, 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 4 .

[0193] Meanwhile, use simultaneously processed T cells (T) and CAR-T not incubated with the antibody (Unstained Ctrl) as controls.

[0194] From Figure 4 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 the VHH-A5 antibody. The infection efficiency of CAR-T cells corresponding to the VHH-A5 antibody is 58.65%.

[0195] Example 10 Toxicity study of CAR-T cells on target cells

[0196] In this example, the Real-Time Cell Analysis (RTCA) technology was used to dynamically detect the toxicity of the CAR-T cells prepared in Example 9 to target cells in real time, 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, place the E-plate into the RTCA instrument and check whether 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 for a few minutes to stabilize the baseline.

[0199] 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.

[0200] 4) Cell inspection and counting: Check the cell morphology and health status, and count the cells using a cell counting chamber.

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

[0202] 6) Real-time monitoring: Place the E-plate into the RTCA instrument, start the real-time monitoring program, and record impedance or capacitance data.

[0203] 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 9.

[0204] 8) Monitor 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. The results are shown in Figure 5A 、 5B and 5C.

[0205] FromFigure 5A-5C It can be seen that the CAR-T cells constructed with VHH-A5 of the present invention have no obvious killing activity (Lysis) against CD70-negative 293T cells; at different effector-to-target ratios, their killing activity (Lysis) against CD70-positive tumor cells 786-O is above 70%; at different effector-to-target ratios, their killing activity (Lysis) against CD70-positive tumor cells ACHN is above 55%. This shows that the CAR-T cells constructed in the present invention have no obvious killing effect on CD70-negative 293T cells, but have obvious killing activity against 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.

[0206] Example 11 Study on the Secretion of IFN-γ by CAR-T Cells

[0207] In this example, the secretion of the CAR-T cell factor 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) was: the CAR-T cells in Example 9 were cultured alone at 37°C for 24 h.

[0211] The second group (293T) was: the CAR-T cells in Example 9 and CD70-negative 293T cells were co-cultured at 37°C for 18 h.

[0212] The second group (293T-CD70) was: the CAR-T cells in Example 9 and CD70-positive target cells (293T-CD70) were co-cultured at 37°C for 24 h, and the effector-to-target ratio was 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 taken and stored at -80°C for later detection.

[0214] 11.2 Detection of IFN-γ

[0215] 11.2.1 Reagents: Human IFN-γ ELISA Kit (Linkage Biotechnology, product number: EK180-96) was used for detection. Before detection, all reagents and samples were restored to 25°C, and 1× wash solution, 1× detection buffer, and detection antibody were prepared according to the instructions.

[0216] 11.2.2 Preparation of Standard Products and Samples Standard Products: The standard product stock solution was diluted 2-fold with 5% 1640 medium, and there were a total of 8 dilution gradients, including zero concentration.

[0217] Sample: Dilute the sample proportionally using 5% 1640 medium.

[0218] 11.2.3 Detection steps

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

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

[0221] (3) Add the sample: Add 100 μL of the cell culture supernatant obtained in step 11.1 of this example to the sample wells.

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

[0223] (5) Incubate: Seal the plate with a sealing film, shake at 300 rpm, and incubate at 25 °C for 2 h.

[0224] (6) Wash: 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.

[0225] (7) Add the enzyme and incubate: Add 100 μL of the diluted streptavidin peroxidase (diluted 1:100) to each well.

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

[0227] (9) Wash: Repeat step (6).

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

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

[0230] (12) Detection and reading: Use an 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. 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 6 .

[0231] From Figure 6It can be seen 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 CD70-negative 293T cells, while a relatively high content of IFN-γ factor is detected in the co-culture of CAR-T cells and 293T-CD70 cells (CD70+) that are CD70-positive.

[0232] The IFN-γ factor is an effective pro-inflammatory agent. In addition to stimulating pro-inflammatory macrophages and inducing apoptosis of Treg cells, it also has cell inhibitory, anti-proliferative, and pro-apoptotic effects on tumor cells, plays an important role in cellular immunity and anti-tumor responses, and is beneficial for CAR-T cells to exert a killing effect on tumor cells.

[0233] Comparative Example

[0234] In Example 1, 2 candidate antibodies, namely VHH-17 antibody and VHH-28 antibody, were simultaneously screened. CAR-T cells containing VHH-17 antibody and VHH-28 antibody were constructed respectively by using the same method as in Examples 4 to 9 of the present application, and the target cell cytotoxicity study was carried out by using the same method as in Example 10. The results are shown in Figure 5A - Figure 5C 。

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

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

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

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

[0239] From Figure 5A to Figure 5CIt can be seen that at different effector-to-target ratios, the killing activities of CAR-T cells constructed with VHH-17 antibody and VHH-28 antibody against CD70-positive 786-O cells are both within 40%; at different effector-to-target ratios, the killing activities of CAR-T cells constructed with VHH-17 antibody and VHH-28 antibody against CD70-positive ACHN cells are within 60%.

[0240] In summary, the present invention protects a specific antibody targeting tumor necrosis factor receptor superfamily 7 factor, which has a high affinity for CD70 antigen and an EC 50 value of 169.7 ng / mL; the present invention also protects a chimeric antigen receptor containing the specific antibody and a CAR-T cell expressing the chimeric antigen receptor. The CAR-T cell has a high killing activity against CD70-positive tumor cells, up to 100%; in addition, the cytokine IFN-γ secreted by the CAR-T cell is above 2000 pg / mL. The above results indicate that the specific antibody, chimeric antigen receptor, CAR-T cell and their related products of the present invention can be effectively applied to immunotherapy and tumor treatment, which is of great significance for the development of drugs for immunotherapy and tumor treatment.

[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 idea disclosed by the present invention should still be covered by the claims of this application.

Claims

1. A specific antibody targeting tumor necrosis factor receptor superfamily member 7, wherein the specific antibody comprises a heavy chain variable region, characterized in that, the heavy chain variable region comprises complementarity determining regions CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID No.2, SEQ ID No.4, and SEQ ID No.6, respectively.

2. The specific antibody according to claim 1, characterized in that, the heavy chain variable region further comprises framework regions FR1, FR2, FR3, and FR4 having the amino acid sequences shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, and SEQ ID No.7, respectively.

3. The specific antibody according to claim 2, characterized in that, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.

8.

4. The specific antibody according to claim 1, characterized in that, the specific antibody is a nanobody.

5. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor comprises an antigen-binding domain, a hinge region, and a transmembrane domain, and the antigen-binding domain comprises the specific antibody according to any one of claims 1-4.

6. The chimeric antigen receptor according to claim 5, characterized in that, it comprises one or more of the following technical features: A1) The transmembrane domain is selected from one or more of CD8α, CD28, or DAP10; A2) The hinge region is selected from the CD8α hinge region; A3) The chimeric antigen receptor sequentially comprises the specific antibody, the hinge region, and the transmembrane domain from the N-terminus to the C-terminus; A4) The chimeric antigen receptor further comprises a signal peptide and / or a signal transduction domain.

7. The chimeric antigen receptor according to claim 6, characterized in that, the signal peptide is selected from the CD8α signal peptide; and / or, the signal transduction domain is selected from immunoreceptor tyrosine-based activation motifs and / or costimulatory molecules.

8. A substance, characterized in that, the substance is selected from one or more of the following: B1) A polynucleotide that encodes the specific antibody according to any one of claims 1-4, or the chimeric antigen receptor according to any one of claims 5-7; B2) A vector that contains the polynucleotide as described in B1); B3) A modified cell that contains the vector as described in B2); or a polynucleotide as described in B1) is integrated into the genome; B4) A recombinant virus that is obtained by packaging the vector as described in B2) with a virus; B5) A viral vector system that contains the vector as described in B2) and an auxiliary plasmid or a host cell; B6) An antibody-drug conjugate that contains the specific antibody according to any one of claims 1-4, or the chimeric antigen receptor according to any one of claims 5-7, and a conjugate conjugated thereto; B7) A pharmaceutical composition, comprising a therapeutically effective amount of a specific antibody as described in any one of claims 1-4, or a chimeric antigen receptor as described in any one of claims 5-7, or a polynucleotide as described in B1), or a vector as described in B2), or a modified cell as described in B3), or an antibody-drug conjugate as described in B6). A8) A product, comprising a specific antibody as described in any one of claims 1-4, a chimeric antigen receptor as described in any one of claims 5-7, or a polynucleotide as described in B1), or a vector as described in B2), or a modified cell as described in B3), or a recombinant virus as described in B4), or a viral vector system as described in B5), or an antibody-drug conjugate as described in B6).

9. An application Characterized in that The application is selected from one or more of the following: 1) The application of a specific antibody as described in any one of claims 1-4, or a chimeric antigen receptor as described in any one of claims 5-7, or a polynucleotide as described in B1) or a vector as described in B2) or a modified cell as described in B3) in claim 8 in the preparation of a product for in vitro detection of CD70. 2) The application of a specific antibody as described in any one of claims 1-4, or a chimeric antigen receptor as described in any one of claims 5-7, or a polynucleotide as described in B1) or a vector as described in B2) or a modified cell as described in B3) or an antibody-drug conjugate as described in B6) or a pharmaceutical composition as described in B7) in the preparation of a drug for preventing or treating a disease associated with CD70 expression. 3) The application of a specific antibody as described in any one of claims 1-4, or a chimeric antigen receptor as described in any one of claims 5-7, or a polynucleotide as described in B1) or a vector as described in B2) or a modified cell as described in B3) or an antibody-drug conjugate as described in B6) or a pharmaceutical composition as described in B7) in the preparation of a drug for regulating the activity or level of CD70 protein. 4) The application of a specific antibody as described in any one of claims 1-4, or a chimeric antigen receptor as described in any one of claims 5-7, or a product as described in B5) in claim 8 in the in vitro non-diagnostic and non-therapeutic detection of the activity level of CD70 protein.

10. A method Characterized in that The method is selected from one or more of the following: 1) A method for preparing a specific antibody as described in any one of claims 1-4 or a chimeric antigen receptor as described in any one of claims 5-7, comprising the following steps: a) transforming a polynucleotide as described in B1) or a vector as described in B2) in claim 8 into a host cell, or directly culturing a modified cell as described in B3) in claim 8; b) expressing the polynucleotide; c) recovering the specific antibody or the chimeric antigen receptor from the culture. 2) A method for preparing the engineered cells as described in B3) of claim 8, comprising the following steps: introducing the specific antibody as described in any one of claims 1-4, or the chimeric antigen receptor as described in any one of claims 5-7, or the polynucleotide as described in B1) of claim 8 or the vector as described in B2) into target cells; 3) A method for non-diagnostic and non-therapeutic detection of CD70 protein or nucleic acid molecule encoding the same in a test sample, comprising the following steps: contacting the test sample with the specific antibody as described in any one of claims 1-4, or the chimeric antigen receptor as described in any one of claims 5-7, or the pharmaceutical composition as described in B7) of claim 8; detecting the formation of a complex of the CD70 protein or nucleic acid molecule encoding the same with the specific antibody or the chimeric antigen receptor.

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