Anti-CD70 monoclonal antibody, hybridoma cell strain and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-CD70 monoclonal antibody, a hybridoma cell strain and application thereof. The invention provides a monoclonal antibody. Amino acid sequences of three CDR regions of a heavy chain of the monoclonal antibody respectively have amino acid sequences shown as SEQ ID NO: 11, 12 and 13; the amino acid sequences of the three CDR regions of the light chain of the gene respectively have the amino acid sequences shown as SEQ ID NO: 6, 7 and 8. The anti-CD70 monoclonal antibody provided by the invention can specifically recognize a CD70 recombinant protein expressed by eukaryotic expression and a CD70 protein expressed on a cell membrane. The CD70 detection kit constructed by the anti-CD70 monoclonal antibody is high in sensitivity and good in specificity, and has a better application effect in various detections including enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to anti-CD70 monoclonal antibodies, hybridoma cell lines and applications thereof. Background Art

[0002] CD70 is a type II transmembrane protein belonging to the tumor necrosis factor family. It is composed of 193 amino acids and has a molecular weight of 50 kDa. It is mainly expressed on the surface of activated T cells, B cells, natural killer cells and other immune cells, and plays a certain role in maintaining immune homeostasis. The CD70 receptor is CD27. The interaction between CD70 and CD27 can promote the activation, proliferation and differentiation of T cells and B cells and regulate immune responses. Under normal circumstances, CD70 is mainly expressed in activated lymphocytes. Under pathological conditions, CD70 is highly expressed in a variety of tumor tissues. For example, CD70 is abnormally expressed at high levels in diffuse large B-cell lymphoma, mantle cell lymphoma, peripheral T-cell lymphoma, acute myeloid leukemia, lung cancer, renal cell carcinoma, central nervous system glioma and other cancers. CD70 expression is associated with tumor immune escape, enhanced invasiveness and poor prognosis. It can be used as a new biomarker for early diagnosis of cancer, a new target for clinical diagnosis and treatment, and monitoring of disease prognosis. Currently, the clinical indications are blood tumors and renal cell carcinoma; the preclinical indications include breast cancer, ovarian cancer, glioblastoma and other cancers. CD70 can be used as a potential target for tumor treatment, and its research and clinical application prospects are broad.

[0003] Traditional monoclonal antibody preparation technology, namely hybridoma technology, involves fusing myeloma cells with immune B lymphocytes to form hybridoma cells that can secrete highly pure monoclonal antibodies. This technology can produce homogeneous, highly specific antibodies against specific antigens - monoclonal antibodies. Up to now, there are no drugs targeting CD70 on the market at home and abroad, but according to literature estimates, the number of patients with representative CD70-positive tumors in China has increased by more than 100,000 per year, showing a good market application prospect for therapeutic drugs for this target. In the field of tumor immunotherapy, the potential of CD70 antibodies lies in their ability to mediate endocytosis, making them a preferred target for antibody-drug conjugates (ADCs). In addition, CD70 antibodies can kill CD70-positive tumor cells through mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC).

[0004] At present, some anti-CD70 antibodies are in the preclinical or clinical research stage. However, the existing anti-CD70 antibodies still have some shortcomings. For example, some antibodies have low affinity and are difficult to effectively bind to the CD70 molecules on the surface of tumor cells, thus affecting their anti-tumor effects. Low specificity is also a prominent problem. Some antibodies may cross-react with other similar molecules, leading to increased side effects. Furthermore, poor stability makes it easy for antibodies to lose their activity during storage and transportation, limiting their practical application.

[0005] In summary, the development of an anti-CD70 antibody with high affinity, high specificity and good stability has extremely important clinical significance and application value. At the same time, establishing an efficient and simple method for preparing anti-CD70 antibodies to reduce costs and improve production efficiency is also an urgent problem to be solved in the current biomedical field. Summary of the invention

[0006] In view of this, the present invention provides anti-CD70 monoclonal antibodies, hybridoma cell lines and their applications. The anti-CD70 monoclonal antibodies provided by the present invention can specifically recognize CD70 recombinant proteins expressed in eukaryotes and CD70 proteins expressed on cell membranes. The CD70 detection kit constructed with anti-CD70 monoclonal antibodies has high sensitivity and good specificity, and has good application effects in a variety of tests including enzyme-linked immunosorbent assay, flow cytometry and immunofluorescence.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a monoclonal antibody,

[0009] (1) the amino acid sequences of the three CDR regions of its heavy chain have the amino acid sequences shown in SEQ ID NOs: 11, 12 and 13, respectively; and

[0010] (2) the amino acid sequences of the three CDR regions of its light chain have the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively; or

[0011] (3) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1) or (2), and having the same or similar function as the amino acid sequence described in (1) or (2); or

[0012] (4) An amino acid sequence that is at least 80% homologous to the sequence described in (1), (2) or (3).

[0013] In some embodiments of the present invention, in the above monoclonal antibody, the sequence of SEQ ID NO: 11 is: DYYMY.

[0014] In some embodiments of the present invention, in the above monoclonal antibody, the sequence of SEQ ID NO: 12 is: TSSDGGIYTYYPDSVKG.

[0015] In some embodiments of the present invention, in the above monoclonal antibody, the sequence of SEQ ID NO: 13 is: SYDYSWFPY.

[0016] In some embodiments of the present invention, in the above monoclonal antibody, the sequence of SEQ ID NO: 6 is: RASSSVSYMH.

[0017] In some embodiments of the present invention, in the above monoclonal antibody, the sequence of SEQ ID NO: 7 is: ATSNPAS.

[0018] In some embodiments of the present invention, in the above monoclonal antibody, the sequence of SEQ ID NO: 8 is: QQWSSNPLT.

[0019] In some embodiments of the present invention, in the above monoclonal antibodies,

[0020] (5) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 10; and

[0021] (6) whose light chain variable region has the amino acid sequence shown in SEQ ID NO: 5; or

[0022] (7), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (5) or (6), and having the same or similar function as the amino acid sequence described in (5) or (6); or

[0023] (8) An amino acid sequence that is at least 80% homologous to the sequence described in (5), (6) or (7).

[0024] In some embodiments of the present invention, in the above-mentioned monoclonal antibody, the sequence of SEQ ID NO: 10 is: EVQLQESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATSSDGGIYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAGSYDYSWFPYWGQGTLVTVSA.

[0025] In some embodiments of the present invention, in the above monoclonal antibody, the sequence of SEQ ID NO: 5 is: DIVLTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNPASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK.

[0026] In some embodiments of the present invention, in the above-mentioned monoclonal antibody, the subtype of the monoclonal antibody is IgG1; and the light chain is of kappa type.

[0027] In some embodiments of the present invention, the above monoclonal antibody is selected from:

[0028] (I), transforming and / or transfecting host cells with the above expression vector;

[0029] (II) a conjugate prepared by coupling the above monoclonal antibody with a solid medium or a semisolid medium;

[0030] (III) the above monoclonal antibody which is chemically or biologically labeled;

[0031] ( ), a conjugate prepared by coupling the chemically labeled or biologically labeled monoclonal antibody to a solid medium or a semi-solid medium.

[0032] The present invention also provides a nucleic acid molecule encoding the above monoclonal antibody.

[0033] (9), whose heavy chain has the nucleotide sequence shown in SEQ ID NO:9; and

[0034] (10), whose light chain has the nucleotide sequence shown in SEQ ID NO: 4; or

[0035] A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (11), (9) or (10); or

[0036] (12) a sequence having at least 80% homology to the nucleotide sequence described in (9), (10) or (11); or

[0037] (13) A complementary sequence of the nucleotide sequence described in (9), (10), (11) or (12).

[0038] In some embodiments of the present invention, in the above-mentioned nucleic acid molecule, the sequence of SEQ ID NO:9 is: GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTATATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCAGTAGTGATGGTGGTGGTATTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAGGATCATATGATTACTCCTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0039] In some embodiments of the present invention, in the above-mentioned nucleic acid molecule, the sequence of SEQ ID NO:4 is: GACATTGTGCTCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCCGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0040] The present invention also provides an expression vector, comprising: the above nucleic acid molecule.

[0041] The present invention also provides a biomaterial comprising at least one of the following:

[0042] (I), transforming and / or transfecting host cells with the above expression vector;

[0043] (II) a conjugate prepared by coupling the above monoclonal antibody with a solid medium or a semisolid medium;

[0044] (III) the above monoclonal antibody which is chemically or biologically labeled;

[0045] ( ), a conjugate prepared by coupling the chemically labeled or biologically labeled monoclonal antibody to a solid medium or a semi-solid medium.

[0046] The present invention also provides a method for preparing the monoclonal antibody, which comprises culturing the host cells in the biomaterial to induce the expression of the monoclonal antibody;

[0047] Or cultivate the hybridoma cell line with the preservation number of CCTCC NO: C202438 to obtain the monoclonal antibody.

[0048] The present invention also provides the use of the above monoclonal antibody, the above nucleic acid molecule, the above expression vector, the above biological material and / or the monoclonal antibody obtained by the above preparation method in any of the following items:

[0049] (I) preparing a product for detecting CD70 protein; and / or

[0050] (II), preparing a product for localizing CD70 protein; and / or

[0051] (III) preparing a product for labeling tumor cells.

[0052] The present invention also provides a reagent or a kit, comprising: the above monoclonal antibody, the above nucleic acid molecule, the above expression vector, the above biological material and / or the monoclonal antibody obtained by the above preparation method.

[0053] In some embodiments of the present invention, in the above-mentioned reagents or kits, the kit includes: one or more of a CD70 enzyme-linked immunosorbent assay kit, a CD70 immunofluorescence assay kit, and a CD70 flow cytometry assay kit.

[0054] In some embodiments of the present invention, in the above reagents or kits, the CD70 enzyme-linked immunosorbent assay kit further includes: any one or a combination of at least two of a blocking solution, an enzyme-labeled secondary antibody, a washing solution, a color developing solution or a stop solution.

[0055] In some embodiments of the present invention, in the above reagents or kits, the anti-CD70 monoclonal antibody is modified with a fluorescent group, and the fluorescent group is any one of FITC, Cy3, Cy5 and Alexa Fluor 488.

[0056] The present invention also provides a drug or drug combination, comprising: the above monoclonal antibody, the above nucleic acid molecule, the above expression vector, the above biological material and / or the monoclonal antibody obtained by the above preparation method.

[0057] The beneficial effects of the present invention include:

[0058] (1) The anti-CD70 monoclonal antibody provided by the present invention has strong specificity, high sensitivity and a wide range of applications. The anti-CD70 monoclonal antibody has a high binding ability with the CD70 protein. Under the same dilution conditions, the anti-CD70 monoclonal antibody secreted by the hybridoma cell line 19-10-E11 has a higher positive OD value for the CD70 protein reaction, indicating that its potency is higher than that of the commercial antibody.

[0059] (2) The anti-CD70 monoclonal antibody provided by the present invention is an IgG1 subtype antibody, which can specifically recognize the CD70 protein on the cell membrane in cell immunofluorescence detection and flow cytometry detection, and has a high affinity.

[0060] (3) The anti-CD70 monoclonal antibody provided by the present invention has different CDR amino acid composition and sequence from commercially available antibodies, thus enriching the types of antibodies.

[0061] Biological Deposit Description

[0062] Hybridoma cell line 19-10-E11, deposit date: November 20, 2024, deposit number: CCTCC NO: C202438, deposit unit name: China Center for Type Culture Collection, deposit center address: Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0064] Figure 1 The plasmid map of pLVX-CD70-mCherry in Preparation Example 1 of the present invention is shown;

[0065] Figure 2 The cell fluorescence image of Hela stable cell line overexpressing human CD70 is shown;

[0066] Figure 3 shows the plasmid map of pCMV3-CD70-mFc-N-His in Preparation Example 2 of the present invention;

[0067] Figure 4 The Coomassie brilliant blue staining of CD70-mFc recombinant protein purified by Ni column affinity is shown; where: M is the protein molecular weight standard; 1: cell culture supernatant; 2: loading flow-through; 3: 10 mM imidazole washing solution; 4: 20 mM imidazole washing solution; 5: 250 mM imidazole elution solution;

[0068] Figure 5 The figure shows the results of indirect ELISA between the antibodies produced by all hybridoma cell lines secreting anti-CD70 monoclonal antibodies and the eukaryotic expressed human CD70 recombinant protein;

[0069] Figure 6 Figure 2 shows the SDS-PAGE electrophoresis detection diagram of the purified ascites anti-CD70 monoclonal antibody; wherein, M is the protein molecular weight standard; 1 and 2: anti-CD70 monoclonal antibody in the reduced state, 6 μg;

[0070] Figure 7 The subtype identification results of the anti-CD70 monoclonal antibody secreted by the hybridoma cell line 19-10-E11 are shown;

[0071] Figure 8 Shows the sequencing results of the PCR products of the heavy chain and light chain variable regions and the structural analysis of the heavy chain and light chain variable regions;

[0072] Fig. 9 The results of detecting the specificity of anti-CD70 monoclonal antibody by indirect ELISA are shown; wherein, 1: anti-CD70 monoclonal antibody; 2: negative control; 3: positive control antibody YA1409;

[0073] Fig.10 The figure shows the result of cell immunofluorescence experiment using CD70 immunofluorescence detection kit;

[0074] Fig.11 Graph showing the results of a cell flow cytometry analysis experiment using a CD70 flow cytometry detection kit. DETAILED DESCRIPTION

[0075] The invention discloses an anti-CD70 monoclonal antibody, a hybridoma cell line and applications thereof.

[0076] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0077] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0078] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0079] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0080] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0081] The invention provides a hybridoma cell strain 19-10-E11, whose deposit number is CCTCC NO: C202438.

[0082] The present invention also provides the use of the hybridoma cell line 19-10-E11 in preparing anti-CD70 protein monoclonal antibodies.

[0083] The present invention also provides an anti-CD70 monoclonal antibody, comprising a light chain variable region and a heavy chain variable region;

[0084] The light chain variable region includes CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 6 to 8;

[0085] The heavy chain variable region includes CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 11-13.

[0086] In some specific embodiments of the present invention, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 5; and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 10.

[0087] In some specific embodiments of the present invention, the anti-CD70 monoclonal antibody is secreted and produced by the hybridoma cell line 19-10-E11 or its successor cell line.

[0088] In some specific embodiments of the present invention, the anti-CD70 monoclonal antibody subtype is IgG1.

[0089] The present invention also provides a nucleic acid molecule encoding the anti-CD70 monoclonal antibody.

[0090] In some specific embodiments of the present invention, the nucleic acid molecule encoding the light chain variable region has a nucleotide sequence as shown in SEQ ID NO:4; the nucleic acid molecule encoding the heavy chain variable region has a nucleotide sequence as shown in SEQ ID NO:9.

[0091] The present invention also provides a method for preparing the anti-CD70 monoclonal antibody, comprising: immunizing mice with an incomplete adjuvant for 1 to 2 weeks, and then intraperitoneally injecting 1 to 2×10 6 The hybridoma cell line 19-10-E11 with a preservation number of CCTCC NO: C202438 was prepared, and the mouse ascites was collected 10 to 14 days later and purified to obtain the anti-CD70 monoclonal antibody.

[0092] In some specific embodiments of the invention, the mouse comprises a BALB / c mouse.

[0093] The present invention also provides the use of the anti-CD70 monoclonal antibody in any of the following items:

[0094] (I) preparing an immunoassay product for detecting CD70 protein; and / or

[0095] (II), preparing a product for localizing CD70 protein; and / or

[0096] (III) preparing products for labeling tumor cells.

[0097] In some specific embodiments of the present invention, the product includes a kit, a reagent or a chip.

[0098] The present invention also provides a reagent or a reagent combination, comprising the anti-CD70 monoclonal antibody.

[0099] The present invention also provides a kit, comprising the anti-CD70 monoclonal antibody.

[0100] The present invention also provides a chip, comprising the anti-CD70 monoclonal antibody.

[0101] In some specific embodiments of the present invention, the kit includes a CD70 enzyme-linked immunosorbent assay kit, a CD70 immunofluorescence assay kit, and a CD70 flow cytometry assay kit.

[0102] In some specific embodiments of the present invention, the CD70 enzyme-linked immunosorbent assay kit further includes: any one or a combination of at least two of a blocking solution, an enzyme-labeled secondary antibody, a washing solution, a color developing solution or a stop solution.

[0103] In some specific embodiments of the present invention, the anti-CD70 monoclonal antibody is modified with a fluorescent group, and the fluorescent group is any one of FITC, Cy3, Cy5, and Alexa Fluor 488.

[0104] In some specific embodiments of the present invention, the anti-CD70 monoclonal antibody in the CD70 flow cytometry detection kit can bind to the CD70 protein on the cell membrane. In the flow cytometry detection, the anti-CD70 monoclonal antibody is used as a primary antibody to detect cells expressing the CD70 protein.

[0105] The method for preparing the anti-CD70 monoclonal antibody of the present invention mainly comprises the following steps:

[0106] (1) Using the eukaryotic expression platform, a eukaryotic expression plasmid was constructed. After sequencing verification, it was transiently expressed in HEK293T cells. Western blot confirmed that the protein could be expressed. The recombinant human CD70-mFc protein was obtained by culture, transiently expressed, and purified in HEK293F cells.

[0107] (2) BALB / c mice were immunized with human CD70-mFc recombinant protein. After the serum titer was qualified, the mouse spleen cells were fused with mouse myeloma cells SP2 / 0. After multiple screening and subcloning, a hybridoma cell line capable of producing monoclonal antibodies that specifically recognize human CD70 protein was obtained.

[0108] (3) Cultivating hybridoma cell lines, preparing ascites containing anti-CD70 monoclonal antibodies by intraperitoneal inoculation of mice, performing affinity purification to obtain anti-CD70 monoclonal antibodies, and determining the activity of anti-CD70 monoclonal antibodies by ELISA.

[0109] In the present invention, the anti-CD70 monoclonal antibody in the CD70 flow cytometry detection kit can bind to the CD70 protein on the cell membrane. In the flow cytometry detection, the anti-CD70 monoclonal antibody is used as a primary antibody to detect cells expressing the CD70 protein.

[0110] In Preparation Examples 1 to 8 and Examples 1 to 6 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0111] The present invention will be further described below in conjunction with embodiments:

[0112] Preparation Example 1 Preparation of human CD70 overexpressing Hela cell line

[0113] The process is as follows:

[0114] Construction of human CD70 overexpression plasmid: The designed CD70-mCherry gene sequence was fully synthesized by Beijing Qingke Biotechnology Co., Ltd., and the target gene fragment was obtained by double restriction digestion with XbaI and MluI, and inserted into the XbaI and MluI sites of the pLVX-IRES-mCherry vector to obtain the following: Figure 1 The recombinant lentiviral vector pLVX-CD70-mCherry is shown, wherein the amino acid sequence of the CD70 protein is shown in SEQ ID NO:1.

[0115] SEQ ID NO: 1: MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP.

[0116] The recombinant lentiviral vector overexpressing CD70 was combined with the virus packaging auxiliary plasmids pMD2G and pSPAX2 to co-transfect 293T cells to obtain the viral supernatant; after ultra-fast concentration and purification, the viral supernatant was infected with Hela cells, and monoclonal cells were picked by limiting dilution method for culture, and the EGFP fluorescence expression was observed by fluorescence microscopy. The results are as follows Figure 2 As shown, a human CD70-overexpressing Hela cell line was successfully constructed and named Hela-CD70-mCherry.

[0117] Preparation Example 2 Preparation of human CD70-mFc recombinant protein immunogen

[0118] The preparation process of the human CD70-mFc recombinant protein immunogen is as follows:

[0119] A signal peptide, a His tag and mFc suitable for eukaryotic protein secretion were added to the N-terminus of the extracellular domain of human CD70 protein, wherein mFc refers to the Fc protein fragment of mouse IgG. The target gene was connected to the eukaryotic expression vector pCMV3-C-His by homologous recombination to obtain the following: Figure 3 The eukaryotic recombinant plasmid pCMV3-CD70-mFc-N-His is shown. The amino acid sequence of the extracellular domain of human CD70 protein (Gln39-Pro193) and the complete sequence with added mFc are shown in SEQ ID NO: 2.

[0120] SEQ ID NO:2:PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI QHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP.

[0121] The constructed eukaryotic expression plasmid pCMV3-CD70-mFc-N-His was transiently expressed in 293T cells. After 72 hours, the cell expression supernatant was collected and verified by Western blot to confirm that the protein was expressed. Then the culture was expanded, the plasmid was extracted, and the plasmid DNA was transiently transfected into HEK293F cells. Protein expression was performed in HEK293F cells. Samples were counted at different time points, the cell state was observed, the cell viability was detected, and the expression of the target protein was detected by Western blot. The cell culture was harvested at 96 hours, centrifuged at 10000 rpm for 30 minutes, and the liquid was filtered with a 0.45 μm disposable sterile filter. The supernatant was collected for purification to obtain human CD70-mFc recombinant protein.

[0122] A Ni column was used for affinity purification, and the purified protein was subjected to SDS-PAGE gel electrophoresis to detect its protein size and purity. The results are as follows Figure 4 As shown, the size of human CD70-mFc recombinant protein under reducing conditions is about 55 kDa, consistent with the expected band position, and the purity is greater than 95%.

[0123] Preparation Example 3 Preparation of human CD70 recombinant protein

[0124] The preparation process of the human CD70 recombinant protein is as follows:

[0125] A signal peptide and six histidines suitable for eukaryotic protein secretion were added to the N-terminus of the extracellular domain of human CD70 protein. The target gene was connected to the eukaryotic expression vector pVAX1 by homologous recombination to construct the eukaryotic recombinant plasmid pVAX1-CD70. The amino acid sequence of the extracellular domain region (Gln39-Pro193) of human CD70 protein and the complete sequence with six histidines added are shown in SEQ ID NO:3.

[0126] SEQ ID NO:3: HHHHHHQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP.

[0127] The constructed eukaryotic expression plasmid pVAX1-CD70 was transiently expressed in 293T cells. After 72 hours, the cell expression supernatant was collected and verified by Western blot to confirm that the protein was expressed. Then the culture was expanded, the plasmid was extracted, and the plasmid DNA was transiently transfected into HEK293F cells. Protein expression was performed in HEK293F cells. Samples were counted at different time points, the cell state was observed, the cell viability was detected, and the expression of the target protein was detected by Western blot. The cell culture was harvested at 96 hours, centrifuged at 10000 rpm for 30 minutes, and the liquid was filtered with a 0.45 μm disposable sterile filter. The supernatant was collected and affinity purified using a Ni column to obtain human CD70 recombinant protein.

[0128] Preparation Example 4 Preparation of hybridoma cell lines

[0129] The method for preparing a hybridoma cell line comprises the following steps:

[0130] (1) Immunization of mice

[0131] Prepare 5 6-week-old female Balb / c mice, collect blood from the tail vein, and centrifuge to obtain serum as a negative control. The human CD70-mFc recombinant protein purified in Preparation Example 2 was immunized into mice according to the scheme in Table 1. In general, 100 μg of purified human CD70-mFc recombinant protein emulsified with Freund's complete adjuvant was used for the first immunization of mice by intraperitoneal injection. After the first immunization, each mouse received three more injections of 100 μg of purified human CD70-mFc recombinant protein emulsified with Freund's incomplete adjuvant by intraperitoneal and subcutaneous multi-point injection at intervals of 2 weeks. On the 3rd day after the fourth immunization, blood was collected from the tail vein, and serum was obtained by centrifugation. The serum antibody titer was detected by ELISA, and mice with higher titers were selected for shock immunization, intraperitoneal injection, and fusion was performed 3 days later.

[0132] Table 1 Animal immunization scheme

[0133] Days operate dose Adjuvant Inoculation site 1 Primary immunization 100 μg CFA Intraperitoneal injection 14 First booster vaccination 100 μg IFA Intraperitoneal and subcutaneous injection 28 Second booster immunization 100 μg IFA Intraperitoneal and subcutaneous injection 42 The third booster immunization 100 μg IFA Intraperitoneal and subcutaneous injection 56 Shock immunity 100 μg none Intraperitoneal injection

[0134] (2) Cell fusion

[0135] Mouse spleen cells with higher titer were mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1, where mouse spleen cells were about 1×10 8 and SP2 / 0 cells are about 2×10 7 , centrifuged at 1000 rpm for 5 min, and discarded the culture medium; washed once with serum-free DMEM culture medium, centrifuged at 1000 rpm for 5 min, and discarded the culture medium; fused using 50% PEG as a fusion agent, placed the centrifuge tube containing cells in a 37°C water bath, slowly added 1 mL of 50% PEG solution while stirring the cells, let it stand for 1 min, slowly added 10 mL of serum-free DMEM culture medium, centrifuged at 1000 rpm for 5 min, and discarded the supernatant. Add DMEM culture medium containing 20% ​​fetal bovine serum and 1×HAT, and inoculate the cells into a 96-well cell culture plate, 200 μL per well, and culture in a 37°C, 5% CO2 cell culture incubator.

[0136] (3) Screening of hybridoma cells

[0137] The fused cells were cultured in an incubator for 10 to 14 days, with half the medium replaced every 5 days. When the fused cells grew to cover 1 / 3 of the bottom well area, the culture supernatant was taken and the hybridoma cell lines secreting anti-CD70 antibodies were screened by indirect immunofluorescence method. The operation steps of the indirect immunofluorescence method are as follows: the CD70 overexpression Hela cell stable transfection cell line (CD70 high expression) and Hela cells (CD70 negative) obtained in Preparation Example 1 are inoculated into 96-well cell culture plates, 100 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 cell culture incubator overnight; the supernatant is discarded, and each well is rinsed with PBS solution 3 times; the PBS solution is discarded, and the cells are fixed with PBS solution containing 4% paraformaldehyde, and incubated at room temperature (20°C ± 5°C) for 15 min; the fixative is discarded, and each well is rinsed with PBS solution 3 times; the PBS solution is discarded, and a PBS solution containing 1% BSA is added to block for 30 min; the blocking solution is discarded, and the supernatant of the fused cell culture is added as the primary antibody, 50 μL per well, and incubated at room temperature (20°C ± 5°C) for 1 hour; the supernatant is discarded, and each well is rinsed with PBS solution 3 times, each time for 5 min; the secondary antibody (Alexa Fluor® 488-labeled goat anti-mouse IgG (H+L)), incubate at room temperature (20℃±5℃) in the dark for 1 hour; discard the secondary antibody, add PBS solution to each well for 3 times, 5 min each time; observe directly under an inverted fluorescence microscope, green fluorescence appears in the CD70 overexpressing Hela cell stably transfected cell line incubated with hybridoma cell supernatant, and no green fluorescence is observed in Hela cells, which are determined to be positive clones that can be subcloned.

[0138] (4) Construction of hybridoma cell lines

[0139] The positive hybridoma cells were subcloned and sorted by the limiting dilution method. After three subcloning and indirect immunofluorescence experiment screening, 12 hybridoma cell lines that can stably secrete anti-human CD70 monoclonal antibodies were obtained. The clone numbers were 19-6-B3, 19-7-F9, 19-10-E11, 20-4-G10, 21-10-B10, 24-1-F11, 24-1-G3, 24-4-B3, 24-5-E7, 24-6-D7, 24-7-G3, and 24-7-G6. The antibodies produced were then detected and compared by the indirect ELISA method. The results are as follows: Figure 5 Under the same experimental conditions, the titers of four antibodies were the best, which were produced by hybridoma cell line 19-7-F9, hybridoma cell line 19-10-E11, hybridoma cell line 24-4-B3, and hybridoma cell line 24-7-G3, and the antibody clone numbers were 19-7-F9, 19-10-E11, 24-4-B3, and 24-7-G3, respectively. Figure 5N is a negative control, P is a positive control, and combined with the results of cell immunofluorescence experiment and cell flow cytometry, the clone number of the hybridoma cell line with the best titer, the strongest specificity and the widest application is 19-10-E11.

[0140] The operating steps of the indirect ELISA method are as follows: the ELISA plate was coated with 1 μg / mL human CD70 recombinant protein, 100 μL / well, overnight at 4°C; the coating solution was discarded the next day, and the plate was washed three times with PBST (pH 7.4, containing 0.1% Tween-20, 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.01 M potassium dihydrogen phosphate), and 150 μL of 5% BSA was added to each well, and the plate was blocked at 37°C for 2 hours; after the blocking solution was discarded, the plate was washed three times with PBST, and then incubated with the primary antibody, 100 μL of the diluted antibody solution was added to each well, and the antibody produced by 12 positive hybridoma cells was diluted with 1% BSA to a concentration of 0.5 μg / mL, and the mouse serum with antibody titer after immunization was used as the positive control P, and the mouse serum without immunization was used as the negative control N, and the plate was incubated at 37°C for 1 hour, and washed three times with PBST; 100 μL of the corresponding 1: 5000 diluted (H+L), incubate with secondary antibody and then wash, incubate at 37℃ for 1 hour, wash three times with PBST; add 100 μL TMB colorimetric solution to each well, develop at 37℃ for 10 min, add stop solution, and measure the absorbance at 450 nm with an enzyme reader.

[0141] (5) Deposit of hybridoma cell line 19-10-E11

[0142] The hybridoma cell with clone number 19-10-E11 was preserved and named hybridoma cell line 19-10-E11, and deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: C202438 and the deposit date of November 20, 2024.

[0143] Preparation Example 5 Preparation of anti-CD70 monoclonal antibody

[0144] The anti-CD70 monoclonal antibody is produced by the hybridoma cell line 19-10-E11 described in Preparation Example 4.

[0145] Three 10-week-old female Balb / c mice were prepared and intraperitoneally injected with 500 μL of Freund's incomplete adjuvant per mouse. One week later, hybridoma cell line 19-10-E11 was intraperitoneally injected with 1-2×10 6Cells / mouse. After 10-14 days, the mouse ascites was collected according to the swelling of the mouse abdomen. The collected ascites was centrifuged at 4℃ 12000 rpm for 10 min, and the yellow clear liquid in the middle was collected. The antibody was purified by Protein A / G affinity chromatography, the antibody concentration was determined by Nanodrop, and the purity was identified by SDS-PAGE gel electrophoresis. SDS-PAGE gel electrophoresis Figure 6 As shown in the figure, the purity of the purified anti-CD70 monoclonal antibody is >95%, and IgG heavy chain with a molecular weight of 50 kDa and light chain with a molecular weight of 25 kDa can be seen. M is the protein molecular weight standard; 1 and 2: anti-CD70 monoclonal antibody in a reduced state, 6 μg.

[0146] Preparation Example 6 Preparation of CD70 ELISA kit

[0147] The CD70 enzyme-linked immunosorbent assay kit includes the anti-CD70 monoclonal antibody obtained in Preparation Example 5, a blocking solution and an enzyme-labeled secondary antibody, a washing solution, a TMB colorimetric solution, and a stop solution. The blocking solution is 5% BSA; the enzyme-labeled secondary antibody is (H+L) and (H+L); the washing solution is PBST buffer (pH 7.4, containing 0.1% Tween-20, 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M potassium dihydrogen phosphate); the stop solution is 2 mol / L H2SO4.

[0148] Preparation Example 7 Preparation of CD70 Immunofluorescence Detection Kit

[0149] The CD70 immunofluorescence detection kit includes a fluorescent group-modified anti-CD70 monoclonal antibody, a cell fixative, a blocking solution, a washing solution, and a cell nucleus dye, wherein the fluorescent group is any one of FITC, Cy3, Cy5, and Alexa Fluor 488. The operation process of fluorescent group-modified anti-CD70 monoclonal antibody is described by taking FITC labeling as an example: FITC solution is added dropwise to the anti-CD70 monoclonal antibody solution obtained in Preparation Example 5 under magnetic stirring, and then stirred continuously at room temperature (20°C ± 5°C) for 4 to 6 hours, and then centrifuged. The supernatant is the FITC-labeled anti-CD70 monoclonal antibody, and the labeled antibody is further purified by dialysis to remove unbound free fluorescein.

[0150] Preparation Example 8 Preparation of CD70 Flow Cytometry Detection Kit

[0151] The CD70 flow cytometry detection kit includes the anti-CD70 monoclonal antibody obtained in Preparation Example 5, an antibody diluent, a cell fixative, a washing solution, and a fluorescent secondary antibody. The antibody diluent is a PBS buffer solution containing 3% BSA (pH 7.4, containing 3% BSA, 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M potassium dihydrogen phosphate); the cell fixative is a 1% paraformaldehyde solution (pH 7.4, containing 1% paraformaldehyde, 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M potassium dihydrogen phosphate); the washing solution is a PBS buffer solution (pH 7.4, containing 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, 0.01 M potassium dihydrogen phosphate); the fluorescent secondary antibody is .

[0152] Example 1 Identification of subtypes of anti-CD70 monoclonal antibodies

[0153] In this example, the subtype of the anti-CD70 monoclonal antibody secreted by the hybridoma cell line 19-10-E11 described in Preparation Example 4 was identified by ELISA.

[0154] The specific steps are as follows:

[0155] The anti-CD70 monoclonal antibody secreted by hybridoma cell line 19-10-E11 was detected using the mouse monoclonal antibody subtype identification kit from proteintech. The specific process is as follows: the supernatant of hybridoma cell line 19-10-E11 was diluted 1:50 and 50 μL was added to the sample wells of the ELISA plate strips. 50 μL of HRP-labeled secondary antibodies of each subtype antibody were added to each well. After gently mixing on a mixer, incubate at room temperature (20℃±5℃) for 1 hour, discard the liquid in the well, wash the plate 3 times with 1×PBST, pat dry on absorbent paper, add the prepared color development solution to the well, color at room temperature (20℃±5℃) in the dark for 10~20 min, then add the stop solution to each well, and read the OD value by an enzyme reader. 450 . Figure 7 The subtype identification results of the anti-CD70 monoclonal antibody secreted by the hybridoma cell line 19-10-E11 provided in this example show that the anti-CD70 monoclonal antibody is IgG1 and the light chain is kappa type.

[0156] Example 2 Amplification and analysis of the variable region gene of the anti-CD70 monoclonal antibody

[0157] First, the total RNA was extracted from the hybridoma cell line 19-10-E11 described in Preparation Example 4 using the Trizol method: 1×106 10 positive hybridoma cells, discard the supernatant, wash once with PBS buffer, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL Trizol reagent to the cell pellet, mix repeatedly with a pipette and transfer to a 1.5 mL EP tube, and leave at room temperature for 5 min to ensure that the cells are fully lysed. Add 200 μL chloroform, shake vigorously to mix thoroughly, place at room temperature for 5 min, centrifuge at 4°C, 12000 rpm for 15 min, aspirate about 300 μL of the upper liquid and transfer it to a new 1.5 mL EP tube, add 0.5 mL isopropanol, gently invert several times to mix, place at room temperature for 10 min, then centrifuge at 4°C, 12000 rpm for 10 min, gelatinous RNA precipitation can be seen at the bottom of the tube, carefully aspirate the supernatant, add 1 mL 75% ethanol, gently invert the centrifuge tube to suspend the precipitation, then centrifuge at 4°C, 12000 rpm for 5 min, discard the supernatant, dry the EP tube at room temperature for 5 min to fully evaporate the ethanol, add RNase-free double distilled water to dissolve the precipitation, and obtain total RNA. Use a reverse transcription kit to reverse transcribe and synthesize cDNA using the extracted total RNA as a template.

[0158] Degenerate primers were designed to amplify the light chain variable region and heavy chain variable region of monoclonal antibody 19-10-E11. The sequencing results of the heavy chain and light chain variable region PCR products and the structural analysis of the heavy chain and light chain variable regions are shown in Figure 2. Figure 8 The obtained monoclonal antibody 19-10-E11 light chain variable region has a size of 318 bp, a nucleotide sequence as shown in SEQ ID NO:4, and an amino acid sequence as shown in SEQ ID NO:5, including CDR1 as shown in SEQ ID NO:6, CDR2 as shown in SEQ ID NO:7, and CDR3 as shown in SEQ ID NO:8. The monoclonal antibody 19-10-E11 heavy chain variable region has a size of 354 bp, a nucleotide sequence as shown in SEQ ID NO:9, and an amino acid sequence as shown in SEQ ID NO:10, including CDR1 as shown in SEQ ID NO:11, CDR2 as shown in SEQ ID NO:12, and CDR3 as shown in SEQ ID NO:13.

[0159] SEQ ID NO:4:GACATTGTGCTCACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCCGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA;

[0160] SEQ ID NO:5:DIVLTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNPASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK;

[0161] SEQ ID NO:6:RASSSVSYMH;

[0162] SEQ ID NO:7:ATSNPAS;

[0163] SEQ ID NO:8:QQWSSNPLT;

[0164] SEQ ID NO:9:GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTATATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCAGTAGTGATGGTGGTATTTACACCT ACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAGGATCATATGATTACTCCTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA;

[0165] SEQ ID NO:10: EVQLQESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATSSDGGIYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAGSYDYSWFPYWGQGTLVTVSA;

[0166] SEQ ID NO: 11: DYYMY;

[0167] SEQ ID NO:12: TSSDGGIYTYYPDSVKG;

[0168] SEQ ID NO: 13: SYDYSWFPY.

[0169] Example 3 Detection of human CD70 recombinant protein using CD70 ELISA kit

[0170] This example uses the CD70 enzyme-linked immunosorbent assay kit described in Preparation Example 6 to detect the sample:

[0171] The purified human CD70 recombinant protein in Preparation Example 3 was used to coat the ELISA plate, and the specificity of the anti-CD70 monoclonal antibody secreted by the hybridoma cell line 19-10-E11 was detected by indirect ELISA. The positive control antibody was the CD70 antibody (Rabbit IgG) of MedChemExpress, clone number YA1409 (Cat. No.: HY-P81664).

[0172] The specific operation steps are as follows: coat the ELISA plate with 1 μg / mL human CD70 recombinant protein, 100 μL / well, overnight at 4°C; discard the coating solution the next day, wash three times with PBST, add 150 μL of 5% BSA to each well, and block at 37°C for 2 hours; discard the blocking solution, wash three times with PBST, and then incubate with primary antibodies, dilute the anti-CD70 monoclonal antibody and control antibody YA1409 with 1% BSA to a concentration of 0.5 μg / mL, and the negative control is 1% BSA, add 100 μL to each well, incubate at 37°C for 1 hour, and wash three times with PBST; add 100 μL of the corresponding 1:5000 diluted (H+L) and (H+L), incubate with secondary antibody and then wash, incubate at 37℃ for 1 hour, wash three times with PBST; add 100 μL TMB colorimetric solution to each well, develop at 37℃ for 10 min, add stop solution, and measure the absorbance at 450 nm with an enzyme reader.

[0173] The results of the ELISA experiment are as follows Fig. 9 As shown, 1: anti-CD70 monoclonal antibody; 2: negative control; 3: positive control antibody YA1409. The anti-CD70 monoclonal antibody secreted by the hybridoma cell line 19-10-E11 and the positive control antibody YA1409 can specifically recognize human CD70 recombinant protein, and the results are both strongly positive.

[0174] Example 4 Determination of affinity constants of anti-CD70 monoclonal antibodies

[0175] In this example, the affinity constant of the anti-CD70 monoclonal antibody described in Preparation Example 5 was determined.

[0176] The ELISA plate was coated with 1 μg / mL human CD70 recombinant protein obtained in Preparation Example 3, 100 μL / well, overnight at 4°C; the next day, the coating solution was discarded, washed 3 times with PBST, 150 μL of 5% BSA was added to each well, and blocked at 37°C for 2 hours; after discarding the blocking solution, the plate was washed 3 times with PBST, and then incubated with the primary antibody, and the monoclonal antibody purified in Preparation Example 5 (initial concentration of 0.5 mg / mL) was diluted 2-fold from 1:200, for a total of 13 dilutions, 1 well was left as a blank control, 100 μL was added to each well, incubated at 37°C for 1 hour, and washed 3 times with PBST; (H+L) was diluted 1:5000, 100 μL was added to each well, incubated at 37°C for 1 hour, and washed 3 times with PBST; 100 μL of TMB colorimetric solution was added to each well, color developed at 37°C for 10 min, and after adding the stop solution, the absorbance value at a wavelength of 450 nm was measured with an enzyme marker. The test results are shown in Table 2. The dilution factor A corresponding to half of the maximum binding OD value was found, and the affinity constant was calculated using the following formula: affinity constant = (150000×A) / original antibody concentration.

[0177] Table 2 Affinity constant determination of anti-CD70 monoclonal antibodies

[0178]

[0179] The affinity constant of the anti-CD70 monoclonal antibody of the present invention is calculated by the above formula to be approximately 7.68×10 9 The present invention provides an anti-CD70 monoclonal antibody having a high affinity to the CD70 protein.

[0180] Example 5 Detection of intracellular CD70 using CD70 immunofluorescence detection kit

[0181] In this example, the CD70 immunofluorescence detection kit described in Preparation Example 7 was used to detect a variety of cells.

[0182] Hela cells, Hela-CD70-mCherry cells, 786-O cells and A498 cells were inoculated in 24-well plates with cell slides, respectively, and cultured in an incubator overnight; washed three times with PBS buffer (pH 7.4, containing 0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M disodium hydrogen phosphate, and 0.01 M potassium dihydrogen phosphate), fixed with 4% paraformaldehyde for 15 min, washed three times with PBS buffer, and blocked with 5% BSA solution at 37°C for 2 hours; after discarding the blocking solution, washed three times with PBS buffer, and then incubated with primary antibodies, diluted FITC-labeled anti-CD70 monoclonal antibody with 1% BSA, incubated at 4°C in the dark overnight, washed three times with PBS buffer, added DAPI solution for cell nucleus staining, washed three times with PBS buffer, dried the liquid on the slide with absorbent paper, and sealed with sealing solution containing anti-fluorescence quencher, observed and photographed under a fluorescence microscope, and the results are as follows Fig.10 shown.

[0183] Depend on Fig.10 It can be seen that the anti-CD70 monoclonal antibody provided by the present invention can specifically recognize the CD70 protein.

[0184] Example 6 Detection of intracellular CD70 using a flow cytometry detection kit

[0185] In this example, the flow cytometry detection kit described in Preparation Example 8 was used to detect a variety of cells.

[0186] Hela cells, Hela-CD70-mCherry cells, 786-O cells and A498 cells were treated separately to obtain single cell suspensions, anti-CD70 monoclonal antibodies diluted in PBS buffer containing 3% BSA were added, and the suspensions were incubated at room temperature (20℃±5℃) or 4℃ for 30 min, and the cells were washed 3 times with ice-cold PBS buffer. Fluorescent secondary antibodies diluted in PBS buffer containing 3% BSA were added, and the cells were resuspended. The suspensions were incubated at room temperature (20℃±5℃) or 4℃ in the dark for 30 min, and the cells were washed 3 times with ice-cold PBS buffer. The cells were resuspended with PBS buffer and analyzed by flow cytometry. If the cells cannot be put on the machine in time, the cells can be resuspended with 1% paraformaldehyde and stored at 4℃, but this process is not recommended to exceed 16 hours. The test results are as follows: Fig.11 shown.

[0187] Depend on Fig.10 It can be seen that the antibody has almost no binding with the surface antigen of Hela cells (CD70 negative), but has good binding with the surface antigen of other CD70 positive cells, and the binding rates with the surface antigen of Hela-CD70-mCherry cells, 786-O cells and A498 cells are 93.5%, 84.6% and 93.0%, respectively. The anti-CD70 monoclonal antibody provided by the present invention can specifically recognize CD70 protein, and has important application prospects in the preparation of CD70 detection kits.

[0188] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the scope of protection of the present invention.

[0189] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody, characterized in that (1) the amino acid sequences of the three CDR regions of its heavy chain have the amino acid sequences shown in SEQ ID NOs: 11, 12 and 13, respectively; and (2) the amino acid sequences of the three CDR regions of its light chain have the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively; or (3) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1) or (2), and having the same or similar function as the amino acid sequence described in (1) or (2); or (4) An amino acid sequence that is at least 80% homologous to the sequence described in (1), (2) or (3).

2. The monoclonal antibody according to claim 1, characterized in that (5) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 10; and (6) whose light chain variable region has the amino acid sequence shown in SEQ ID NO: 5; or (7), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (5) or (6), and having the same or similar function as the amino acid sequence described in (5) or (6); or (8) An amino acid sequence that is at least 80% homologous to the sequence described in (5), (6) or (7).

3. The monoclonal antibody according to any one of claims 1 to 3, characterized in that Selected from: (I), a monoclonal antibody produced by a hybridoma cell line with a deposit number of CCTCC NO: C202438; (II), a monoclonal antibody that binds to an epitope capable of binding to a monoclonal antibody produced by a hybridoma cell line having a deposit number of CCTCC NO: C202438; (III) a monoclonal antibody that competes with the monoclonal antibody produced by the hybridoma cell line deposited with CCTCC NO: C202438 in a competitive binding assay; ( ), a monoclonal antibody having an antigen-binding fragment of the monoclonal antibody described in any one of (I) to (III), wherein the fragment retains the ability to specifically bind to CD70 or a variant thereof.

4. A nucleic acid molecule encoding a monoclonal antibody according to any one of claims 1 to 3, characterized in that: (9), whose heavy chain has the nucleotide sequence shown in SEQ ID NO:9; and (10), whose light chain has the nucleotide sequence shown in SEQ ID NO: 4; or A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases of the nucleotide sequence described in (11), (9) or (10); or (12) a sequence having at least 80% homology to the nucleotide sequence described in (9), (10) or (11); or (13) A complementary sequence of the nucleotide sequence described in (9), (10), (11) or (12).

5. An expression vector, characterized in that include: The nucleic acid molecule according to claim 4.

6. Biomaterial, characterized in that Include at least one of the following: (I), transforming and / or transfecting a host cell with the expression vector according to claim 5; (II) A conjugate prepared by coupling the monoclonal antibody according to any one of claims 1 to 3 with a solid medium or a semisolid medium; (III) the monoclonal antibody according to any one of claims 1 to 3 which is chemically labeled or biologically labeled; ( ), a conjugate prepared by coupling the chemically labeled or biologically labeled monoclonal antibody according to any one of claims 1 to 3 with a solid medium or a semi-solid medium.

7. The method for preparing a monoclonal antibody according to any one of claims 1 to 3, characterized in that: Cultivating the host cell in the biomaterial according to claim 6 to induce the expression of the monoclonal antibody; Or cultivate the hybridoma cell line with the preservation number of CCTCC NO: C202438 to obtain the monoclonal antibody.

8. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the biological material according to claim 6 and / or the monoclonal antibody obtained by the preparation method according to claim 7 in any of the following items; (I) preparing a product for detecting CD70 protein; and / or (II), preparing a product for localizing CD70 protein; and / or (III) preparing a product for labeling tumor cells.

9. A reagent or a kit, characterized in that include: The monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the biological material according to claim 6 and / or the monoclonal antibody obtained by the preparation method according to claim 7.

10. A drug or a drug combination, characterized in that include: The monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the biological material according to claim 6 and / or the monoclonal antibody obtained by the preparation method according to claim 7.

Images