Anti-CD70 nano antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, discloses an anti-CD70 nano antibody and application thereof, particularly discloses an anti-CD70 nano antibody, and defines a specific amino acid sequence of the anti-CD70 nano antibody. The anti-CD70 nano antibody obtained by immunizing alpaca is high in affinity and strong in specificity, can be used for immunoblotting, enzyme-linked immunosorbent assay kits and flow cytometry detection and counting, and can also be used for treating diseases such as tumors and autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-CD70 nano antibody and an application thereof. Background Art

[0002] In recent years, immunotherapy based on specific targets has become a hot topic in the field of tumor treatment. Cluster of differentiation 70 (CD70) is a member of the tumor necrosis factor receptor superfamily and a type II transmembrane glycoprotein. It was originally used as a marker for Hodgkin's lymphoma together with CD30, and the Ki-24 antibody was cloned. According to the naming rules, Ki-24 was named CD70. As early as 1993, GOODWIN discovered that CD27 ligand (CD27L) is a type II transmembrane glycoprotein. A series of research results show that CD27L and CD70 are the same molecule. CD70 binding to CD27 can induce the activation of NF-κB and c-Jun kinase signaling pathways, thereby exerting a series of physiological effects, such as promoting cell survival, gene expression and pro-inflammatory response. Under normal circumstances, CD70 is only transiently expressed in activated T cells and B cells and mature dendritic cells. Recent studies have found that CD70 is abnormally expressed in a variety of hematological malignancies and solid tumors, especially in lymphoma, renal cell carcinoma, glioblastoma, melanoma, nasopharyngeal carcinoma, malignant pleural mesothelioma, hepatocarcinoma, and tumors induced by Epstein-Barr virus (EBV) infection. The expression level of CD70 varies in different types of tumors, such as more than 80% in non-Hodgkin's lymphoma, Hodgkin's lymphoma, primary melanoma, thymic squamous cell carcinoma, and anaplastic astrocytoma, while it is less than 30% in mantle cell lymphoma and non-small cell lung cancer. CD70 is highly expressed in a variety of hematological malignancies and solid tumor tissues, and low / no expression in normal tissues, making CD70 a striking target for tumor immunotherapy. Therefore, drugs developed by targeting CD70 are a very promising strategy for tumor treatment.

[0003] INAGUMA et al. found that CD70 can enhance the migration and invasion ability of tumors by regulating the expression of MET protein and activating the MET-ERK signaling pathway. Some studies have also shown that the mechanism of CD70 expression in tumor cells may be that CD70 binds to CD27 expressed by T cells, activating the apoptosis protein Siva to cause B cells or T cells to produce cytotoxic effects and apoptosis, thereby causing immune escape. In addition, the CD27 and CD70 signaling pathways can mediate paracrine growth signals through B cell-B cell interactions, which contributes to the occurrence and metastasis of lymphoma.

[0004] Nanobodies (VHH) have the same structural domains as ordinary antibody VH, namely 4 conserved framework regions (FR1 / 2 / 3 / 4) and 3 complementary determining regions (CDR1 / 2 / 3). The molecular weight of nanobodies is only 10% of that of traditional antibodies, and they retain the complete antigen binding ability of HCAbs, and have the characteristics of strong specificity, good affinity and high stability. Since nanobodies do not have Fc segments and cannot produce cytotoxic effects such as ADCC / CDC like traditional antibodies, VHH antibodies are often fused with Fc segments to construct Fc-VHH fusion antibodies to increase ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) activity. Compared with ordinary antibodies, these forms of nanobodies can be widely used in the treatment of various diseases. Summary of the invention

[0005] The purpose of the first aspect of the present invention is to provide an anti-CD70 nanobody.

[0006] The second aspect of the present invention is to provide a fusion antibody.

[0007] The purpose of the third aspect of the present invention is to provide a biological material related to the anti-CD70 nanobody of the first aspect of the present invention or the fusion antibody of the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide an immunoconjugate.

[0009] The fifth aspect of the present invention aims to provide a medicine.

[0010] The purpose of the sixth aspect of the present invention is to provide the use of the anti-CD70 nanobody of the first aspect of the present invention, the fusion antibody of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immunoconjugate of the fourth aspect of the present invention or the drug of the fifth aspect of the present invention.

[0011] The seventh aspect of the present invention aims to provide a test kit or a medicine box.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] In a first aspect of the present invention, an anti-CD70 nanobody is provided, wherein the anti-CD70 nanobody comprises a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein:

[0014] The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:48, and SEQ ID NO:53;

[0015] The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:41, SEQ ID NO:49, and SEQ ID NO:54;

[0016] The amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:42, and SEQ ID NO:50.

[0017] In some embodiments of the present invention, the CDR of the anti-CD70 nanobody comprises:

[0018] CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14 and CDR3 shown in SEQ ID NO:15; or

[0019] CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:20; or

[0020] CDR1 shown in SEQ ID NO:23, CDR2 shown in SEQ ID NO:24 and CDR3 shown in SEQ ID NO:25; or

[0021] CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:24 and CDR3 shown in SEQ ID NO:29; or

[0022] CDR1 shown in SEQ ID NO:32, CDR2 shown in SEQ ID NO:24 and CDR3 shown in SEQ ID NO:33; or

[0023] CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:24 and CDR3 shown in SEQ ID NO:37; or

[0024] CDR1 shown in SEQ ID NO:40, CDR2 shown in SEQ ID NO:41 and CDR3 shown in SEQ ID NO:42; or

[0025] CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:24 and CDR3 shown in SEQ ID NO:37; or

[0026] CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49 and CDR3 shown in SEQ ID NO:50; or

[0027] CDR1 shown in SEQ ID NO:53, CDR2 shown in SEQ ID NO:54 and CDR3 shown in SEQ ID NO:50.

[0028] In some embodiments of the invention, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0029] In some embodiments of the present invention, the amino acid sequence of the anti-CD70 nanobody is as shown in SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:51, or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:51 and having the same or similar function.

[0030] A second aspect of the present invention provides a fusion antibody comprising a first domain and a second domain;

[0031] The first domain is the anti-CD70 nanobody of the first aspect of the present invention;

[0032] The second domain has the effect of extending the half-life in vivo and / or has a binding effect on effector cells.

[0033] In some embodiments of the present invention, the second domain includes (but is not limited to):

[0034] Immunoglobulin Fc region (such as human immunoglobulin Fc region); and / or

[0035] Serum albumin (such as human HSA) or a fragment thereof, a domain that binds to serum albumin (such as an anti-serum albumin antibody, including a nanobody), polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and / or

[0036] A molecule that has affinity for a T cell surface molecule and / or is capable of binding to a surface molecule present on a T cell (eg, CD3).

[0037] In some embodiments of the present invention, the human immunoglobulin Fc region includes mutations for changing Fc-mediated effector functions, and the effector functions include a combination of one or more of CDC activity, ADCC activity, and ADCP activity.

[0038] In some embodiments of the present invention, the immunoglobulin is a combination of one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM.

[0039] In some embodiments of the present invention, the IgG is selected from one or more combinations of IgG1, IgG2, IgG3 or IgG4 subtypes.

[0040] In some embodiments of the present invention, the amino acid sequence of the immunoglobulin Fc region is as shown in SEQ ID NO:55; or an amino acid sequence that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO:55 and has the same or similar function.

[0041] In some embodiments of the present invention, the first domain is connected to the N-terminus or C-terminus of the second domain.

[0042] In some embodiments of the present invention, the first domain and the second domain are hinge-connected (such as direct fusion of the anti-CD70 nanobody and the hinge region of Fc) or connected via a connecting peptide.

[0043] In some embodiments of the present invention, the connecting peptide is a flexible linker.

[0044] In some embodiments of the present invention, the flexible linker amino acid sequence includes but is not limited to GSAS, (GGCAGCGCCAGC) n 、(GGCGGCGGCAGC) n 、(GGCGGCGGCGGCAGC) n、YAPVDV、(GGGS) n 、(GGSG) n 、(GGGGS) n 、(G) n , where 1≤n≤5, and n is an integer.

[0045] In some embodiments of the present invention, the amino acid sequence of the fusion antibody is as shown in SEQ ID NOs: 1-8.

[0046] The third aspect of the present invention provides a biomaterial related to the anti-CD70 nanobody of the first aspect of the present invention or the fusion antibody of the second aspect of the present invention; the biomaterial is any one of a1) to a12):

[0047] a1) a nucleic acid molecule encoding the anti-CD70 nanobody of the first aspect of the invention or the fusion antibody of the second aspect of the invention;

[0048] a2) an expression cassette containing the nucleic acid molecule described in a1);

[0049] a3) a recombinant vector containing the nucleic acid molecule described in a1);

[0050] a4) a recombinant vector containing the expression cassette described in a2);

[0051] a5) a recombinant microorganism containing the nucleic acid molecule described in a1);

[0052] a6) a recombinant microorganism containing the expression cassette described in a2);

[0053] a7) a recombinant microorganism containing the recombinant vector described in a3);

[0054] a8) a recombinant microorganism containing the recombinant vector described in a4);

[0055] a9) a transgenic cell line containing the nucleic acid molecule described in a1);

[0056] a10) a transgenic cell line containing the expression cassette described in a2);

[0057] a11) a transgenic cell line containing the recombinant vector described in a3);

[0058] a12) A transgenic cell line containing the recombinant vector described in a4).

[0059] In some embodiments of the present invention, the nucleic acid molecule encoding the anti-CD70 nanobody of the first aspect of the present invention is shown as SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:44, SEQ ID NO:47, and SEQ ID NO:52.

[0060] In some embodiments of the invention, the transgenic animal cell line does not contain reproductive material.

[0061] In some embodiments of the present invention, the expression cassette comprises a 5' transcription control region, an open reading frame encoding the fusion antibody of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR) and a transcription termination signal.

[0062] In some embodiments of the present invention, the 5' transcription control region comprises a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter, may be used), a transcription start site, an enhancer and / or a silencer element.

[0063] In some embodiments of the invention, the 3'UTR may encode an AU-rich element, which is a common regulator of mRNA stability through the 3'-5' exosome pathway and is usually located in the 3'UTR. The AU-rich element may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements having the sequence AUAUAU.

[0064] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0065] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0066] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0067] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0068] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0069] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0070] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0071] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new species of plants or animals.

[0072] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli, Streptomyces, Bacillus subtilis and other bacteria well known in the art that can be used to express target proteins.

[0073] In some embodiments of the present invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell and an insect cell.

[0074] The fourth aspect of the present invention provides an immunoconjugate, comprising:

[0075] (A) the anti-PD-L1 Nanobody of the first aspect of the invention or the fusion antibody of the second aspect of the invention; and

[0076] (B) a functional molecule connected to (A) (including but not limited to covalently linked, coupled, attached, adsorbed).

[0077] In some embodiments of the present invention, (B) includes cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions based on chimeric antigen receptor technology, transmembrane regions (such as the transmembrane regions of CD8 or CD28) and intracellular signaling regions (such as CD3ζ chain, FcεRIγ tyrosine activation motif, intracellular signaling regions of co-stimulatory signal molecules CD27, CD28, CD137, CD134, MyD88, CD40, etc.), or a combination thereof.

[0078] In some embodiments of the present invention, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker.

[0079] In some embodiments of the present invention, the tumor-suppressing molecule is an anti-tumor cytokine (such as IL-12, IL-15, IFN-beta, TNFalpha) or an anti-tumor toxin.

[0080] In some embodiments of the present invention, the detectable marker is selected from a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0081] In some embodiments of the present invention, (B) is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents or enzymes capable of producing detectable products, radionuclides, biological toxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0082] The fifth aspect of the present invention provides a drug comprising the anti-CD70 nanobody of the first aspect of the present invention, the fusion antibody of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, or the immunoconjugate of the fourth aspect of the present invention.

[0083] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0084] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

[0085] In some embodiments of the present invention, the drug further comprises a combination drug, including but not limited to immune effector molecules, cells, cytotoxic substances, and multikinase inhibitors.

[0086] The sixth aspect of the present invention provides the use of the anti-CD70 nanobody of the first aspect of the present invention, the fusion antibody of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immunoconjugate of the fourth aspect of the present invention, or the drug of the fifth aspect of the present invention in any one of (1) to (4):

[0087] (1) Preparation of preparations, test kits or pharmaceutical kits for diagnosing, treating or preventing cancer;

[0088] (2) preparing a preparation or kit for treating or preventing an autoimmune disease;

[0089] (3) Quantitative / qualitative detection of CD70 expression;

[0090] (4) Prepare reagents / kits for quantitative / qualitative detection of CD70 expression.

[0091] It can also be used for targeted delivery of traditional chemotherapy drugs (traditional Chinese medicine monomers, microRNA, etc.).

[0092] In some embodiments of the present invention, the cancer described in (1) includes: the cancer described in (1) includes: at least one of lymphoma (such as non-Hodgkin's lymphoma, Hodgkin's lymphoma, mantle cell lymphoma), lung cancer (such as non-small cell lung cancer), renal cell carcinoma, glioblastoma, melanoma (such as primary melanoma), nasopharyngeal carcinoma, malignant pleural mesothelioma, anaplastic astrocytoma, thymic squamous cell carcinoma, liver cancer and tumors induced by Epstein-Barr virus infection.

[0093] In some embodiments of the present invention, the autoimmune disease in (2) includes at least one of lupus erythematosus, sepsis, arthritis, pancreatitis and type I diabetes.

[0094] In some embodiments of the present invention, the kit / reagent kit described in (4) includes reagents for immunoblotting, enzyme-linked immunosorbent assay, and flow cytometry counting.

[0095] The seventh aspect of the present invention provides a kit or a medicine box, comprising the anti-CD70 nanobody of the first aspect of the present invention, the fusion antibody of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immunoconjugate of the fourth aspect of the present invention, or the drug of the fifth aspect of the present invention.

[0096] In some embodiments of the present invention, the kit can be used for immunoblotting, enzyme-linked immunosorbent assay, and flow cytometry counting.

[0097] The beneficial effects of the present invention are:

[0098] The anti-CD70 nanoantibodies obtained by immunizing alpacas in the present invention have high affinity and strong specificity, can be used for immunoblotting, enzyme-linked immunosorbent assay kits, flow cytometry detection and counting, and can also be used for the treatment of tumors, autoimmune diseases, chronic infectious diseases and the like, and have the potential to be developed as immune checkpoint inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 Schematic diagram of the structure of the fusion antibody of the present invention.

[0100] Figure 2 Map of the antigen lentiviral vector used for the construction of CD70 target antigen cell line (A) and flow cytometry detection report of the construction of CD70 target antigen cell line (B).

[0101] Figure 3 These are the results of flow cytometry testing of the affinity of the supernatants of fusion antibodies FP865, FP866, FP867 and FP868 for target antigen cells.

[0102] Figure 4 These are the results of flow cytometry detection of the affinity of the supernatants of fusion antibodies FP869, FP870, FP871, FP872, FP873 and FP874 for target antigen cells.

[0103] Figure 5 The flow cytometric test results of the affinity of each fusion antibody to non-target antigen cell serum.

[0104] Figure 6 A flow cytometer for quantitatively detecting the binding ability of fusion antibodies FP865, FP866, FP867 or FP868 to target cells.

[0105] Figure 7 Flow cytometric reporter for quantitative detection of the binding ability of fusion antibodies FP869, FP870, FP871 and FP872 to target cells.

[0106] Figure 8 Flow cytometric reporter for quantitative detection of the binding ability of fusion antibodies FP873 and FP847 to target cells.

[0107] Fig. 9 To quantitatively detect the binding ability of each fusion antibody and target cell Anti-CD70 - MFI.

[0108] Fig.10 To quantitatively detect the binding ability of each fusion antibody and target cell Anti-CD70 - the proportion of positive cells.

[0109] Fig.11 Screening for the blocking function of Anti-CD70 antibodies.

[0110] Fig.12 Bar graph for Anti-CD70 antibody blocking function screening. DETAILED DESCRIPTION

[0111] The present invention is further described in detail below through specific examples.

[0112] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0113] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0114] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0115] Example 1 Plasmid Construction

[0116] Multiple candidate nanobody sequences were obtained by immunizing alpacas with the amino acid sequence of the CD70 target antigen. The VHH region nucleic acid sequence of the nanobody was connected to the human IgG Fc nucleic acid sequence using multi-fragment homologous recombination technology. The connecting region was hinge. The plasmid after homologous recombination was identified by enzyme digestion and then sequenced and analyzed. The fusion antibody sequence structure Figure 1 Ten fusion antibodies, namely C7-1-3 (FP865), C7-1-44 (FP866), C7-1-49 (FP867), C7-1-50 (FP868), C7-1-58 (FP869), C7-1-95 (FP870), C7-1-105 (FP871), C7-2-138 (FP872), C72-158 (FP873), and C72-168 (FP874), were constructed, and their amino acid sequences are shown in SEQ ID NOs: 1 to 10.

[0117] The amino acid sequence of VHH of the fusion antibody C7-1-3 (FP865) is shown in SEQ ID NO: 11, the nucleotide sequence is shown in SEQ ID NO: 12, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 13 to 15, respectively.

[0118] QLQLVESGGGLVQPGGSLRLSCAAFGFTLDGYAIGWFRQAPGKEREGVSCISSGDSTNFPDSVKGRFTIFRDNVKNTVYLQMNSLKPEDTAVYYCAATGIYFCGDYADFWGQGTQVTVS (SEQ ID NO: 11);

[0119] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTTTGGATTCACTTTGGATGGTTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATT AGTAGTAGTGGTGATAGCACAAACTTCCCAGACTCCGTGAAGGGCCGATTCACCATCTTCAGAGACAACGTCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCTACAGGGATCTACTTTTGTGGAGACTATGCCGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:12);

[0120] CDR1: GFTLDGYA (SEQ ID NO: 13); CDR2: ISSSGDST (SEQ ID NO: 14); CDR3: AATGIYFCGDYADFW (SEQ ID NO: 15).

[0121] The amino acid sequence of VHH of the fusion antibody C7-1-44 (FP866) is shown in SEQ ID NO: 16, the nucleotide sequence is shown in SEQ ID NO: 17, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 18 to 20, respectively.

[0122] QVQLVESGGGLVQPGDSLRLSCTASGFPFSKYAMGWVRELPVKGQEWVSGIYTDGRTYYEDSVKGRFTISRDNAKNTVYLQMDSLRPEDTGVYFCATPGMEGGLASKWSWYALEYWGKGTHVTVS(SEQ ID NO:16);

[0123] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGGGACTCTCTGAGGCTCTCCTGTACAGCCTCTGGATTCCCCTTCAGTAAATATGCCATGGGCTGGGTCCGCGAGCTTCCAGTTAAGGGGGCAAGAATGGGTGTCCGGTATTTATACTGA TGGTAGGACTTACTATGAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAAACACGGTGTATCTGCAAATGGACAGCCTGAGACCTGAGGACACGGGCGTGTATTTCTGTGCGACCCCCGGGATGGAAGGCGGATTGGCGTCGAAGTGGTCTTGGTACGCGCTGGAGTACTGGGGCAAAGGGACCCACGTCACCGTCTCC(SEQ ID NO:17);

[0124] CDR1: GFPFSKYA (SEQ ID NO: 18); CDR2: IYTDGRT (SEQ ID NO: 19); CDR3: ATPMGMEGGLASKWSWYALEY (SEQ ID NO: 20).

[0125] The amino acid sequence of VHH of the fusion antibody C7-1-49 (FP867) is shown in SEQ ID NO:21, the nucleotide sequence is shown in SEQ ID NO:22, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:23 to 25, respectively.

[0126] QLQLVESGGGLVQPGGSLRLSCAASGATVDFYAMHWFRQAPGKEHEEVSCISSSGGSTNYADSVKGRFTISRDNAQNAVYLQMNSLKPEDTAVYYCAASPWCDQRALDAWGQGTLVTVS(SEQ ID NO:21);

[0127] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGGCTCTCCTGTGCAGCCTCTGGCGCCACTGTAGATTTTTATGCCATGCACTGGTTCCGCCAGGCGCCAGGGAAGGAGCATGAGGAGGTCTCATGTAT TAGTAGTAGTGGTGGGAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACGATCTCCAGAGACAACGCCCAGAACGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCGGCCTCCCCCATGGTGCGACCAGAGGGCTTTGGACGCATGGGGCCAGGGGGACCCTGGTCACTGTCTCC(SEQ ID NO:22);

[0128] CDR1: GATVDFYA (SEQ ID NO:23); CDR2: ISSSGGST (SEQ ID NO:24); CDR3: AASPWCDQRALDA (SEQ ID NO:25).

[0129] The amino acid sequence of VHH of the fusion antibody C7-1-50 (FP868) is shown in SEQ ID NO:26, the nucleotide sequence is shown in SEQ ID NO:27, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:28, 24 and 29 respectively.

[0130] QLQLVESGGGLVQPGGSLRLSCAASGGTLDNYAIHWFRQAPGEELEGVSCISSSGGSTNYADSVKDRFTISRDDAKNTAFLQMNSLKPEDTAVYYCAASPWCDNRALDAWGQGTQVTVS (SEQ ID NO: 26);

[0131] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACTTTGGATAATTATGCCATACACTGGTTCCGCCAGGCCCCAGGGGAGGAACTTGAGGGGGTCTCATGTAT TAGTAGTAGTGGTGGTAGCACAAACTATGCAGACTCCGTGAAGGACCGATTCACCATCTCCAGAGACGACGCTAAGAACACGGCGTTTTCTGCAAATGAACAGTCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCCTCCCCATGGTGCGACAATAGGGCTTTGGACGCATGGGGCCAGGGGGACCCAGGTCACTGTCTCC(SEQ ID NO:27);

[0132] CDR1: GGTLDNYA (SEQ ID NO:28); CDR2: ISSSGGST (SEQ ID NO:24); CDR3: AASSRLDPRVRTTDMDY (SEQ ID NO:39).

[0133] The amino acid sequence of VHH of the fusion antibody C7-1-58 (FP869) is shown in SEQ ID NO:30, the nucleotide sequence is shown in SEQ ID NO:31, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:32, 24 and 33 respectively.

[0134] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTNYADSVQGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCAAAGIGPCGSYMDPLGSWGQGTQVTVS(SEQ ID NO:30);

[0135] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAG TAGTGGTGGTAGCACAAACTATGCAGACTCCGTGCAGGGCCGATTCGCCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTACTACTGTGCAGCAGCGGGGATTGGGCCTTTGTGGTAGTTACATGGACCCCCTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:31);

[0136] CDR1: GFTLDYYA (SEQ ID NO: 32); CDR2: ISSSGGST (SEQ ID NO: 24); CDR3: AAAGIGPCGSYMDPLGS (SEQ ID NO: 33).

[0137] The amino acid sequence of VHH of the fusion antibody C7-1-95 (FP870) is shown in SEQ ID NO:34, the nucleotide sequence is shown in SEQ ID NO:35, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:36, 24 and 37 respectively.

[0138] QVQLVESGGGLVQPGGSLRLSCAASGSTLDDYAIGWFRQAPGKEREGVSCISSSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATGYYYCGDYADYWGQGTQVTVS(SEQ ID NO:34);

[0139] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCGGCCTCTGGATCCACTTTGGATGATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATT AGTAGTAGTGGTGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCTACAGGGTATTACTGTGGAGACTATGCCGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:35);

[0140] CDR1: GSTLDDYA (SEQ ID NO:36); CDR2: ISSSGGST (SEQ ID NO:24); CDR3: AATGYYYCGDYADY (SEQ ID NO:37).

[0141] The amino acid sequence of VHH of fusion antibody C71-105 (FP871) is shown in SEQ ID NO:38, the nucleotide sequence is shown in SEQ ID NO:39, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:40 to 42, respectively.

[0142] QLQLVESGGGLVQAGGSLRLSCAASGDTINIARFNWHRQAPGKQRELVAIITAAGRTNYADSVQGRFTISRDNAKNTVYLQMGSLKPEDTAVYSCNAESWDYKTYWGQGIQVTVS(SEQ ID NO:38);

[0143] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGACACCATCAATATCGCTCGCTTCAACTGGCACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGC AATTATTACTGCTGCCGGTCGCACGAACTACGCAGACTCCGTACAGGGCCGATTCACCATCTCCAGAGACAACGCAAAGAACACGGTATATCTGCAGATGGGCAGCCTGAAGCCTGAGGACACGGCCGTCTATAGCTGTAATGCAGAGAGTTGGGACTACAAGACGTACTGGGGCCAGGGGATCCAGGTCACCGTCTCC(SEQ ID NO:39);

[0144] CDR1: GDTINIAR (SEQ ID NO:40); CDR2: ITAAGRT (SEQ ID NO:41); CDR3: NAESWDYKTY (SEQ ID NO:42).

[0145] The amino acid sequence of VHH of the fusion antibody C7-2-138 (FP872) is shown in SEQ ID NO:43, the nucleotide sequence is shown in SEQ ID NO:44, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:45, 24 and 37 respectively.

[0146] QVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSCISSSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATGYYYCGDYADYWGQGTQVTVS(SEQ ID NO:43);

[0147] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATGATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATT AGTAGTAGTGGTGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCTACAGGGTATTACTGTGGAGACTATGCCGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:44);

[0148] CDR1: GFTLDDYA (SEQ ID NO: 45); CDR2: ISSSGGST (SEQ ID NO: 24); CDR3: AATGYYYCGDYADY (SEQ ID NO: 37).

[0149] The amino acid sequence of VHH of the fusion antibody C72-158 (FP873) is shown in SEQ ID NO:46, the nucleotide sequence is shown in SEQ ID NO:47, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:48 to 50, respectively.

[0150] QVQLVESGGGLVQPGGSLRLSCAASGFTLDNYAIGWFRQAPGKDLEGVSCISSSDGSTNYAGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASLPGCDQRAQDYWGQGTQVTVS(SEQ ID NO:46);

[0151] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATAATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGACCTTGAGGGGGTCTCATGTATT AGTAGTAGTGATGGTAGCACAAACTATGCAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCATCCCTACCTGGATGCGACCAAAGGGCACAGGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCC(SEQ ID NO:47);

[0152] CDR1: GFTLDNYA (SEQ ID NO:48); CDR2: ISSSDGST (SEQ ID NO:49); CDR3: AASLPGCDQRAQDYSEQ ID NO:50).

[0153] The amino acid sequence of VHH of fusion antibody C72-168 (FP874) is shown in SEQ ID NO:51, the nucleotide sequence is shown in SEQ ID NO:52, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs:53, 54 and 50 respectively.

[0154] QVQLVESGGGLVQPGGSLRLSCAASRFTLDHYAIAWFRRAPGKEREGVSCFSSSDGSTYYANSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATISGNWCFLEENRYFEDWGQGTLVTVS(SEQ ID NO:51);

[0155] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTAGATTCACTTTGGATCATTATGCCATAGCTTGGTTCCGTCGGGCCCCAGGGAAGGAGCGCGAGGGGGTGTCATGTTTTAGTAGTAGTGATGGTAGCACATACTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGACTATATCTGGTAACTGGTGCTTCCTCGAGGAAAACAGGTATTTCGAAGATTGGGGCCAGGGCACCCTGGTCACTGTCTCC(SEQ ID NO:52);

[0156] CDR1: RFTLDHYA (SEQ ID NO:53); CDR2: FSSSDGST (SEQ ID NO:54); CDR3: AASLPGCDQRAQDY (SEQ ID NO:50).

[0157] The amino acid sequence of human IgG Fc is shown in SEQ ID NO:55, and the nucleotide sequence is shown in SEQ ID NO:56.

[0158] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:55);

[0159] (SEQ ID NO:56).

[0160] The specific experimental process of plasmid construction of each antibody is as follows:

[0161] (1) Single fragment amplification reaction system preparation and procedure

[0162] The single fragment amplification reaction system was prepared as shown in Table 1, and the reaction was carried out according to the reaction procedure shown in Table 2. The primers for PCR amplification of the VHH region of the Anti-CD70 nanobody are shown in Table 3, and the size of the amplified fragment is 408 bp.

[0163] hIgG1-Fc fragment amplification primers:

[0164] F-IGg1: CTGAGCTGCTGGGAGGAccg (SEQ ID NO:57) (70% GC Tm=68);

[0165] R-IGgl:AAAAGGCGCAACCCGCTAGCtcatttacccggagacagggagagag (SEQ ID NO: 58) (54% GC Tm=67).

[0166] Table 1 Single fragment amplification reaction system

[0167] 5×Q5 Reaction Buffer 10μL 10mM dNTPs 1μL Q5 High-Fidelity DNA Polymerase 0.5μL Template plasmid 0.1~2ng Primer F 1μM Primer R 1μM <![CDATA[UltraPure TM Distilled Water]]> to 50μL

[0168] Table 2 Reaction procedure

[0169]

[0170] Table 3 PCR amplification primers for Anti-CD70 nanobody VHH region

[0171]

[0172] (2) Multi-fragment homologous recombination preparation system and procedure

[0173] The above double fragments were homologously recombined with the lentiviral vector backbone 8044 bp recovered after double digestion with NotI and NheI. The system was prepared as shown in Table 4.

[0174] Prepare the multi-fragment homologous recombination reaction system as shown in Table 4. For multi-fragment recombination reaction, perform the reaction at 50°C for 15 min; then cool to 4°C or immediately place on ice for cooling.

[0175] Table 4 Multi-fragment homologous recombination reaction system

[0176] Backbone plasmid digestion and purification product 50~100ng Target gene amplification recovery product 1:1 molar ratio of the same backbone plasmid digestion and purification product 2×ClonExpress Mix 5μL <![CDATA[UltraPure TM Distilled Water]]> To 10μL

[0177] (3) Construction of lentiviral vector for overexpression of target antigen gene

[0178] The CD70 target antigen amino acid sequence (SEQ ID NO: 66) was added to the CD70 PCR amplification reaction system according to Table 1, and the single-fragment PCR program was run according to Table 2, wherein the annealing temperature was set to 65°C. The lentiviral plasmid of the CD70 overexpression cell line was finally constructed (the plasmid map is shown in Figure 2 A). PCR amplification primers are as follows:

[0179] F-EF1-CD70: catttcaggtgtcgtgaagcggccgcgccaccatgctgcgtcggcgg (SEQ ID NO: 67);

[0180] P2A-CD70:gAAgTTaGTAGCTCCggatccggctatttcttgtccatcatc (SEQ ID NO: 68).

[0181] Example 2 Plasmid Extraction

[0182] (1) Plasmid transformation of the vector confirmed to be correct by sequence alignment was performed at 37°C for 14 h;

[0183] (2) Pick a single clone and culture it in a small amount of culture solution, with a culture volume of 4 mL Amp+LB (100 mg / L Amp+), at 37°C and 220 rpm for 10 h.

[0184] (3) Large-scale culture of bacterial liquid: The next day, the seed liquid was inoculated into 250 mL Amp+LB (100 mg / L Amp+) and cultured at 37°C and 220 rpm for 14 h.

[0185] (4) Use Tiangen Plasmid Extraction Kit for plasmid extraction: a. Add the bacterial solution to the collection bottle (500 mL), centrifuge at 4000 rpm for 30 min, and remove the supernatant as much as possible. b. Column equilibration step: Add 2.5 mL of equilibration solution BL to the adsorption column CP6 (the adsorption column is placed in a 50 mL collection tube). c. Add 10 mL of solution P1 to the bacterial precipitate to resuspend the bacteria. Add 10 mL of solution P2 to the bacterial suspension, gently invert 6 to 8 times to fully lyse the bacteria. Add 10 mL of solution P4 to the centrifuge tube, gently invert 6 to 8 times, and mix thoroughly until the solution appears white dispersed flocculent precipitate. d. Centrifuge the lysate at 3580 × g for 30 min. g. Carefully pour the supernatant lysate into the filter CS1, slowly push the push handle to filter, and collect the filtrate in a clean 50 mL tube. h. Add 3 mL of red endotoxin-free solution ER respectively, and after inversion and mixing, the solution will appear uniform and transparent yellow. i. Add 0.3 times the volume of isopropanol in the above filtrate, mix by inverting, and transfer to the adsorption column CP6. j. Centrifuge at 8228×g for 2 minutes at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube. k. Add 10mL of buffer ED to the adsorption column CP6, centrifuge at 8228×g for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. Repeat this step for low-copy plasmids. m. Add 10mL of rinse solution PW to the adsorption column CP6, centrifuge at 8228×g for 2 minutes, discard the waste liquid in the collection tube, put the adsorption column back into the collection tube, and repeat the operation steps. o. Put the adsorption column CP6 back into the collection tube, centrifuge at 8228×g for 5 minutes, then open the adsorption column CP6 and leave it at room temperature for a few minutes to completely dry the residual rinse solution in the adsorption material. p. Place the adsorption column CP6 in a clean 50mL collection tube, add 1-2mL of elution buffer TB to the middle part of the adsorption membrane, place it at room temperature for 5 minutes, and then centrifuge it at 8228×g for 5 minutes. q. Take 1μL of plasmid DNA to determine the concentration (adjust BufferTE to zero), divide the plasmid DNA into 1.5mL centrifuge tubes, mark the name, concentration, batch number and volume with a marker, and store at -20℃.

[0186] Example 3 Production of Antibodies

[0187] (1) Observe the 293T cells used for packaging under a microscope and select cells with a cell density of 80% to 95%.

[0188] (2) Prepare 10% packaging medium: open a new bottle of 500 mL DMEM medium, add 55 mL FBS, then add 550 μL 1 mol / L sodium pyruvate solution and 550 μL 25 mmol / L chloroquine phosphate solution, and mix well.

[0189] (3) Take out the selected cells from the incubator, pour the old culture medium in the culture bottle into the waste tank, add 17 mL of 10% packaging medium, and then return it to the carbon dioxide incubator to adapt.

[0190] (4) According to the number of culture bottles to be transfected, calculate the plasmid to be added (prepared in Example 2, 30 μg of plasmid per bottle) and 0.125 mol / L calcium chloride solution to prepare a DNA-CaCl2 mixed solution. Then take an equal volume of 2×HBS solution and add it dropwise to the DNA-CaCl2 mixed solution, vortexing the DNA-CaCl2 mixed solution while adding. After the addition is complete, let it stand at room temperature for 20 minutes until a white precipitate is formed.

[0191] (5) Remove the cells obtained in step (3) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, label the virus name and operation date on the bottle, return it to the incubator, and replace the medium with 5% packaging medium every 4 to 6 hours.

[0192] To prepare 5% packaging medium, open a new bottle of 500 mL DMEM medium and add the cell adhesion promoting factor complex ITX-100×gibco 51500056 and 27.5 mL gibco KnockOut TM Serum replacement (Cat. No. 10828028), add 550 μL 1 mol / L sodium pyruvate solution and 550 μL 25 mmol / L chloroquine phosphate solution, and mix well.

[0193] (6) Take out the 293T cells obtained in step (5) from the incubator, discard the old culture medium, add 25 mL of 5% packaging medium to each bottle, and then return to the incubator for continued culture. After 72 hours of culture, harvest the supernatant to obtain the fusion antibody.

[0194] Example 4 Preparation of target antigen overexpression lentivirus

[0195] (1) Observe the 293T cells used for packaging under a microscope and select cells with a cell density of 80% to 95%.

[0196] (2) Prepare 10% packaging medium: open a new bottle of 500 mL DMEM medium, add 55 mL FBS, then add 550 μL 1 mol / L sodium pyruvate solution and 550 μL 25 mmol / L chloroquine phosphate solution, and mix well.

[0197] (3) Take out the selected cells from the incubator, pour the old culture medium in the culture bottle into the waste tank, add 17 mL of 10% packaging medium, and then return it to the carbon dioxide incubator to adapt.

[0198] (4) According to the number of culture bottles to be packaged, calculate the amount of expression plasmid, packaging auxiliary plasmid, envelope plasmid and 0.125 mol / L calcium chloride solution to be added to prepare a DNA-CaCl2 mixed solution. Then take an equal volume of 2×HBS solution and add it dropwise to the DNA-CaCl2 mixed solution, vortexing the DNA-CaCl2 mixed solution while adding. After the addition is completed, let it stand at room temperature for 20 minutes until a white precipitate is formed.

[0199] (5) Remove the cells obtained in step (1) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, mark the virus name and operation date on the bottle, return it to the incubator, and replace the medium with 5% packaging medium after 4 to 6 hours.

[0200] (6) Prepare 5% packaging medium: add 27.5 mL of FBS and 27.5 mL of serum replacement to a 500 mL bottle of DMEM medium, add 550 μL of 1 mol / L sodium pyruvate solution and 550 μL of 25 mmol / L chloroquine phosphate solution, and mix well.

[0201] (7) Take out the 293T cells obtained in step (5) from the incubator, discard the old culture medium, add 25 mL of 5% packaging medium to each bottle, and then return to the incubator to continue culturing. Collect the virus after 42 to 48 hours of culture.

[0202] Example 5 Construction of target antigen overexpression cell line

[0203] Resuscitation of Jurkat cell line: Take out the Jurkat cells to be revived from the liquid nitrogen tank, thaw quickly in a 37°C constant temperature water bath, and use a 10mL pipette to transfer the cell suspension to RM1640 medium with 10mL 10% FBS in advance, centrifuge at 1000rpm for 5min, discard the supernatant after centrifugation, resuspend the cell pellet in fresh 10mL 10% FBS RM1640, transfer to T25, and culture in a 37°C CO2 incubator. Subculture every two days at a ratio of 1:3;

[0204] The cells that were cultured stably for 3 generations starting from the recovery were inoculated into a 24-well plate at 0.8E5 / well. The harvested lentiviral supernatant (Example 4) was transduced at 10 μL, 20 μL, and 40 μL. The expression of EGFP was detected by flow cytometry after 72 hours, and the positive rate was greater than 85%. The positive rate of the CD70 target antigen overexpressing cells involved in the present invention was 86.6%. The cell line (i.e., CD70jurkat) was successfully constructed and can be used for subsequent antibody supernatant affinity property detection.

[0205] The results of flow cytometry of cell lines constructed with CD70 Jurkat target antigen are as follows Figure 2In middle B, the results showed that the GFP positivity rate was 61.85%, because the green light protein promoter gene was a weak promoter, and the positivity rate of antigen CD70 was 97%, and the target antigen cell line was successfully constructed.

[0206] Example 6 Antibody supernatant specificity test

[0207] (1) Collect 1 mL of the antibody supernatant obtained 72 h after transfection (Example 3) in a 1.5 mL centrifuge tube.

[0208] (2) Centrifugation at 12000 rpm for 5 min to remove cell debris and clarify using a 0.22 μm filter for later use;

[0209] (3) Cell staining: CD70 Jurkat cells (Example 5) and Jurkat cells to be stained by flow cytometry were taken, washed with sodium chloride / PBS, and 50 μL and 200 μL of the antibody supernatant in step (2) were added, and incubated at 4° C. for 30 min in a dark environment;

[0210] (4) Wash the stained cells with sodium chloride, centrifuge, discard the supernatant, add mouse anti-human anti-hunan-IG1-FC-APC Biolegend 410712-100tests flow cytometry antibody, incubate at 4°C for 60 min, and protect from light;

[0211] (5) Wash the cells to be tested with sodium chloride, add flow cytometry buffer after centrifugation, and test on the flow cytometry machine.

[0212] The flow cytometry results showed that C7-1-3 (FP865), C7-1-44 (FP866), C7-1-49 (FP867), C7-1-50 (FP868), C7-1-58 (FP869), C7-1-95 (FP870), C7-1-105 (FP871), C7-2-138 (FP872), C7-2-158 (FP873), and C7-2-168 (FP874) specifically bound to CD70 jurkat ( Figure 3-4 and Table 5). Similarly, after co-incubation with Jurkat cells, mouse anti-human anti-hunan-IG1-FC-APC was used for secondary antibody incubation for 1 hour before flow cytometry detection, such as Figure 5 As shown, all fusion antibodies failed to bind to Jurkat cells.

[0213] Table 5 Flow cytometry results of affinity test of fusion antibody supernatant to target antigen cells

[0214]

[0215] Example 7 ELISA quantitative detection of antibody supernatant

[0216] ELISA was used to quantitatively detect the expression ability of fusion antibodies C7-1-3 (FP865), C7-1-44 (FP866), C7-1-49 (FP867), C7-1-50 (FP868), C7-1-58 (FP869), C7-1-95 (FP870), C7-1-105 (FP871), C7-2-138 (FP872), C72-158 (FP873), and C72-168 (FP874), as follows:

[0217] Take out the ELISA plate from the aluminum foil bag after equilibration at room temperature for 20 minutes. Set up standard wells and sample wells, add 50 μL of human IgG Fc fragment standard of different concentrations to each standard well; add 50 μL of the sample to be tested (fusion antibodies FP878, FP879, FP880, FP881, FP882, FP883, FP884 and FP885) to the sample wells (gradient dilution, 1:100 / 1:1000 / 1:10000), and do not add to the blank wells. In addition to the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard wells and sample wells, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 60 minutes. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate). Add 50μL of substrate A and B to each well, incubate at 37℃ in the dark for 15min. Add 50μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450nm within 15min.

[0218] The results are shown in Table 6. Among the fusion antibodies, the ones with the strongest expression ability are C7-1-50 (FP868), C7-1-95 (FP870), C7-1-105 (FP871), C7-2-138 (FP872), and C7-2-158 (FP873), and the one with the middle expression is C7-1-3 (FP865).

[0219] Table 6 Elisa detection of protein content in each supernatant

[0220]

[0221]

[0222] Example 8 Quantification and Binding Ability Detection of Anti-CD70 Fusion Antibody

[0223] Cell staining: Take CD70-jurkat cells and Jurkat cells to be stained by flow cytometry, wash with sodium chloride / PBS, dilute each fusion antibody as shown in Table 7, and stain the CD70-jurkat cells, incubate at 4°C for 30 minutes, and protect from light; wash the above stained cells with sodium chloride, centrifuge, discard the supernatant, add anti-hunan-IG1-FC-APC flow cytometry antibody, incubate at 4°C for 60 minutes, and protect from light; wash the cells to be detected with sodium chloride, centrifuge, add flow cytometry buffer, and detect on the machine.

[0224] Table 7 Preparation of fusion antibody dilution

[0225]

[0226] After ELISA quantification, the supernatants of each antibody were subjected to gradient flow cytometry staining at 100 ng, 25 ng, 6.25 ng, 1.56 ng, 0.391 ng, and 0.098 ng. The flow cytometry results are shown in Figure 6 to Figure 8 As shown in Table 8, all fusion antibodies were able to bind to Jurkat-CD70 target cells. As the antibody concentration decreased after dilution, the number and intensity of target cell binding decreased. Analysis showed that the antibodies with the strongest fluorescence after binding to target cells were C7-1-3 (FP865), C7-2-158 (FP873), and C7-2-168 (FP874) (Table 8 and Fig. 9 ); the antibodies with the largest number of target cells bound per unit mass were C7-1-3 (FP865), C7-1-58 (FP869), C7-1-95 (FP870), and C7-2-138 (FP872) (Table 9 and Fig.10 ). Taking all factors into consideration, C7-1-3 (FP865) has the strongest affinity.

[0227] Table 8 Quantitative detection and target cell Anti-CD70 binding ability (MFI)

[0228]

[0229]

[0230] Table 9 Quantitative detection and target cell Anti-CD70 binding ability (positive cell ratio)

[0231]

[0232] Example 9 Anti-CD70 fusion antibody blocking ability detection

[0233] Construct jurkat-CD27-RFP and jurkat-CD70-EGFP cell lines; utilize the affinity property of co-stimulatory molecule receptor ligand to genetically synthesize a lentiviral vector containing the CD27 gene, construct jurkat-CD27-RFP cells in the manner of Example 5, and take the above cells for cell counting; according to the counting results, dilute the cell density to 1E+06 / mL, take 200 μL of each cell in a 1.5 mL centrifuge tube, add 1 μg of purified antibody, add RM1640 without antibody to the Blank group, and the diluent is 50 μL 1% FBS 1640 buffer. Incubate at 37°C for 1 hour and detect on a flow cytometer.

[0234] The results are as follows Figure 11-12 As shown, since the Blank group does not contain antibody supernatant, it can be seen from the flow cytometry that jurkat-CD27-RFP and jurkat-CD70-EGFP are bound, and the binding positivity rate is 21%; after adding anti-CD70 fusion antibody, the experimental group with antibody binding ability greater than CD27 molecules showed a decrease in the co-positivity rate of jurkat-CD27-RFP and jurkat-CD70-EGFP, among which C7-1-44 (FP866), C7-1-49 (FP867), C7-1-50 (FP868), C7-1-105 (FP871) and C72-168 (FP874) had the best blocking effect.

[0235] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An anti-CD70 nanobody, comprising a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein: The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:48, and SEQ ID NO:53; The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:41, SEQ ID NO:49, and SEQ ID NO:54; The amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:42, and SEQ ID NO:

50.

2. The anti-CD70 nanobody according to claim 1, characterized in that The amino acid sequence of the anti-CD70 nanobody is as shown in SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:51, or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:51 and having the same or similar function.

3. A fusion antibody comprising a first domain and a second domain; The first domain is the anti-CD70 nanobody according to claim 1 or 2; The second domain has the effect of extending the half-life in vivo and / or has a binding effect on effector cells.

4. The fusion antibody according to claim 3, characterized in that: The second domain comprises: Immunoglobulin Fc region; and / or Serum albumin or a fragment thereof, a domain that binds serum albumin, polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and / or a molecule having affinity for a T cell surface molecule and / or capable of binding to a surface molecule present on a T cell; Preferably, the immunoglobulin is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, and IgM; Preferably, the first domain and the second domain are connected by a hinge or by a connecting peptide.

5. A biological material related to the anti-CD70 nanobody according to claim 1 or 2 or the fusion antibody according to claim 3 or 4; the biological material is any one of a1) to a12): a1) a nucleic acid molecule encoding the anti-CD70 nanobody according to claim 1 or 2 or the fusion antibody according to claim 3 or 4; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic cell line containing the nucleic acid molecule described in a1); a10) a transgenic cell line containing the expression cassette described in a2); a11) a transgenic cell line containing the recombinant vector described in a3); a12) A transgenic cell line containing the recombinant vector described in a4).

6. An immunoconjugate comprising: (A) the anti-CD70 nanobody according to claim 1 or 2 or the fusion antibody according to claim 3 or 4; as well as (B) Functional molecule linked to (A).

7. The immunoconjugate according to claim 6, characterized in that The (B) comprises cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, tumor-suppressing molecules, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof; Preferably, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker; Preferably, the tumor-inhibiting molecule is an anti-tumor cytokine or an anti-tumor toxin.

8. A drug comprising the anti-CD70 nanobody according to claim 1 or 2, the fusion antibody according to claim 3 or 4, the biomaterial according to claim 5, or the immunoconjugate according to claim 6 or 7.

9. Use of the anti-CD70 nanobody according to claim 1 or 2, the fusion antibody according to claim 3 or 4, the biomaterial according to claim 5, the immunoconjugate according to claim 6 or 7, or the drug according to claim 8 in any one of (1) to (4): (1) Preparation of preparations, test kits or pharmaceutical kits for diagnosing, treating or preventing cancer; (2) preparing a preparation or kit for treating or preventing an autoimmune disease; (3) Quantitative / qualitative detection of CD70 expression; (4) preparing reagents / kits for quantitative / qualitative detection of CD70 expression; Preferably, the cancer described in (1) includes: At least one of lymphoma, lung cancer, renal cell carcinoma, glioblastoma, melanoma, nasopharyngeal carcinoma, malignant pleural mesothelioma, anaplastic astrocytoma, thymic squamous cell carcinoma, hepatocarcinoma and tumors induced by Epstein-Barr virus infection; Preferably, the autoimmune disease in (2) comprises at least one of lupus erythematosus, sepsis, arthritis, pancreatitis and type I diabetes.

10. A test kit or a medicine kit comprising the anti-CD70 nanobody according to claim 1 or 2, the fusion antibody according to claim 3 or 4, the biomaterial according to claim 5, the immunoconjugate according to claim 6 or 7, or the drug according to claim 8.

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