Anti-cd70 nanobodies and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an anti-CD70 nanobody and application thereof, and specifically discloses an anti-CD70 nanobody, and limits the specific amino acid sequence thereof. The anti-CD70 nanobody obtained by immunizing a llama has high affinity and strong specificity, and can be used for immunoblotting, an enzyme-linked immunoreaction kit, flow cytometry counting, and also can be used for treating diseases such as tumors and autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-CD70 nanobody and its application. Background Technology

[0002] In recent years, immunotherapy based on specific targets has become a hot topic in the field of cancer treatment. Cluster of differentiation 70 (CD70) is a member of the tumor necrosis factor receptor superfamily and is a type II transmembrane glycoprotein. Initially used alongside CD30 as a marker for Hodgkin's lymphoma, it was cloned into a Ki-24 antibody. Following naming conventions, Ki-24 was named CD70. As early as 1993, Goodwin discovered that CD27 ligand (CD27L) is a type II transmembrane glycoprotein, and a series of studies have shown that CD27L and CD70 are the same molecule. The binding of CD70 to CD27 can induce the activation of the 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 responses. Under normal circumstances, CD70 is only transiently expressed in activated T cells and B cells, as well as mature dendritic cells. Recent studies have found that CD70 is abnormally expressed in various hematologic malignancies and solid tumors, particularly in lymphoma, renal cell carcinoma, glioblastoma, melanoma, nasopharyngeal carcinoma, malignant pleural mesothelioma, liver cancer, and tumors induced by Epstein-Barr virus (EBV) infection. The expression level of CD70 varies among different types of tumors; for example, it is expressed at levels exceeding 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. The high expression of CD70 in various hematologic malignancies and solid tumors, and its low / absent expression in normal tissues, makes CD70 a compelling target for tumor immunotherapy. Therefore, drugs developed targeting CD70 represent a promising strategy for cancer treatment.

[0003] INAGUMA et al. found that CD70 can enhance tumor migration and invasion by regulating MET protein expression and activating the MET-ERK signaling pathway. Other studies have shown that the mechanism of CD70 expression in tumor cells may involve CD70 binding to CD27 expressed on T cells, initiating the apoptosis protein Siva, which induces cytotoxicity in B cells or T cells, leading to apoptosis and immune escape. Furthermore, the CD27 and CD70 signaling pathways can mediate paracrine growth signals through B cell-B cell interactions, contributing to the development and metastasis of lymphoma.

[0004] Nanobodies (VHHs) share the same structural domains as conventional VH antibodies, namely four conserved framework regions (FR1 / 2 / 3 / 4) and three complement-determining regions (CDR1 / 2 / 3). Nanobodies have a molecular weight only 10% that of traditional antibodies, retaining the complete antigen-binding capacity of HCAbs, and exhibiting high specificity, good affinity, and high stability. Because nanobodies lack an Fc fragment, they cannot produce ADCC / CDC cytotoxic effects like conventional antibodies. Therefore, VHH antibodies are often fused with an Fc fragment to construct Fc-VHH fusion antibodies to enhance ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) activities. Compared to conventional antibodies, these forms of nanobodies can be widely used in the treatment of various diseases. Summary of the Invention

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

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

[0007] The object of a third aspect of the present invention is to provide biomaterials 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] A fourth aspect of the present invention is to provide an immunoconjugate.

[0009] The fifth aspect of this invention is to provide a medicine.

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

[0011] The seventh aspect of this invention is to provide a reagent kit or medicine box.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides an anti-CD70 nanobody, the anti-CD70 nanobody comprising a complementarity-determining region (CDR), the CDR comprising complementarity-determining region CDR1, complementarity-determining region CDR2, and complementarity-determining region CDR3, wherein...

[0014] The amino acid sequence of the complementarity-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 complementarity-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 complementarity-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 includes:

[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 present invention, CDR1, CDR2 and CDR3 are separated by the backbone 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 that is functionally identical or similar to the amino acid sequences 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 after substitution, deletion, or addition of one or more amino acids.

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

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

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

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

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

[0035] Serum albumin (such as human HSA) or fragments thereof, serum albumin-binding domains (such as anti-serum albumin antibodies, including nanobodies), polyethylene glycol, polyethylene glycol-liposome complexes, or combinations thereof; and / or

[0036] Molecules that have an affinity for T cell surface molecules and / or are able to bind to surface molecules (such as CD3) present on T cells.

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

[0038] In some embodiments of the present invention, the immunoglobulin is 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 Fc region of the immunoglobulin is as shown in SEQ ID NO:55; or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:55 after substitution, deletion or addition of one or more amino acids.

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

[0042] In some embodiments of the present invention, the first and second domains are hinged together (e.g., the anti-CD70 nanobody is directly fused to the hinge region of Fc) or connected via a linker peptide.

[0043] In some embodiments of the present invention, the linker 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) nYAPVDV, (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 NO:1-8.

[0046] A third aspect of the present invention provides biomaterials 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; said biomaterials are any one of a1) to a12):

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

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

[0049] a3) A recombinant vector containing the nucleic acid molecules described in a1);

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

[0051] a5) Recombinant microorganisms containing the nucleic acid molecules described in a1);

[0052] a6) Recombinant microorganisms containing the expression cassette described in a2);

[0053] a7) Recombinant microorganisms containing the recombinant vector described in a3);

[0054] a8) Recombinant microorganisms containing the recombinant vector described in a4);

[0055] a9) Transgenic cell lines containing the nucleic acid molecules described in a1);

[0056] a10) Transgenic cell lines containing the expression cassette described in a2);

[0057] a11) Transgenic cell lines containing the recombinant vector described in a3);

[0058] a12) Transgenic cell lines containing the recombinant vector described in a4).

[0059] In some embodiments of the present invention, the nucleic acid molecules encoding the anti-CD70 nanobody of the first aspect of the present invention are shown in 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 present invention, the transgenic animal cell line does not contain reproductive material.

[0061] In some embodiments of the present invention, the expression cassette includes 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' transcriptional control region includes a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeper" promoter may be used), a transcription start site, an enhancer, and / or a silencing element.

[0063] In some embodiments of the invention, the 3'UTR may encode AU-rich elements, which are common regulators of mRNA stability via the 3'-5' exogenous pathway and are typically located in the 3'UTR. AU-rich elements may comprise one or more repeats of the sequence AUUUA. It may also comprise one or more so-called US2B elements having the sequence AUAUAU.

[0064] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

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

[0066] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

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

[0068] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, 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 plant or animal varieties.

[0072] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which are capable of expressing the target protein.

[0073] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

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

[0075] (A) The anti-CD70 nanobody of the first aspect of the present invention or the fusion antibody of the second aspect of the present invention; and

[0076] (B) Functional molecules that are connected to (A) (including but not limited to covalent connection, coupling, attachment, and adsorption).

[0077] In some embodiments of the present invention, (B) includes cytotoxins, radioisotopes, bioactive proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting immune cell surface markers, 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 the CD3ζ chain, FcεRIγ tyrosine activation motif, intracellular signaling regions of co-stimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, CD40, etc.), or combinations thereof.

[0078] In some embodiments of the present invention, the molecules that target tumor surface markers are antibodies or ligands that bind to tumor surface markers.

[0079] In some embodiments of the present invention, the tumor-inhibiting molecules are anti-tumor cytokines (such as IL-12, IL-15, IFN-beta, TNFalpha) or anti-tumor toxins.

[0080] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, 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 (computed tomography) contrast agents or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

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

[0083] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

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

[0085] In some embodiments of the present invention, the drug further includes combination drugs, including but not limited to immune effector molecules, cells, cytotoxic substances, and multi-kinase inhibitors.

[0086] A 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) To prepare preparations, kits or boxes for the diagnosis, treatment or prevention of cancer;

[0088] (2) To prepare preparations or kits for the treatment or prevention of autoimmune diseases;

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

[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 (Chinese herbal monomers, microRNA, etc.).

[0092] In some embodiments of the present invention, the cancer described in (1) includes: at least one of the following: lymphoma (such as non-Hodgkin lymphoma, Hodgkin 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 described 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 / kit described in (4) includes reagents for immunoblotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry for counting.

[0095] A seventh aspect of the present invention provides a kit or pharmaceutical kit comprising an anti-CD70 nanobody of the first aspect of the present invention, a fusion antibody of the second aspect of the present invention, a biomaterial of the third aspect of the present invention, an immunoconjugate of the fourth aspect of the present invention, or a 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 (ELISA), and flow cytometry for cell counting.

[0097] The beneficial effects of this invention are:

[0098] The anti-CD70 nanobody obtained by immunizing alpacas in this invention has high affinity and strong specificity. It can be used for immunoblotting, enzyme-linked immunosorbent assay kits, flow cytometry detection and counting, and can also be used for the treatment of diseases such as tumors, autoimmune diseases and chronic infections. It has the potential to be developed as an immune checkpoint inhibitor. Attached Figure Description

[0099] Figure 1 This is a schematic diagram of the structure of the fusion antibody of the present invention.

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

[0101] Figure 3 The results of flow cytometry analysis of the cell affinity of the supernatant of fusion antibodies FP865, FP866, FP867 and FP868 for the target antigens are presented.

[0102] Figure 4 The results of flow cytometry analysis of the cell affinity of the supernatant of fusion antibodies FP869, FP870, FP871, FP872, FP873 and FP874 for the target antigens are presented.

[0103] Figure 5 The results of flow cytometry analysis of the affinity of each fusion antibody for non-target antigens in cell clearing are shown.

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

[0105] Figure 7 Flow cytometry report to quantitatively detect the binding capacity of fusion antibodies FP869, FP870, FP871 and FP872 to target cells.

[0106] Figure 8 Flow cytometry report for quantitative detection of the binding capacity of fusion antibodies FP873 and FP847 to target cells.

[0107] Figure 9 To quantitatively detect the binding ability of each fusion antibody to Anti-CD70 on target cells—MFI.

[0108] Figure 10 To quantitatively detect the binding ability of each fusion antibody to Anti-CD70 in target cells—the proportion of positive cells.

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

[0110] Figure 12 A bar chart showing the number of antibody screenings for blocking function of Anti-CD70. Detailed Implementation

[0111] The present invention will be further described in detail below through specific embodiments.

[0112] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0113] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0114] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0115] Example 1 Plasmid Construction

[0116] Multiple candidate nanobody sequences were obtained by immunizing alpacas with the CD70 target antigen amino acid sequence. The VHH region nucleic acid sequence of the nanobody was ligated with the human IgG Fc nucleic acid sequence using multi-fragment homologous recombination technology. The ligation region was the hinge. The plasmids after homologous recombination were identified by enzyme digestion and then sequenced to determine the fusion antibody sequence structure. Figure 1 As shown in the figure. Ten fusion antibodies were constructed, 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), and their amino acid sequences are shown in SEQ ID NO: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 its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 NO: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 its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:32, 24 and 33 respectively.

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

[0135] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATTAGTAG 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 NO:36, 24 and 37 respectively.

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

[0139] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCGGCCTCTGGATCCACTTTGGATGATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATT AGTAGTAGTGGTGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCTACAGGGTATTACTGTGGAGACTATGCCGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC(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 the 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 its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 NO:45, 24 and 37 respectively.

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

[0147] ATGGAAACCCCCGCCCAGCTGCTGTTTCTGCTGCTGCTTTGGCTGCCTGACACCACCCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTGGATGATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATGTATT AGTAGTAGTGGTGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCTACAGGGTATTACTGTGGAGACTATGCCGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC(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 its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 the 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 its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 procedures for constructing plasmids for each antibody are as follows:

[0161] (1) Preparation and procedure of single-fragment amplification reaction system

[0162] Prepare the single-fragment amplification reaction system as shown in Table 1, and perform the reaction 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 amplified fragment size is 408 bp.

[0163] hIgG1-Fc fragment amplification primers:

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

[0165] R-IGg1:AAAAGGCGCAACCCGCTAGCtcatttacccggagacagggag (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 Primers for PCR amplification of the VHH region of Anti-CD70 nanobody

[0171]

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

[0173] The above two fragments were homologously recombinated with the 8044bp lentiviral vector backbone recovered after double digestion with NotI and NheI. The system preparation is shown in Table 4.

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

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

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

[0177] (3) Construction of lentiviral vectors for overexpressing target antigen genes

[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, with the annealing temperature set to 65℃. The final lentiviral plasmid for the CD70 overexpression cell line was constructed (plasmid map shown in Table 2). Figure 2 (A). The 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) Transform the vector with the correct sequence alignment into a plasmid at 37°C for 14 hours.

[0183] (2) Pick single colonies, culture a small amount of bacterial solution (4 mL Amp+LB, 100 mg / L Amp+), at 37℃ and 220 rpm for 10 h.

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

[0185] (4) Plasmid extraction using the Tiangen plasmid extraction kit: a. Add the bacterial suspension to a collection bottle (500 mL), centrifuge at 4000 rpm for 30 min, and remove as much supernatant as possible. b. Column equilibration: Add 2.5 mL of equilibration buffer BL to the adsorption column CP6 (place the adsorption column in a 50 mL collection tube). c. Resuspend the bacterial cells in 10 mL of solution P1. Add 10 mL of solution P2 to the bacterial suspension, gently invert 6–8 times to fully lyse the cells. Add 10 mL of solution P4 to the centrifuge tube, gently invert 6–8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. d. Centrifuge the lysate at 3580 × g for 30 min. g. Carefully pour the supernatant lysate into 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, invert and mix until the solution is a uniform, transparent yellow. i. Add 0.3 times the volume of the filtrate above in isopropanol, mix thoroughly by inverting, and transfer to adsorption column CP6. j. Centrifuge at 8228×g for 2 min at room temperature, discard the waste liquid in the collection tube, and return adsorption column CP6 to the collection tube. k. Add 10 mL of buffer ED to adsorption column CP6, centrifuge at 8228×g for 2 min, discard the waste liquid in the collection tube, and return adsorption column to the collection tube. Repeat this step for low copy plasmids. m. Add 10 mL of wash buffer PW to adsorption column CP6, centrifuge at 8228×g for 2 min, discard the waste liquid in the collection tube, and return adsorption column to the collection tube. Repeat the steps. o. Return adsorption column CP6 to the collection tube, centrifuge at 8228×g for 5 min, then open the cap of adsorption column CP6 and place it at room temperature for several minutes to thoroughly dry any remaining wash liquid in the adsorption material. p. Place the CP6 adsorption column in a clean 50 mL collection tube, add 1–2 mL of elution buffer TB to the center of the adsorption membrane, incubate at room temperature for 5 min, then centrifuge at 8228 × g for 5 min at room temperature. q. Take 1 μL of plasmid DNA to determine the concentration (zero with Buffer TE), aliquot the plasmid DNA into 1.5 mL centrifuge tubes, label them with the name, concentration, batch number, and volume, and store at -20℃.

[0186] Example 3 Antibody Production

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

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

[0189] (3) Take the selected cells out of the incubator, pour the old culture medium in the culture flask into the waste liquid tank, add 17 mL of 10% packaged culture medium, and then put it back into the carbon dioxide incubator to adapt.

[0190] (4) Calculate the required amount of plasmid (prepared in Example 2, 30 μg plasmid per bottle) and 0.125 mol / L calcium chloride solution to prepare a DNA-CaCl2 mixture, based on the number of culture flasks to be transfected. Then, add an equal volume of 2×HBS solution dropwise to the DNA-CaCl2 mixture while vortexing the mixture. After the addition is complete, allow it to stand at room temperature for 20 minutes until a white precipitate forms.

[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 bottle with the virus name and operation date, put it back into the incubator, and change the medium with 5% packaged culture medium for 4-6 hours.

[0192] To prepare 5% packaged culture medium, open a new 500mL bottle of DMEM medium and add the cell adhesion-promoting factor complex ITX-100×gibco 51500056 and 27.5mL of gibco KnockOut. TM Serum substitute (catalog number 10828028) was added to 550 μL of 1 mol / L sodium pyruvate solution and 550 μL of 25 mmol / L chloroquine phosphate solution, and mixed well.

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

[0194] Example 4: Preparation of Lentiviral Virus Overexpressing Target Antigen

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

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

[0197] (3) Take the selected cells out of the incubator, pour the old culture medium in the culture flask into the waste liquid tank, add 17 mL of 10% packaged culture medium, and then put it back into the carbon dioxide incubator to adapt.

[0198] (4) Calculate the required number of expression plasmids, packaging helper plasmids, coating plasmids, and 0.125 mol / L calcium chloride solution to be added, and prepare a DNA-CaCl2 mixture. Then, add an equal volume of 2×HBS solution dropwise to the DNA-CaCl2 mixture while vortexing the mixture. After the addition is complete, let it stand at room temperature for 20 minutes until a white precipitate forms.

[0199] (5) Remove the cells obtained in step (1) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, and label the bottle with the virus name and operation date. Return the bottle to the incubator and change the medium with 5% packaged culture medium for 4-6 hours.

[0200] (6) Prepare 5% packaged culture medium. In a 500mL bottle of DMEM culture medium, add 27.5mL of FBS and 27.5mL of serum substitute, add 550μL of 1mol / L sodium pyruvate solution and 550μL of 25mmol / 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 25mL of 5% packaged culture medium to each bottle, and then put them back into the incubator to continue culturing. Harvest the cells after culturing for 42-48 hours.

[0202] Example 5: Construction of target antigen overexpression cell lines

[0203] Resuscitating the Jurakat cell line: Remove the Jurakat cells to be resuscitated from the liquid nitrogen tank, thaw them rapidly in a 37°C constant temperature water bath, and then use a 10mL pipette to transfer the cell suspension to RM1640 medium containing 10mL of 10% FBS. Centrifuge at 1000rpm for 5min, discard the supernatant after centrifugation, resuspend the cell pellet in 10mL of fresh RM1640 medium containing 10% FBS, transfer it to T25, and incubate in a 37°C CO2 incubator. Passage the cells every two days at a ratio of 1:3.

[0204] Cells that have been stably cultured for 3 generations since revival were seeded into 24-well plates at a density of 0.8 E5 / well. The harvested lentiviral supernatant (Example 4) was transduced at 10 μL, 20 μL, and 40 μL. After 72 hours, the expression of EGFP was detected by flow cytometry. The positive rate was greater than 85%, and the positive rate of the CD70 target antigen overexpressing cells involved in this invention was 86.6%. The cell line (i.e., CD70jurkat) was successfully constructed and can be used for subsequent antibody supernatant affinity testing.

[0205] Flow cytometry results of CD70 jurkat target antigen-constructed cell lines are as follows: Figure 2In the B cell line, the GFP positivity rate was 61.85%, which was attributed to the weak promoter of the chlorophyll protein promoter gene. The positivity rate of the CD70 antigen was 97%, indicating that 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) into a 1.5 mL centrifuge tube.

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

[0209] (3) Cell staining: Take CD70 jurkat cells (Example 5) and jurkat cells to be stained by flow cytometry, wash with sodium chloride / PBS, add 50 μL and 200 μL of antibody supernatant from step (2), incubate at 4°C for 30 min, and protect from light;

[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℃ for 60 min, and protect from light;

[0211] (5) Wash the cells to be tested with sodium chloride, centrifuge, add flow cytometry buffer, and then perform the test.

[0212] 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 bind to CD70 jurkat. Figures 3-4 (and Table 5). Similarly, after co-incubation with Jurakat cells, secondary antibody incubation with mouse anti-human anti-hunan-IG1-FC-APC was performed for 1 hour, followed by flow cytometry analysis. Figure 5 As shown, none of the fusion antibodies bound to jurkat cells.

[0213] Table 5. Flow cytometry results of the affinity of the fusion antibody supernatant for the target antigen cells.

[0214]

[0215] Example 7: ELISA Quantitative Detection of Antibody Supernatant

[0216] The expression levels of the 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) were quantitatively detected by ELISA, as follows:

[0217] Remove the ELISA plate from the aluminum foil bag after equilibration to room temperature for 20 minutes. Set up standard wells and sample wells. Add 50 μL of human IgG Fc fragment standards of different concentrations to each standard well. Add 50 μL of the test samples (fusion antibodies FP878, FP879, FP880, FP881, FP882, FP883, FP884, and FP885) (serial dilution, 1:100 / 1:1000 / 1:10000) to each sample well. Do not add any to the blank wells. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each standard and sample well. Seal the reaction wells with sealing film and incubate at 37°C in a water bath or incubator for 60 minutes. Discard the liquid, blot dry on absorbent paper, add 350 μL of washing buffer to each well, let stand for 1 min, discard the washing buffer, blot dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used). Add 50 μL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well, and within 15 min, measure the OD value of each well at a wavelength of 450 nm.

[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), while the one with the middle expression level is C7-1-3 (FP865).

[0219] Table 6 Protein content in each supernatant as determined by ELISA

[0220]

[0221]

[0222] Example 8: Detection of binding ability of anti-CD70 fusion antibody after quantification

[0223] Cell staining: CD70-jurkat cells and jurkat cells to be stained by flow cytometry were washed with sodium chloride / PBS. After washing, the fusion antibodies were diluted as shown in Table 7 and used to stain CD70-jurkat cells. The cells were incubated at 4°C for 30 min in the dark. The stained cells were washed with sodium chloride, centrifuged, and the supernatant was discarded. Anti-hunan-IG1-FC-APC flow cytometry antibody was added and the cells were incubated at 4°C for 60 min in the dark. The cells to be tested were washed with sodium chloride, centrifuged, and then flow cytometry buffer was added for flow cytometry analysis.

[0224] Table 7 Preparation of Fusion Antibody Diluent

[0225]

[0226] After ELISA quantification, the antibody supernatants were subjected to gradient flow cytometry staining at concentrations of 100 ng, 25 ng, 6.25 ng, 1.56 ng, 0.391 ng, and 0.098 ng. The flow cytometry results are as follows: Figures 6-8 As shown, all fusion antibodies were able to bind to jurakat-CD70 target cells. With decreasing antibody concentration after dilution, both the number and intensity of binding to target cells decreased. Analysis revealed that the antibodies exhibiting 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 10). Figure 9 The antibodies that bound the most target cells per unit mass were C7-1-3 (FP865), C7-1-58 (FP869), C7-1-95 (FP870), and C7-2-138 (FP872) (Table 9 and 10). Figure 10 Considering all factors, C7-1-3 (FP865) has the strongest affinity attribute.

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

[0228]

[0229]

[0230] Table 9. Quantitative detection and target cell Anti-CD70 binding capacity (percentage of positive cells)

[0231]

[0232] Example 9: Detection of the blocking ability of anti-CD70 fusion antibody

[0233] Jurkat-CD27-RFP and Jurkat-CD70-EGFP cell lines were constructed. Utilizing the affinity properties of the co-stimulatory molecule receptor ligand, a lentiviral vector containing the CD27 gene was synthesized. Jurkat-CD27-RFP cells were constructed according to the method described in Example 5. Cell counting was performed on these cells. Based on the counting results, the cell density was diluted to 1E+06 / mL. 200 μL of each cell type was placed in a 1.5 mL centrifuge tube. 1 μg of purified antibody was added. The Blank group was supplemented with antibody-free RM1640. The dilution buffer was 50 μL of 1% FBS 1640. The cells were incubated at 37°C for 1 h and then analyzed by flow cytometry.

[0234] The results are as follows Figures 11-12 As shown, the Blank group, which did not contain antibody supernatant, showed binding of jurkat-CD27-RFP and jurkat-CD70-EGFP in the flow cytometry, with a positive binding rate of 21%. After adding anti-CD70 fusion antibody, the experimental groups with antibody binding ability greater than that of CD27 molecules showed a decrease in the co-positive rate of jurkat-CD27-RFP and jurkat-CD70-EGFP. Among them, the C7-1-44 (FP866), C7-1-49 (FP867), C7-1-50 (FP868), C7-1-105 (FP871), and C72-168 (FP874) showed the best blocking effect.

[0235] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An anti-CD70 nanobody, wherein the anti-CD70 nanobody comprises a complementarity-determining region (CDR1), a complementarity-determining region (CDR2), and a complementarity-determining region (CDR3), wherein, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-CD70 nanobody are shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; or The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-CD70 nanobody are shown in SEQ ID NO:32, SEQ ID NO:24, and SEQ ID NO:33, respectively; or The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-CD70 nanobody are shown in SEQ ID NO:36, SEQ ID NO:24, and SEQ ID NO:37, respectively; or The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-CD70 nanobody are shown in SEQ ID NO:45, SEQ ID NO:24, and SEQ ID NO:37, respectively.

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:21, SEQ ID NO:30, SEQ ID NO:34 or SEQ ID NO:43, or is an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:34 or SEQ ID NO:43 after substitution, deletion or addition of one or more amino acids.

3. A fusion antibody, comprising a first domain and a second domain; The first structural domain is the anti-CD70 nanobody as described in claim 1 or 2; The second domain has the effect of prolonging the in vivo half-life; The second domain is the immunoglobulin Fc region.

4. The fusion antibody according to claim 3, characterized in that, The immunoglobulin is selected from one or more combinations of IgG, IgA1, IgA2, IgD, IgE, and IgM.

5. The fusion antibody according to claim 4, characterized in that, The first and second domains are hinged together or connected by a linker peptide.

6. Biomaterials relating to the anti-CD70 nanobody of claim 1 or 2 or the fusion antibody of any one of claims 3 to 5; wherein the biomaterial is any one of a1) to a12): a1) A nucleic acid molecule encoding the anti-CD70 nanobody as described in claim 1 or 2 or the fusion antibody as described in any one of claims 3 to 5; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

7. A drug comprising the anti-CD70 nanobody of claim 1 or 2, the fusion antibody of any one of claims 3 to 5, or the biomaterial of claim 6.

8. The use of the anti-CD70 nanobody according to claim 1 or 2, the fusion antibody according to any one of claims 3 to 5, or the biomaterial according to claim 6 in the preparation of reagents or kits for quantitative or qualitative detection of CD70.

9. A kit comprising the anti-CD70 nanobody of claim 1 or 2, the fusion antibody of any one of claims 3 to 5, the biomaterial of claim 6, or the drug of claim 7.

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