Anti-HER2 nano antibody, pharmaceutical composition, detection reagent and application thereof

Abstract

The invention provides an anti-HER2 nano antibody, a pharmaceutical composition containing the nano antibody, and a detection reagent. The nano antibody disclosed by the invention can specifically recognize HER2, has relatively high affinity to HER2, and can be used for preparing a diagnostic reagent or a therapeutic drug aiming at HER2 positive tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to anti-HER2 nano antibodies, pharmaceutical compositions, detection reagents and applications thereof. Background Art

[0002] Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase located on the cell membrane surface and is a member of the epidermal growth factor receptor family. When overexpressed, HER2 heterodimerizes with other family members (EGFR, HER3, HER4), leading to autophosphorylation of tyrosine residues in the intracellular domain, initiating multiple intracellular signaling pathways leading to cell proliferation and tumorigenesis. A large number of studies have shown that about 20% of breast cancers have activation of related signaling pathways caused by HER2 overexpression; in addition, HER2 is also overexpressed in a variety of solid tumors, including 20% ​​of gastric cancer and gastroesophageal junction cancer, 17%-44% of salivary gland cancer, 27% of ovarian cancer, 18-80% of uterine cancer, 21% of cervical cancer, 2% of non-small cell lung cancer, 20% of bile duct cancer, 26% of pancreatic cancer, 5% of colon cancer, 12.4% of bladder cancer and 10% of prostate cancer. According to the 2020 Global Cancer Statistics, there are approximately 2.26 million new cases of breast cancer and approximately 685,000 deaths each year, surpassing lung cancer to become the world's leading cancer. Based on this estimate, there are more than 400,000 cases of HER2-positive breast cancer alone. Together with other types of HER2-positive solid tumors, the total number of patients with HER2-positive tumors is huge, and the disease burden is heavy. Therefore, it is of great significance to continue to develop new drugs for HER2-positive tumors.

[0003] HER2 targeted therapy is an effective means of treating HER2-positive tumors. Trastuzumab and Pertuzumab, two monoclonal antibodies that have been on the market for many years, are still the cornerstone drugs of HER2 targeted therapy. Trastuzumab is the first humanized monoclonal antibody drug approved by the U.S. Food and Drug Administration (FDA) that targets HER2 for the treatment of HER2-positive breast cancer. It binds to the fourth part (ECDⅣ) of the HER2 extracellular domain (ECD) and interferes with the activation of the downstream phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) signaling pathway by blocking non-ligand-dependent HER2 heterodimerization, thereby effectively inhibiting the proliferation of HER2-positive cells. Trastuzumab can also activate antibody-dependent cell-mediated cytotoxicity (ADCC) to kill tumor cells. Pertuzumab specifically binds to the dimerization-related domain ECDⅡ of HER2 and interferes with the proliferation of HER2-positive tumors by inhibiting ligand-dependent HER2 heterodimerization.

[0004] Monoclonal antibodies have a large molecular weight and poor tissue penetration, making it difficult to increase the effective concentration of antibodies in the local tumor, resulting in unsatisfactory therapeutic effects; traditional monoclonal antibodies have high immunogenicity, which affects the therapeutic effect; in addition, the production process of monoclonal antibodies is relatively complicated, resulting in high clinical use costs. Therefore, many unfavorable conditions of traditional monoclonal antibodies limit their widespread application in clinical tumor treatment. Nanobodies, also known as single-domain antibodies (sdAbs), are a new type of antibody discovered in recent years. They are essentially a single heavy chain variable region (Variable domain of heavy chain of heavy-chain antibody, VHH) that specifically binds to antigens. Nanobodies are currently the smallest units that can be artificially prepared that are fully functional, stable, and can specifically recognize antigens. The molecular weight of nanoantibodies is usually composed of 110-130 amino acids, with a molecular weight of only about 15kDa. They have better tissue penetration, and at the same time, the small molecular weight brings relatively weak immunogenicity. After artificial modification, nanoantibodies can obtain similar or even higher affinity than traditional monoclonal antibodies. Nanoantibodies are easy to express recombinantly, and the production cost is lower. They are also easy to transform and modify, and can carry radionuclides to become carriers of medical images.

[0005] However, there is currently a lack of high-affinity nanobodies targeting HER2 that can inhibit the growth of HER2-positive tumors in the art. Summary of the invention

[0006] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a nanobody for solving the problem of the lack of HER2-targeted, HER2-positive tumor growth inhibition in the prior art. The nanobody is nanobody A2H4, and the amino acid sequences of CDR1-3 of VHH of the nanobody A2H4 have at least 90% homology with SEQ ID No.1-3 respectively; or the nanobody is nanobody H2C11, and the amino acid sequences of CDR1-3 of VHH of the nanobody H2C11 have at least 90% homology with SEQ ID No.4-6 respectively; or the nanobody is nanobody A9H5, and the amino acid sequences of CDR1-3 of VHH of the nanobody A9H5 have at least 90% homology with SEQ ID No.7-9 respectively; or the nanobody is nanobody G1H2, and the amino acid sequences of CDR1-3 of VHH of the nanobody G1H2 have at least 90% homology with SEQ ID No.10, 8, 11 respectively.

[0007] The present invention provides a research and development basis for the development of antibody-drug conjugates based on the HER2 target.

[0008] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody A2H4 are shown in SEQ ID No. 1 to 3, respectively.

[0009] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody H2C11 are shown in SEQ ID No. 4 to 6, respectively.

[0010] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody A9H5 are shown in SEQ ID No. 7 to 9, respectively.

[0011] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody G1H2 are shown in SEQ ID No. 10, 8, and 11, respectively.

[0012] The present invention also provides a nanobody chimeric protein, wherein the nanobody chimeric protein is a chimeric protein of a nanobody and Fc; the nanobody chimeric protein is nanobody A2G5-Fc, and the amino acid sequences of CDR1 to 3 of the VHH of the nanobody A2G5 have at least 90% homology with SEQ ID No.12 to 14, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No.21; or the nanobody chimeric protein is nanobody A9B5-Fc, and the amino acid sequences of CDR1 to 3 of the VHH of the nanobody A9B5 have at least 90% homology with SEQ ID No.17, 8, and 18, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No.21; or the nanobody chimeric protein is nanobody H2F5-Fc, and the amino acid sequences of CDR1 to 3 of the VHH of the nanobody H2F5 have at least 90% homology with SEQ ID No.4, 15, and 16, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No.21; or the nanobody chimeric protein is nanobody G1E4-Fc, the amino acid sequences of CDR1 to 3 of VHH of the nanobody G1E4 have at least 90% homology with SEQ ID No.19, 8, and 20, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No.21.

[0013] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody A2G5 are shown in SEQ ID No. 12 to 14, respectively.

[0014] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody A9B5 are shown in SEQ ID No. 17, 8, and 18, respectively.

[0015] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody H2F5 are shown as SEQ ID No. 4, 15, and 16, respectively.

[0016] Preferably, the amino acid sequences of CDR1 to 3 of VHH of the Nanobody G1E4 are shown in SEQ ID No. 19, 8, and 20, respectively.

[0017] The present invention also provides an expression vector, which recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody A2H4 according to claim 1; or the expression vector recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody H2C11 according to claim 1; or the expression vector recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody A9H5 according to claim 1; or the expression vector recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody G1H2 according to claim 1.

[0018] Preferably, the expression vector includes a plasmid vector and a viral vector.

[0019] The present invention also provides a recombinant, which comprises the expression vector; or the genome of the recombinant is recombined with a nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody A2H4 according to claim 1; or the genome of the recombinant is recombined with a nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody H2C11 according to claim 1; or the genome of the recombinant is recombined with a nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody A9H5 according to claim 1; or the genome of the recombinant is recombined with a nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody G1H2 according to claim 1.

[0020] Preferably, the recombinant is a recombinant Escherichia coli, yeast, insect cell or mammalian cell.

[0021] The present invention also provides an expression vector, which recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody A2G5 and the Fc amino acid sequence in the nanobody A2G5-Fc; or the expression vector recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody A9B5 and the Fc amino acid sequence in the nanobody A9B5-Fc; or the expression vector recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody H2F5 and the Fc amino acid sequence in the nanobody H2F5-Fc; or the expression vector recombines the nucleic acid encoding the CDR1-3 and amino acid sequence of the VHH of the nanobody G1E4 and the Fc amino acid sequence in the nanobody G1E4-Fc.

[0022] Preferably, the expression vector includes a plasmid vector and a viral vector.

[0023] The present invention also provides a recombinant, which comprises the expression vector; or the genome of the recombinant is recombined with nucleic acid encoding CDR1-3 and its amino acid sequence of VHH of nanobody A2G5 in the nanobody A2G5-Fc and its Fc amino acid sequence; or the genome of the recombinant is recombined with nucleic acid encoding CDR1-3 and its amino acid sequence of VHH of nanobody A9B5 in the nanobody A9B5-Fc and its Fc amino acid sequence; or the genome of the recombinant is recombined with nucleic acid encoding CDR1-3 and its amino acid sequence of VHH of nanobody H2F5 in the nanobody H2F5-Fc and its Fc amino acid sequence; or the genome of the recombinant is recombined with nucleic acid encoding CDR1-3 and its amino acid sequence of VHH of nanobody G1E4 in the nanobody G1E4-Fc and its Fc amino acid sequence.

[0024] Preferably, the recombinant is a recombinant Escherichia coli, yeast, insect cell or mammalian cell.

[0025] The present invention also provides a drug, which comprises an effective amount of the nano antibody A2G5-Fc, A9B5-Fc or H2F5-Fc.

[0026] The present invention also provides a pharmaceutical composition, which comprises an effective amount of the nanobody A2G5-Fc and an effective amount of the nanobody A9B5-Fc; or the pharmaceutical composition comprises an effective amount of the nanobody A2G5-Fc and an effective amount of the nanobody G1E4-Fc; or the pharmaceutical composition comprises an effective amount of the effective amount of the nanobody A2G5-Fc, an effective amount of the nanobody H2F5-Fc and an effective amount of the nanobody G1E4-Fc; or the pharmaceutical composition comprises an effective amount of the nanobody A2G5-Fc and an effective amount of the nanobody H2F5-Fc.

[0027] The present invention also provides a drug combination, which comprises an effective amount of the Nanobody A2G5-Fc and an effective amount of the Nanobody A9B5-Fc; or the drug combination comprises an effective amount of the Nanobody A2G5-Fc and an effective amount of the Nanobody G1E4-Fc; or the drug combination comprises an effective amount of the Nanobody A2G5-Fc, an effective amount of the Nanobody H2F5-Fc and an effective amount of the Nanobody G1E4-Fc; or

[0028] The drug combination comprises an effective amount of the nanobody A2G5-Fc and an effective amount of the nanobody H2F5-Fc.

[0029] The present invention also provides a pharmaceutical composition, which comprises an effective amount of trastuzumab and an effective amount of the nano antibody A2G5-Fc; or the pharmaceutical composition comprises an effective amount of pertuzumab and an effective amount of the nano antibody A2G5-Fc; or the pharmaceutical composition comprises an effective amount of trastuzumab and an effective amount of the nano antibody A9B5-Fc; or the pharmaceutical composition comprises an effective amount of pertuzumab and an effective amount of the nano antibody A9B5-Fc; or the pharmaceutical composition comprises an effective amount of pertuzumab and an effective amount of the nano antibody H2F5-Fc; or the pharmaceutical composition comprises an effective amount of trastuzumab and an effective amount of the nano antibody G1E4-Fc.

[0030] The present invention also provides a drug combination, which comprises an effective amount of trastuzumab and an effective amount of the nano antibody A2G5-Fc; or the drug combination comprises an effective amount of pertuzumab and an effective amount of the nano antibody A2G5-Fc; or the drug combination comprises an effective amount of trastuzumab and an effective amount of the nano antibody A9B5-Fc; or the drug combination comprises an effective amount of pertuzumab and an effective amount of the nano antibody A9B5-Fc; or the drug combination comprises an effective amount of pertuzumab and an effective amount of the nano antibody H2F5-Fc; or the drug combination comprises an effective amount of trastuzumab and an effective amount of the nano antibody G1E4-Fc.

[0031] The present invention also provides a diagnostic reagent for detecting HER2-positive tumors or positive cells, wherein the diagnostic reagent comprises an effective amount of the nano antibody A2H4 and a detection reagent; or the diagnostic reagent comprises an effective amount of the nano antibody H2C11 and a detection reagent; or the diagnostic reagent comprises an effective amount of the nano antibody A9H5 and a detection reagent; or the diagnostic reagent comprises an effective amount of the nano antibody G1H2 and a detection reagent.

[0032] Preferably, the detection reagent is an enzyme-linked immunosorbent assay reagent, a cell immunofluorescence assay reagent, a flow cytometry assay reagent, an isotope tracer, a contrast agent, a nanomagnetic particle, and an imaging agent.

[0033] The present invention also provides a diagnostic reagent for detecting HER2-positive tumors or positive cells, wherein the diagnostic reagent comprises an effective amount of the nano antibody A2G5-Fc and a detection reagent; or the diagnostic reagent comprises an effective amount of the nano antibody A9B5-Fc and a detection reagent; or the diagnostic reagent comprises an effective amount of the nano antibody H2F5-Fc and a detection reagent; or the diagnostic reagent comprises an effective amount of the nano antibody G1E4-Fc and a detection reagent.

[0034] Preferably, the detection reagent is an enzyme-linked immunosorbent assay reagent, a cell immunofluorescence assay reagent, a flow cytometry assay reagent, an isotope tracer, a contrast agent, a nanomagnetic particle, and an imaging agent.

[0035] The present invention also provides the use of the nano antibody, the drug, the drug composition, and the drug combination in the preparation of drugs for preventing and / or treating tumors.

[0036] Preferably, the drug for preventing and / or treating tumors is a drug for preventing and / or treating tumors in which HER2 dimerization occurs.

[0037] Preferably, the drug for preventing and / or treating tumors is a drug for preventing and / or treating gastric cancer and gastroesophageal junction cancer, salivary gland cancer, ovarian cancer, uterine cancer, cervical cancer, non-small cell lung cancer, bile duct cancer, pancreatic cancer, colon cancer, bladder cancer and prostate cancer.

[0038] The present invention also provides the use of the diagnostic reagent in preparing a diagnostic reagent or a diagnostic kit for detecting HER2 positive tumors or positive cells.

[0039] As described above, the anti-HER2 nanoantibodies, pharmaceutical compositions, detection reagents and their applications of the present invention have the following characteristics:

[0040] Beneficial effects:

[0041] 1. The anti-HER2 nanobody of the present invention is easy to express in prokaryotic and eukaryotic systems, and is simple to prepare, which reduces the cost of using the antibody. At the same time, the anti-HER2 nanobody of the present invention has good hydrophilicity and solubility, which can improve its utilization as a targeted diagnosis or targeted therapy drug.

[0042] 2. The anti-HER2 nanoantibody described in the present invention can specifically recognize HER2 and has a high affinity for HER2, and can be used to prepare diagnostic reagents or therapeutic drugs for HER2-positive tumors.

[0043] 3. The anti-HER2 nanoantibodies described in the present invention recognize different epitopes of HER2 and can be used to detect tumor tissues with HER2 mutations, such as gastric cancer and gastroesophageal junction cancer, salivary gland cancer, ovarian cancer, uterine cancer, cervical cancer, non-small cell lung cancer, bile duct cancer, pancreatic cancer, colon cancer, bladder cancer and prostate cancer.

[0044] 4. The anti-HER2 nanoantibody described in the present invention can be effectively internalized after binding to HER2 on the cell surface, and can be used to prepare various antibody-drug conjugates.

[0045] 5. The anti-HER2 nanoantibodies described in the present invention can be used in combination to effectively inhibit the proliferation of HER2-positive tumor cells and can be used to detect or treat HER2-positive tumors.

[0046] 6. The recombinant expression vector, cells and bacteria containing the coding gene of the anti-HER2 nanoantibody or the coding gene of the recombinant antigen protein described in the present invention, as well as the method for preparing the recombinant antigen protein, provide new genetic engineering expression vectors, cells and bacteria for preparing potential drugs for detecting or treating HER2-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Shown is the SDS-PAGE electrophoresis diagram of the purified hHER2(ECD)-Fc and mHER2(ECD) proteins of the present invention;

[0048] Figure 2 Shown is the SDS-PAGE electrophoresis diagram of the Nanobodies A2H4, H2C11, A9H5, and G1H2 proteins of the present invention;

[0049] Figure 3 Shown are the flow cytometry binding abilities of the Nanobodies A2H4, H2C11, A9H5, and G1H2 of the present invention;

[0050] Figure 4 Shown is the electrophoresis diagram of the purified Nanobodies A2H3, A2G5, A9F5, A9B5, H2A6, H2F5, G1H4, and G1E4 proteins of the present invention;

[0051] Figure 5 Shown are enzyme-linked immunosorbent assay (ELISA) reaction diagrams of the Nanobodies of the present invention and hHER2 (ECD), mHER2 (ECD), chimeric proteins hHER2 (ECD)-D1, hHER2 (ECD)-D2, and hHER2 (ECD)-D4;

[0052] Figure 6 Shown is the flow cytometry binding ability detection of the Nanobody of the present invention to SKBR3 cells;

[0053] Figure 7 It is shown as the internalization rate of the Nanobody of the present invention binding to cell surface HER2;

[0054] Figure 8 It shows the inhibition rate of the Nanobody of the present invention in inhibiting gastric cancer cell NCI-N87 cells;

[0055] Fig. 9 Shown is the inhibitory effect of the combined use of the Nanobodies of the present invention on NCI-N87 cells with or without ligand stimulation;

[0056] Fig.10 It shows the inhibitory ability of the Nanobody of the present invention in combination with trastuzumab or pertuzumab on gastric cancer cells NCI-N87;

[0057] Fig.11 It shows the inhibitory ability of the nanobody of the present invention in combination with trastuzumab or pertuzumab on gastric cancer cell NCI-N87 under ligand stimulation. DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0059] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0061] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0062] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0063] Example 1: In vitro recombinant expression and purification of HER2 protein

[0064] 1.1 The DNA sequences of the extracellular segments of human HER2 and mouse HER2 proteins (NM_001382796.1 and NM_001003817.1) were synthesized and cloned into pSCSTa vector and PTT5 vector, respectively, i.e. pSCSTa-hHER2(ECD)-Fc and PTT5-mHER2(ECD)-His. The protein sequences were verified by sequencing. The protein sequences are detailed in the sequence table.

[0065] 1.2 The above two plasmids were amplified in DH5α competent medium respectively, and the plasmids were extracted using AXYGEN endotoxin-free plasmid midi extraction kit.

[0066] 1.3 The above two plasmids were mixed with the transfection reagent PEI in a ratio of 1:3, and allowed to stand in the dark for 20 minutes, and then added dropwise to the suspension cultured Expi 293 cells to complete transfection. 16 hours after transfection, 5% feed was added to the cells, and culture was continued in a carbon dioxide shaking incubator for 3-4 days.

[0067] 1.4 Use a centrifuge to collect the supernatant of 293 cells, and use AKTA protein purification equipment to purify the above two proteins: use Protein A affinity chromatography column to purify hHER2 (ECD) -Fc, and use Ni-NTA chromatography column to purify mHER2 (ECD) -His.

[0068] 1.5 The purified proteins were replaced with PBS buffer using ultrafiltration concentrators, and the protein concentration was determined using a BCA kit. Then, SDS-PAGE was used to determine that the purity of the two proteins reached more than 95%. The SDS-PAGE electrophoresis of the two proteins can be found in Figure 1 The purified protein was used for subsequent antibody screening and other functional experiments.

[0069] Example 2: Screening of anti-HER2 nanobodies based on synthetic nanobody libraries

[0070] 2.1 The antigen protein hHER2 (ECD)-Fc prepared in Example 1 was used to screen the own synthetic nanoantibody library by phage display method (the construction of the library is disclosed in patent application 202011117094.2). After multiple rounds of enrichment, specific single positive clones were screened using enzyme-linked immunosorbent assay (ELISA). The phage supernatant was added to the ELISA plate coated with hHER2 (ECD)-Fc and reacted at room temperature for 1 hour. After washing 12 times with PBST, Anti-M13-HRP (dilution ratio 1:8000) was added and reacted at room temperature for 30 minutes. After washing 15 times with PBST, the color developing solution was added and reacted at room temperature for 3 minutes, and then the stop solution was added. Finally, the wavelength (OD450) at 450nm was read on the microplate reader.

[0071] 2.2 When the ratio of the sample well OD450 to the control well OD450 is greater than 5, it is judged as a positive clone well. After the positive clones are selected by ELISA, sequencing is performed. Since the sequencing results are mostly repeated sequences, 6 nanobody sequences are actually obtained, namely A2H4, H2C11, A9H5, G1H2, A1H4, and G3H2.

[0072] Among them, the amino acid sequences of CDR1 to 3 of VHH of nanobody A2H4 are shown as SEQ ID Nos. 1 to 3, respectively; the amino acid sequences of CDR1 to 3 of VHH of nanobody H2C11 are shown as SEQ ID Nos. 4 to 6, respectively; the amino acid sequences of CDR1 to 3 of VHH of nanobody A9H5 are shown as SEQ ID Nos. 7 to 9, respectively; the amino acid sequences of CDR1 to 3 of VHH of nanobody G1H2 are shown as SEQ ID Nos. 10, 8, and 11, respectively.

[0073] Example 3: Expression and purification of anti-HER2 nanobodies in a prokaryotic system

[0074] 3.1 For the positive clones obtained in Example 2, the corresponding full-length sequence of the nanobody was loaded onto the pET24a vector. The plasmid was transformed into BL21 competent cells, spread on a solid LB culture plate containing ampicillin, and cultured overnight at 37°C. A single colony was selected and inoculated into LB medium containing ampicillin. After culturing on a shaker at 37°C for 8 hours, 2 mL of the bacterial solution was inoculated into 500 mL of LB medium containing ampicillin. When OD600 reached 0.5-0.6, IPTG was added to a final concentration of 1 mM / L, and cultured on a shaker at 22°C for 14 hours.

[0075] 3.2 The cells were collected by centrifugation, lysate was added and blown evenly, ultrasonically disrupted for 3 min, and the supernatant was collected by centrifugation again. Nanobodies were purified using AKTA protein purification equipment and Ni-NTA chromatography columns.

[0076] 3.3 The purified protein was replaced with PBS buffer using an ultrafiltration concentration tube, the protein concentration was determined using a BCA kit, and then the purity of the nanoantibody was determined to be more than 95% using SDS-PAGE. Figure 2 .

[0077] Example 4: Flow cytometry detection of the binding ability of nanobodies to HER2-positive cells

[0078] 4.1 Count the SKBR3 cells in the logarithmic growth phase and place them into a U-shaped plate, with 100,000 cells per well. Add the nanoantibodies to the U-shaped plate after gradient dilution, incubate at 4°C for 60 minutes, and wash the plate twice with FACS buffer (PBS + 2% FBS).

[0079] 4.2 Use FACS buffer to dilute Anti-camelid VHH antibody (iFlour488) 500 times, add it to the U-shaped plate, incubate at 4℃ for 30 minutes, wash the plate twice with FACS buffer. Use 120μL FACS buffer to resuspend the cells and detect them on the machine.

[0080] 4.3 Flow cytometry results showed that the four nanoantibodies A2H4, H2C11, A9H5, and G1H2 could specifically bind to the HER2-positive cell line SKBR3. The binding ability results are shown in Figure 3 .

[0081] Example 5: Mutation modification of anti-HER2 nanobody

[0082] 5.1 Antibody engineering methods were further used to mutate the CDR regions of A2H4, H2C11, A9H5, and G1H2 and construct a new phage-displayed nanoantibody library.

[0083] 5.2 The results of affinity maturation library construction for H2C11 and A2H4 are shown in Table 1.

[0084] Table 1

[0085]

[0086] 5.3 The results of affinity maturation library construction of A9H5 and G1H2 are shown in Table 2.

[0087] Table 2

[0088]

[0089] Example 6: Screening and identification of anti-HER2 nanobody mutants

[0090] 6.1 According to the method of Example 2, the four CDR region mutation libraries of A2H4, H2C11, A9H5 and G1H2 were enriched for multiple rounds, and the positive clones were selected by ELISA method. The judgment standard was that the ratio of OD450 of the sample well to the OD450 of the control well was greater than 10.

[0091] 6.2 Randomly select 20 positive clones from each library for sequencing.

[0092] 6.3 Eight nanobodies (A2H4-H3, A2H4-G5, A9H5-F5, A9H5-B5, H2C11-A6, H2C11-F5, G1H2-H4, G1H2-E4, corresponding to the abbreviations A2H3, A2G5, A9F5, A9B5, H2A6, H2F5, G1H4, G1E4) were randomly selected from the affinity matured positive clones of 6.2 and expressed and purified in prokaryotes according to the method of Example 3. The electrophoresis results after protein purification showed (electrophoresis diagram see Figure 4 ), the purity of the 8 antibodies exceeded 95%.

[0093] Example 7: BLI (Biolayer interferometry) determination of affinity of anti-HER2 nanobody mutants

[0094] 7.1 Immobilize the anti-HER2 nanobody on the Ni-NTA biosensor, and then combine it with hHER2(ECD)-Fc at different molar concentrations to measure the binding and dissociation constants between the nanobody and the HER2 antigen.

[0095] 7.2 The affinity data of the nanobodies were obtained after calculation by BLI software (Table 3). The results showed that the ability of the nanobodies modified by mutation in the CDR region to bind to the HER2 protein was greatly improved, and the nanobodies with the highest affinity in each group were selected for subsequent functional testing: A2H4-G5 (ie A2G5), A9H5-B5 (ie A9B5), H2C11-F5 (ie H2F5), G1H2-E4 (ie G1E4).

[0096] Among them, the amino acid sequences of CDR1 to 3 of VHH of nanobody A2G5 are shown as SEQ ID Nos. 12 to 14, respectively; the amino acid sequences of CDR1 to 3 of VHH of nanobody A9B5 are shown as SEQ ID Nos. 17, 8, and 18, respectively; the amino acid sequences of CDR1 to 3 of VHH of nanobody H2F5 are shown as SEQ ID Nos. 4, 15, and 16, respectively; the amino acid sequences of CDR1 to 3 of VHH of nanobody G1E4 are shown as SEQ ID Nos. 19, 8, and 20, respectively.

[0097] Table 3

[0098]

[0099] Example 8: Determination of the recognition epitope of anti-HER2 nanobodies

[0100] 8.1 Part of the DNA sequence of the extracellular segment of the human HER2 protein was replaced with the homologous DNA sequence corresponding to the extracellular segment of the mouse HER2 protein, and four different chimeric proteins were constructed according to the method of Example 1: hHER2 (ECD) -D1, hHER2 (ECD) -D2, hHER2 (ECD) -D3, and hHER2 (ECD) -D4. After expression and purification, a high-purity chimeric protein with an Fc tag was obtained. The Fc protein sequence is shown in SEQ ID No. 21.

[0101] 8.2hHER2(ECD)-D1 is a chimeric protein formed by replacing amino acids 23 to 217 of hHER2(ECD) with amino acids 23 to 218 of mHER2(ECD). hHER2(ECD)-D2 is a chimeric protein formed by replacing amino acids 218 to 342 of hHER2(ECD) with amino acids 219 to 343 of mHER2(ECD). hHER2(ECD)-D3 is a chimeric protein formed by replacing amino acids 343 to 510 of hHER2(ECD) with amino acids 344 to 511 of mHER2(ECD). hHER2(ECD)-D4 is a chimeric protein formed by replacing amino acids 511 to 652 of hHER2(ECD) with amino acids 512 to 653 of mHER2(ECD). Since the expression level of hHER2(ECD)-D3 was extremely low, three other chimeric proteins were subsequently used to detect the epitope recognition by the nanoantibody.

[0102] 8.3 Five antigens hHER2 (ECD), mHER2 (ECD), hHER2 (ECD)-D1, hHER2 (ECD)-D2, and hHER2 (ECD)-D4 were coated respectively, and the reactivity of the nanobody of the present invention with the chimeric protein was detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 5 , Figure 5 It was shown that the nanobodies recognized different epitopes of HER2: A2G5 and A9B5 recognized amino acids 23 to 217 of hHER2 (ECD), H2F5 recognized amino acids 218 to 342 of hHER2 (ECD), and G1E4 recognized hHER2 (ECD)-D4, which is amino acids 511 to 652 of hHER2 (ECD).

[0103] Example 9: Flow cytometry detection of the binding ability of nanobodies to HER2-positive cells

[0104] 9.1 According to the method of Example 1, the DNA sequence of the nanobody was cloned into the pSCSTa vector to express and purify the high-purity nanobody with an Fc tag.

[0105] 9.2 According to the method of Example 4, the binding ability of the nanobody to SKBR3 cells was determined. Figure 6 , Figure 6 The results showed that the EC50 values ​​(nM) of the four nanobody-Fc chimeric proteins A2G5-Fc, A9B5-Fc, H2F5-Fc, and G1E4-Fc for binding to cells were 15.78, 6.593, 1.805, and 38.20, respectively. In conclusion, the anti-HER2 nanobody mutants of the present invention can effectively bind to HER2-positive cells SKBR3.

[0106] Example 10: Internalization activity of anti-HER2 nanobody binding to cell surface HER2

[0107] 10.1 After digestion, SKBR3 cells were washed twice with pre-cooled FACS buffer and plated in a U-shaped plate with 10 cells per well. 5 The nanoantibodies were incubated with the cells on ice for 1 h and washed three times with FACS buffer to remove non-specifically bound nanoantibodies.

[0108] 10.2 The cells were divided into two groups: one group was placed on ice as the control group; the other group was incubated in an incubator at 37°C for different time periods (0.5h and 4h) as the experimental group. After incubation, the cells were washed twice with FACS buffer.

[0109] 10.3 Add 100 μL of 2% paraformaldehyde solution to each well, incubate at 4°C for 20 min, and wash 3 times with FACS buffer. Add 100 μL of PE-labeled-anti-huFc (dilution 1:500) to each well, incubate at 4°C for 30 min, wash 3 times with FACS buffer, resuspend the cells, and detect by flow cytometry.

[0110] 10.4 Internalization rate (%) = 100% × (MFI value change of experimental group) / (MFI value of control group after background subtraction). The results show ( Figure 7 ), the nanoantibodies of the present invention can be internalized after binding to cell surface HER2, and the internalization rates after binding to cells for 4 hours are higher than those of trastuzumab (T) and pertuzumab (P).

[0111] Example 11: Anti-HER2 Nanobodies Inhibit Tumor Cell Proliferation

[0112] 11.1 Trypsin digestion of gastric cancer cell NCI-N87 cells, carefully blow the cell clusters to make them single cells, then count the cells, and then plate them in 96-well cell culture plates, 4000 cells per well. Grow them in a constant temperature incubator overnight, discard the original culture medium the next day, add the corresponding concentration of anti-HER2 nanoantibody diluted in complete culture medium to each well, set up positive, negative and blank controls, and set two wells for each antibody and concentration.

[0113] 11.2 After 4 days, discard the medium containing antibodies, add 100 μL of pre-diluted 10% CCK8 solution to each well, incubate in a constant temperature cell culture incubator for 1.5 h, use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm, and calculate the inhibition rate of each antibody on NCI-N87 cells.

[0114] 11.3 The calculation formula is: Inhibition rate (%) = [(Ac-As) / (Ac-Ab)] × 100%. Where As represents the experimental well (culture medium containing cells, CCK-8, and the substance to be tested); Ac: control well (culture medium containing cells, CCK-8, and no substance to be tested); Ab: blank well (culture medium without cells and the substance to be tested, CCK-8).

[0115] 11.4 Results Figure 8 The results showed that A2G5-Fc, A9B5-Fc, and H2F5-Fc all had the ability to inhibit the proliferation of HER2-positive tumor cells NCI-N87, and G1E4-Fc had the ability to promote the proliferation of NCI-N87.

[0116] Example 12: Combination use of anti-HER2 nanobodies recognizing different epitopes to inhibit tumor cell proliferation

[0117] 12.1 Since the anti-HER2 high-affinity nanobodies of the present invention recognize different epitopes of the HER2 protein, it is speculated that the combined use of multiple nanobodies may enhance their anti-tumor ability. Therefore, according to the method of Example 11, the inhibitory effect of two or three nanobodies combined with ligand or without ligand stimulation on NCI-N87 cells was determined. The combined use of different nanobodies is indicated by "+".

[0118] 12.2 Results Fig. 9 The results showed that under the condition of no ligand stimulation, A2G5-Fc+A9B5-Fc, A2G5-Fc+G1E4-Fc, and A2G5-Fc+H2F5-Fc+G1E4-Fc all had a certain inhibitory effect on NCI-N87 cells; under the condition of HRG ligand stimulation, A2G5-Fc+A9B5-Fc, A2G5-Fc+H2F5-Fc, A2G5-Fc+G1E4-Fc, and A2G5-Fc+H2F5-Fc+G1E4-Fc all had a significant inhibitory effect on NCI-N87 cells. Results: Under EGF ligand stimulation conditions, A2G5-Fc+A9B5-Fc, A2G5-Fc+H2F5-Fc, A2G5-Fc+G1E4-Fc, and A2G5-Fc+H2F5-Fc+G1E4-Fc all had significant inhibitory effects on NCI-N87 cells, among which the inhibition rate of the combined use of three nanobodies (A2G5-Fc+H2F5-Fc+G1E4-Fc) was as high as over 55%, indicating that the combined use of nanobodies recognizing different epitopes of HER2 of the present invention can synergistically exert anti-tumor function.

[0119] Example 13: Combination of anti-HER2 nanobodies with trastuzumab or pertuzumab to inhibit tumor cell proliferation

[0120] 13.1 According to the method of Example 11, the anti-tumor ability of the Nanobody of the present invention after combined use with the marketed trastuzumab or pertuzumab was determined. The experimental groups were: A2G5-Fc+T, A2G5-Fc+P, A9B5-Fc+T, A9B5-Fc+P, H2F5-Fc+T, H2F5-Fc+P, G1E4-Fc+T, G1E4-Fc+P; the control group was trastuzumab combined with pertuzumab, i.e. T+P. The results are shown in Fig.10 The results showed that A2G5-Fc+T, A2G5-Fc+P, A9B5-Fc+T, A9B5-Fc+P, H2F5-Fc+P, and G1E4-Fc+T all had significant inhibitory effects on gastric cancer cell NCI-N87 cells, among which the inhibition rates of A2G5-Fc+T and A9B5-Fc+T were significantly better than those of T+P, indicating that the nanobody of the present invention can synergize with trastuzumab to produce a better tumor inhibition effect. H2F5-Fc+T and G1E4-Fc+P have a promoting effect on cell proliferation.

[0121] 13.2 Investigate whether A2G5-Fc+T, A2G5-Fc+P, A9B5-Fc+T, and A9B5-Fc+P still have inhibitory ability against NCI-N87 cells under ligand stimulation. The control groups of this experiment were trastuzumab (T), pertuzumab (P), and trastuzumab combined with pertuzumab (T+P). The results are shown in Fig.11 The results showed that under the stimulation of HRG or EGF ligands, the inhibitory abilities of A2G5-Fc+T, A2G5-Fc+P, A9B5-Fc+T, and A9B5-Fc+P on gastric cancer cell NCI-N87 cells were better than trastuzumab and pertuzumab; under the stimulation of HRG, the inhibition rate of A2G5-Fc+P was equivalent to that of T+P; under the stimulation of EGF, the inhibition rates of A2G5-Fc+P and A9B5-Fc+P were equivalent to those of T+P, and the inhibition rate of A9B5-Fc+T was significantly better than that of T+P.

[0122] In summary, the anti-tumor ability of certain anti-HER2 nanoantibodies of the present invention when used in combination with trastuzumab or pertuzumab is significantly better than that of trastuzumab and pertuzumab in combination.

[0123] In summary, the anti-HER2 nanoantibodies, pharmaceutical compositions, detection reagents and their applications of the present invention can provide better anti-tumor efficacy. Therefore, the present invention effectively overcomes various shortcomings of the prior art and has high industrial utilization value.

[0124] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A nanobody, characterized in that The nanobody is nanobody A2H4, and the amino acid sequences of CDR1 to 3 of VHH of the nanobody A2H4 have at least 90% homology with SEQ ID No. 1 to 3, respectively; preferably, the amino acid sequences of CDR1 to 3 of VHH of the nanobody A2H4 are as shown in SEQ ID No. 1 to 3, respectively; or The nanobody is the nanobody H2C11, and the amino acid sequences of CDR1 to 3 of VHH of the nanobody H2C11 have at least 90% homology with SEQ ID No. 4 to 6, respectively; preferably, the amino acid sequences of CDR1 to 3 of VHH of the nanobody H2C11 are as shown in SEQ ID No. 4 to 6, respectively; or The nanobody is nanobody A9H5, and the amino acid sequences of CDR1 to 3 of VHH of the nanobody A9H5 have at least 90% homology with SEQ ID No. 7 to 9, respectively; preferably, the amino acid sequences of CDR1 to 3 of VHH of the nanobody A9H5 are as shown in SEQ ID No. 7 to 9, respectively; or The nanobody is nanobody G1H2, and the amino acid sequences of CDR1 to 3 of VHH of the nanobody G1H2 have at least 90% homology with SEQ ID No. 10, 8, and 11, respectively; preferably, the amino acid sequences of CDR1 to 3 of VHH of the nanobody G1H2 are as shown in SEQ ID No. 10, 8, and 11, respectively.

2. A nanobody chimeric protein, characterized in that The nanobody chimeric protein is a chimeric protein of a nanobody and Fc; The nanobody chimeric protein is nanobody A2G5-Fc, the amino acid sequences of CDR1 to 3 of VHH of nanobody A2G5 have at least 90% homology with SEQ ID No.12 to 14, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No.21; preferably, the amino acid sequences of CDR1 to 3 of VHH of nanobody A2G5 are as shown in SEQ ID No.12 to 14, respectively; or The nanobody chimeric protein is nanobody A9B5-Fc, the amino acid sequences of CDR1 to 3 of VHH of nanobody A9B5 have at least 90% homology with SEQ ID No. 17, 8, 18, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No. 21; preferably, the amino acid sequences of CDR1 to 3 of VHH of nanobody A9B5 are as shown in SEQ ID No. 17, 8, 18, respectively; or The nanobody chimeric protein is nanobody H2F5-Fc, the amino acid sequences of CDR1 to 3 of VHH of nanobody H2F5 have at least 90% homology with SEQ ID No. 4, 15, and 16, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No. 21; preferably, the amino acid sequences of CDR1 to 3 of VHH of nanobody H2F5 are as shown in SEQ ID No. 4, 15, and 16, respectively; or The nanobody chimeric protein is nanobody G1E4-Fc, and the amino acid sequences of CDR1 to 3 of VHH of nanobody G1E4 have at least 90% homology with SEQ ID No. 19, 8, and 20, respectively, and the amino acid sequence of Fc is as shown in SEQ ID No. 21; preferably, the amino acid sequences of CDR1 to 3 of VHH of nanobody G1E4 are as shown in SEQ ID No. 19, 8, and 20, respectively.

3. An expression vector, characterized in that: The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of VHH of the Nanobody A2H4 according to claim 1; or The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody H2C11 according to claim 1; or The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody A9H5 according to claim 1; or The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody G1H2 according to claim 1; Preferably, the expression vector includes a plasmid vector and a viral vector.

4. A recombinant, characterized in that: The recombinant comprises the expression vector according to claim 3; or The genome of the recombinant is recombined with nucleic acid encoding the amino acid sequence of CDR1 to 3 of VHH of the Nanobody A2H4 according to claim 1; or The genome of the recombinant is recombined with nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody H2C11 according to claim 1; or The genome of the recombinant is recombined with nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody A9H5 according to claim 1; or The genome of the recombinant is recombined with nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody G1H2 according to claim 1; Preferably, the recombinant is a recombinant Escherichia coli, yeast, insect cell or mammalian cell.

5. An expression vector, characterized in that: The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody A2G5 and the amino acid sequence of Fc in the Nanobody A2G5-Fc of claim 2; or The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody A9B5 in the Nanobody A9B5-Fc of claim 2 and the amino acid sequence of Fc; or The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody H2F5 and the amino acid sequence of Fc in the Nanobody H2F5-Fc of claim 2; or The expression vector recombines the nucleic acid encoding the amino acid sequence of CDR1 to 3 of the VHH of the Nanobody G1E4 in the Nanobody G1E4-Fc of claim 2 and the amino acid sequence of Fc; Preferably, the expression vector includes a plasmid vector and a viral vector.

6. A recombinant, characterized in that: The recombinant comprises the expression vector according to claim 5; or The genome of the recombinant is recombined with nucleic acids encoding the amino acid sequences of CDR1 to 3 of the VHH of the Nanobody A2G5 and the amino acid sequence of Fc in the Nanobody A2G5-Fc of claim 2; or The genome of the recombinant is recombined with nucleic acids encoding the amino acid sequences of CDR1 to 3 of the VHH of the Nanobody A9B5 in the Nanobody A9B5-Fc of claim 2 and the amino acid sequence of Fc; or The genome of the recombinant is recombined with nucleic acids encoding the amino acid sequences of CDR1 to 3 of the VHH of the Nanobody H2F5 in the Nanobody H2F5-Fc of claim 2 and the amino acid sequence of Fc; or The genome of the recombinant is recombined with nucleic acids encoding the amino acid sequences of CDR1 to 3 of the VHH of the Nanobody G1E4 in the Nanobody G1E4-Fc of claim 2 and the amino acid sequence of Fc; Preferably, the recombinant is a recombinant Escherichia coli, yeast, insect cell or mammalian cell.

7. A drug, characterized in that The drug comprises an effective amount of the nanobody A2G5-Fc, A9B5-Fc or H2F5-Fc according to claim 2.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the Nanobody A2G5-Fc according to claim 2 and an effective amount of the Nanobody A9B5-Fc; or The pharmaceutical composition comprises an effective amount of the Nanobody A2G5-Fc and an effective amount of the Nanobody G1E4-Fc according to claim 2; or The pharmaceutical composition comprises an effective amount of the Nanobody A2G5-Fc, an effective amount of the Nanobody H2F5-Fc and an effective amount of the Nanobody G1E4-Fc according to claim 2; or The pharmaceutical composition comprises an effective amount of the nanobody A2G5-Fc as described in claim 2 and an effective amount of the nanobody H2F5-Fc.

9. A drug combination, characterized in that The drug combination comprises an effective amount of the Nanobody A2G5-Fc according to claim 2 and an effective amount of the Nanobody A9B5-Fc; or The drug combination comprises an effective amount of the Nanobody A2G5-Fc and an effective amount of the Nanobody G1E4-Fc as described in claim 2; or The drug combination comprises an effective amount of the Nanobody A2G5-Fc of claim 2, an effective amount of the Nanobody H2F5-Fc and an effective amount of the Nanobody G1E4-Fc; or The drug combination comprises an effective amount of the nanobody A2G5-Fc as described in claim 2 and an effective amount of the nanobody H2F5-Fc.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of trastuzumab and an effective amount of the nanobody A2G5-Fc according to claim 2; or The pharmaceutical composition comprises an effective amount of Pertuzumab and an effective amount of the Nanobody A2G5-Fc according to claim 2; or The pharmaceutical composition comprises an effective amount of trastuzumab and an effective amount of the Nanobody A9B5-Fc according to claim 2; or The pharmaceutical composition comprises an effective amount of Pertuzumab and an effective amount of Nanobody A9B5-Fc according to claim 2; or The pharmaceutical composition comprises an effective amount of Pertuzumab and an effective amount of the Nanobody H2F5-Fc according to claim 2; or The pharmaceutical composition comprises an effective amount of trastuzumab and an effective amount of the nanobody G1E4-Fc according to claim 2.

11. A drug combination, characterized in that The drug combination comprises an effective amount of trastuzumab and an effective amount of the nanobody A2G5-Fc according to claim 2; or The drug combination comprises an effective amount of Pertuzumab and an effective amount of the Nanobody A2G5-Fc according to claim 2; or The drug combination comprises an effective amount of trastuzumab and an effective amount of the Nanobody A9B5-Fc according to claim 2; or The drug combination comprises an effective amount of Pertuzumab and an effective amount of Nanobody A9B5-Fc according to claim 2; or The drug combination comprises an effective amount of Pertuzumab and an effective amount of the Nanobody H2F5-Fc according to claim 2; or The drug combination comprises an effective amount of trastuzumab and an effective amount of the nanobody G1E4-Fc according to claim 2.

12. A diagnostic reagent for detecting HER2-positive tumors or positive cells, characterized in that: The diagnostic reagent comprises an effective amount of Nanobody A2H4 and a detection reagent as described in claim 1; or The diagnostic reagent comprises an effective amount of the Nanobody H2C11 according to claim 1 and a detection reagent; or The diagnostic reagent comprises an effective amount of Nanobody A9H5 and a detection reagent as described in claim 1; or The diagnostic reagent comprises an effective amount of Nanobody G1H2 and a detection reagent as described in claim 1; Preferably, the detection reagent is an enzyme-linked immunosorbent assay reagent, a cell immunofluorescence assay reagent, a flow cytometry assay reagent, an isotope tracer, a contrast agent, a nanomagnetic particle, and an imaging agent.

13. A diagnostic reagent for detecting HER2-positive tumors or positive cells, characterized in that: The diagnostic reagent comprises an effective amount of the Nanobody A2G5-Fc and a detection reagent as described in claim 2; or The diagnostic reagent comprises an effective amount of Nanobody A9B5-Fc and a detection reagent as described in claim 2; or The diagnostic reagent comprises an effective amount of the Nanobody H2F5-Fc and a detection reagent as described in claim 2; or The diagnostic reagent comprises an effective amount of the Nanobody G1E4-Fc according to claim 2 and a detection reagent; Preferably, the detection reagent is an enzyme-linked immunosorbent assay reagent, a cell immunofluorescence assay reagent, a flow cytometry assay reagent, an isotope tracer, a contrast agent, a nanomagnetic particle, and an imaging agent.

14. Use of the Nanobody according to claim 1 or 2, the drug according to claim 7, the pharmaceutical composition according to claim 8 or 10, or the drug combination according to claim 9 or 11 in the preparation of a drug for preventing and / or treating tumors; Preferably, the drug for preventing and / or treating tumors is a drug for preventing and / or treating HER2 dimerization tumors; Preferably, the drug for preventing and / or treating tumors is a drug for preventing and / or treating gastric cancer and gastroesophageal junction cancer, salivary gland cancer, ovarian cancer, uterine cancer, cervical cancer, non-small cell lung cancer, bile duct cancer, pancreatic cancer, colon cancer, bladder cancer and prostate cancer.

15. Use of the diagnostic reagent according to claim 12 or 13 in the preparation of a diagnostic reagent or a diagnostic kit for detecting HER2-positive tumors or positive cells.

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