A dual-targeting binding protein of human epidermal growth factor receptor 2 and integrin and uses thereof

Abstract

The present application provides a kind of for human epidermal growth factor 2 (HER2) and integrin dual targeting binding protein and its production method and application.Integrin dual targeting binding protein includes HER2 specific nanobody or single chain antibody and integrin polypeptide ligand (RGD).Dual targeting antigen binding protein can target recognition HER2 and integrin protein specifically expressed on the surface of tumor cell, interfere with the expression and signal transduction of HER2 and integrin protein, while inhibiting the expression of EGFR, HER2, HER3, HER4, so as to maximum degree inhibit the growth signal pathway of tumor cell.The dual targeting binding protein can also be used as a drug carrier to deliver drugs directly to the tumor site.The dual targeting binding protein described in the present application can be prepared by E. coli expression system.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a dual-targeting binding protein of human epidermal growth factor receptor 2 and integrin, and its applications. Background Technology

[0002] Integrins are transmembrane proteins widely distributed on cell surfaces. They typically consist of heterodimers composed of 18 α-monomers and 8 β-monomers linked by non-covalent bonds, with at least 24 combinations identified to date. Integrins are major adhesion receptors in cells, acting as signaling molecules, mechanotransmitters, and key drivers of cell migration. Mechanical injury or other bodily diseases can lead to overactivation of integrins, resulting in bleeding disorders, immune dysregulation, inflammatory diseases, and cancer symptoms. Studies have shown that integrins are expressed on the surface of cells in various cancers, participating in the regulation of growth factor receptor signaling and growth factor receptor-dependent cancer cell migration and the formation of metastatic lesions. RGD ligands specifically bind to integrins and are frequently attached as specific targeting groups to the surfaces of liposomes, dendritic polymers, or molecular brushes for targeting tumor tissue, applications in tumor imaging or therapy.

[0003] CN108699164A discloses a dual-target drug carrier, comprising a first target molecule and a second target molecule. The first target molecule is linked to the second target molecule by a connector. The first and second target molecules include arginine-glycine-aspartic acid (RGD), asparagine-glycine-arginine (NGR), cyclic NGR, internalized RGD (iRGD), cystine-glycine-aspartic acid-lysine-arginine-threonine-arginine-glycine-alanine (CGNKRTRGA), gastrin, bombesin, octreotide or derivatives thereof, epidermal growth factor (EGF), anti-EGFR antibody, vascular endothelial growth factor (VEGF), anti-VEGFR antibody, anti-HER2 antibody, hepatocyte growth factor receptor (HGFR), anti-HGFR antibody, tumor necrosis factor (TNF) or anti-TNF antibody. The product specifications describe the preparation of RGD-EGF and RGD4C-EGF or RGD-VEGF and RGD4C-VEGF, and the investigation of their biological activities. Results showed that these dual-target fusion proteins not only possess the ability to bind to relative biomarker molecules but also retain their original biological properties, enabling them to carry tumor therapeutic drugs or other substances (such as therapeutic radionuclides) as tumor-targeting drug carriers. Furthermore, they can also carry drugs capable of molecular imaging, serving as molecular imaging probes for tumor diagnosis.

[0004] HER2 (human epidermal growth factor 2) is a receptor tyrosine kinase belonging to the human epidermal growth factor receptor (HER) family. It is highly expressed on the surface of various cancer cells and is therefore often used as a target for cancer treatment. HER2-targeted therapy is widely used in the treatment of HER2-positive cancers, especially breast cancer, where it has greatly improved the survival of patients with HER2-positive breast cancer. Currently, five HER2-targeted drugs have been approved by the FDA for clinical treatment, including the monoclonal antibodies trastuzumab and pertuzumab, the small molecule tyrosine kinase inhibitors lapatinib and neratinib, and the antibody drug conjugate (ADC) drug trastuzumab emtansine (T-DM1). However, single HER2-targeted therapy often has several potential resistance mechanisms, including (1) heterogeneity within HER2-positive tumor tissues. The HER2 expression levels of tumor cells within HER2-positive tumor tissues vary greatly, leading to off-target effects of single HER2-targeted therapy; (2) activation of other epidermal growth factor receptors (HERs) causing reactivation of the HER2 receptor. In tumor tissue, the presence of ligands activates HER1 (EGFR), HER3, and HER4 receptors. Activated HERs preferentially form heterodimers with HER2, which are more potent oncogenizers. This not only reactivates HER2 but also reactivates the PI3K / AKT and RAS / MAPK signaling pathways, promoting tumor cell proliferation, metastasis, and recurrence. Studies have reported that HER3 expression increases in HER2-positive breast cancer cells after lapatinib treatment, leading to drug resistance. Furthermore, EGFR phosphorylation and the formation of EGFR / HER2 heterodimers contribute to cancer cell resistance to trastuzumab therapy. Therefore, designing novel drug molecules, especially those that can simultaneously reduce the expression of EGFR, HER2, HER3, and HER4, is crucial for treating HER2-positive and HER2-resistant diseases.

[0005] Currently, no single molecule on the market can simultaneously reduce the expression of EGFR, HER2, HER3, and HER4. Only some small molecule inhibitors can partially inhibit the expression of HERs; for example, afatinib is a dual inhibitor of EGFR and HER2 tyrosine kinases. Therefore, in order to maximally inhibit the HER2 signaling pathway and achieve complete inhibition of HERs, thus enabling effective treatment for HER2-positive patients and HER2-resistant tumors, it is essential and important to develop drugs that can simultaneously inhibit the expression of EGFR, HER2, HER3, and HER4. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a fusion protein based on dual targeting of HER2 and integrin. This fusion protein can simultaneously target and recognize overexpressed HER2 and integrin on the surface of tumor cells. On one hand, by specifically regulating the interaction between the HER2 receptor and integrin, it simultaneously interferes with the expression of HER2 itself or other HER receptors such as EGFR, HER3, and HER4, blocking the transduction of the HER family and its downstream signals. On the other hand, through ligand-receptor interactions, it reduces the interaction of integrin with other growth factors, thereby blocking integrin-induced cancer cell survival and proliferation.

[0007] The specific technical solution of this invention is as follows:

[0008] A dual-targeting binding protein of human epidermal growth factor receptor 2 and integrin, consisting of an antibody against human epidermal growth factor receptor 2 and a polypeptide containing an RGD motif linked by a linker peptide or directly.

[0009] Preferably, the polypeptide containing the RGD motif is selected from one or more of RGD, CRGDK, CRGDKGPDC, DRGDC, GCGYGRGDSPG, and the antibody against human epidermal growth factor receptor 2 is selected from one or more of nanobodies, single-chain antibodies, and monoclonal antibodies.

[0010] Further preferably, the gene sequence encoding the polypeptide amino acid sequence containing the RGD motif is shown in SEQ ID No: 1 or SEQ ID No: 2. The gene sequence encoding the antibody amino acid sequence of human epidermal growth factor receptor 2 is shown in SEQ ID No: 3 or SEQ ID No: 4.

[0011] In a specific example of the present invention, the dual-target binding protein is characterized by an amino acid sequence as shown in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8.

[0012] The dual-targeting binding protein of the present invention is characterized in that the two targeting motifs of the dual-targeting binding protein are located at the C-terminus and the N-terminus, respectively, so that one molecule can simultaneously target two receptors on the cell surface.

[0013] Preferably, the amino acid sequence of the dual-target binding protein of the present invention is shown in SEQ ID No: 5 or SEQ ID No: 6.

[0014] Another object of the present invention is to provide a nucleic acid sequence that expresses the dual-target binding protein described in the present invention.

[0015] Another object of this invention is to provide the application of the dual-targeting binding protein described herein in the preparation of drugs for the prevention or treatment of cancer. The cancers are HER2 receptor-positive or integrin receptor-positive cancers, such as breast cancer, gastric cancer, salivary gland cancer, colon cancer, prostate cancer, cervical cancer, ovarian cancer, lung cancer, pancreatic cancer, liver cancer, and bladder cancer.

[0016] The dual-targeting binding protein described in this invention is used to inhibit the expression of the HER family on the cell surface, including EGFR, HER2, HER3, and HER4.

[0017] The dual-targeting binding protein described in this invention is used to inhibit the signal transduction of HER2 molecules and integrin molecules on the cell surface.

[0018] The dual-targeting binding protein described in this invention can be used as a pharmaceutical active ingredient to formulate pharmaceutical preparations with conventional drug carriers in the art for various routes of administration, such as oral, sublingual, inhalation, topical, rectal, and injection (intradermal, subcutaneous, intramuscular, intravenous, and arterial injection). Intravenous injection is preferred.

[0019] The dual-targeting binding protein described in this invention can also be used as a carrier to conjugate drugs or probes. Drugs include paclitaxel and its derivatives, doxorubicin and its derivatives, maytansine and its derivatives, auristatin and its derivatives, calicheamicin and duocarmycins, and other commonly used clinical chemotherapy drugs. Probes include fluorescent imaging probes, nuclear magnetic resonance imaging probes, and radioactive imaging materials.

[0020] The dual-targeting binding protein of the conjugated drug or probe not only specifically recognizes the HER2 receptor and integrin molecule on the cell surface, blocking the signal transduction between them; the conjugated drug or probe is directed to the surface of cancer cells to exert cytotoxic effects or be used for tumor cell imaging.

[0021] The dual-targeting binding protein described in this invention can be prepared by artificial synthesis or by expression using prokaryotic, eukaryotic, or insect expression systems.

[0022] In a specific example of the present invention, the dual-targeting binding protein is expressed by an E. coli expression system and obtained by standard affinity chromatography purification method.

[0023] Advantages of this invention:

[0024] 1. The dual-targeting binding protein of this invention can simultaneously target and recognize HER2 and integrin overexpressed on the surface of tumor cells. On one hand, by specifically regulating the interaction between the HER2 receptor and integrin, it simultaneously interferes with the expression of HER2 itself or other HER receptors such as EGFR, HER3, and HER4, maximally blocking the transduction of the HER family and its downstream signals. On the other hand, through ligand-receptor interactions, it reduces the interaction of integrin with other growth factors, thereby blocking the survival and proliferation of integrin-induced cancer cells. This invention significantly improves the anti-tumor effect of the fusion protein through synergistic interactions between targets.

[0025] 2. The dual-targeting fusion protein of this invention can not only specifically recognize the HER2 receptor, reduce HER2 expression levels, and interfere with downstream HER2 signal transduction, but also specifically recognize integrin molecules, such as reducing α-receptor expression. v β3 expression levels. This invention has found that the biological effects mediated by the dual-target binding protein after recognizing the target exhibit synergistic interactions compared to single-target interactions. The fusion protein not only downregulates HER2 and α... v Expression of β3 can also lead to downregulation of other HER receptors such as EGFR, HER3, and HER4. This indicates that the dual-targeting binding protein described in this invention utilizes interactions between HER families, interactions between integrin families, and interactions between the HER family and the integrin family, via HER2 and α... v Interference with β3 target information achieves the goal of regulating the cellular microenvironment.

[0026] 3. The results of this invention further show that different HER2 antibodies, when conjugated to peptides containing the RGD motif, can inhibit the activity of EGFR, HER2, HER3, and HER4. The dual-targeting binding protein nano-Her2-RGD using nanobodies exhibits varying degrees of inhibitory activity against all four members of the HER family: EGFR, HER2, HER3, and HER4. The dual-targeting binding protein ScFv-Her2-RGD using single-chain antibodies shows more significant inhibitory effects on HER2 and HER3. Attached Figure Description

[0027] Figure 1 Electrophoresis image of nano-Her2-RGD protein, which is the dual-target binding protein in Example 1.

[0028] Figure 2 This is an electrophoresis image of the dual-target binding protein ScFv-Her2-RGD from Example 1.

[0029] Figure 3 This demonstrates the ability of the dual-targeting binding protein nano-Her2-RGD to target cells and be taken up by them.

[0030] Figure 4 This study investigates the regulatory role of the dual-targeting binding protein nano-Her2-RGD in the expression of HER receptor and integrin.

[0031] Figure 5 This study investigates the regulatory role of the dual-targeting binding protein ScFv-Her2-RGD in the expression of HER receptor and integrin.

[0032] Figure 6 This study investigates the regulatory role of the dual-targeting binding protein nano-Her2-RGD in the expression of the HER receptor and integrin gene.

[0033] Figure 7 This study investigates the regulatory role of the dual-targeting binding protein nano-Her2-RGD in HER receptor expression.

[0034] Figure 8 To enhance the antitumor activity of doxorubicin nano-Her2-RGD-DOX conjugated to dual-targeting binding proteins. Detailed Implementation

[0035] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Example 1: Design and preparation of the dual-target binding protein of the present invention

[0037] 1. Dual-targeting binding protein nano-Her2-RGD: The antibody targeting HER2 is a nanobody, the amino acid sequence of which is shown in SEQ ID No:3. Its C-terminus is linked to the integrin polypeptide ligand CRGDKGPDC via a (GGGGS)3 flexible polypeptide to obtain nano-Her2-RGD. The amino acid sequence of the dual-targeting binding protein is shown in SEC ID No:5.

[0038] Shanghai Jereh Biotechnology Co., Ltd. was commissioned to construct a recombinant plasmid containing the dual-targeting binding protein described in this invention. The recombinant plasmid vector was PET28a.

[0039] The recombinant plasmid was introduced into *E. coli* BL21(DE3) using the heat shock method. After incubation at 37°C for one hour, it was plated onto kanamycin agar plates and incubated on a shaker at 37°C for 12 hours. Single colonies were picked and placed in shake tubes containing 5 mL of LB medium and incubated overnight. The resulting culture was then transferred to 400 mL of medium and expanded at 37°C. The IPTG induction conditions were achieved when the bacterial concentration in the culture flask was OD0.05. 600 0.6–0.8. After culturing for 20 hours at 18°C ​​and 200 rpm, bacteria were collected by centrifugation (8000 rpm, 5 min). Cells were washed three times with ultrapure water and resuspended in 20 mM imidazole buffer. Cells were disrupted by sonication, and the supernatant was collected by centrifugation (10000 rpm, 30 min). The target protein was purified using the AKTA system and eluted with a linear gradient of 500 mM imidazole buffer to confirm its location and collection. Protein gel electrophoresis showed clear bands of the target protein with a single composition, such as… Figure 1 As shown.

[0040] 2. Dual-targeting binding protein ScFv-Her2-RGD: The antibody targeting HER2 is a single-chain antibody, and its amino acid sequence is shown in SEQ ID No:4. The full-length amino acid sequence of the dual-targeting binding protein is shown in SEC ID No:6.

[0041] Shanghai Jereh Biotechnology Co., Ltd. was commissioned to construct a recombinant plasmid containing the dual-targeting binding protein described in this invention. The recombinant plasmid vector was PET22b.

[0042] The recombinant plasmid was introduced into competent *E. coli* Shuffle T7 cells using a heat shock method. After incubation at 37°C for one hour, the cells were plated on ampicillin plates and cultured on a shaker at 37°C for 12 hours. Single colonies were picked and cultured overnight in shake tubes containing 5 mL of LB medium. The culture was then expanded to 400 mL of medium at 37°C. The IPTG induction conditions were achieved with a bacterial concentration of OD600 of 0.6–0.8 in the culture flask. Cells were cultured for another 20 hours at 25°C and 200 rpm, and then collected by centrifugation (8000 rpm, 5 min). The cells were washed three times with ultrapure water and resuspended in 20 mM imidazole buffer. Cells were disrupted by sonication and the supernatant was collected by centrifugation (10000 rpm, 30 min). The target protein was purified using the AKTA system and eluted using a linear gradient of 500 mM imidazole buffer. The location of the target protein was confirmed, and the protein was collected. Gel electrophoresis showed clear bands of the target protein. Figure 2 As shown.

[0043] Example 2 examines the dual-targeting effect of the dual-targeting binding protein described in this invention.

[0044] First, the fusion protein nano-Her2-RGD obtained in Example 1 was transferred to PBS buffer using dialysis. A certain amount of Rhodamine B was added, and the mixture was incubated at 4°C in the dark for 8 hours. Finally, the reaction system was placed in a dialysis bag (MW7000Da) and dialyzed with PBS to remove free unreacted Rhodamine B. The dialysis buffer was changed at least four times. The red fluorescently labeled protein was stored at 4°C in the dark. Human breast cancer cells MCF-7 cells were cultured in DMEM medium in a humid environment of 5% CO2 at 37°C. Cells were seeded at a density of 10,000 cells per well in six-well plates, with one coverslip added to each well, and allowed to grow overnight. 200 μL of the labeled fusion protein was added to each well and co-cultured with the cells for 4 hours. Then, the culture medium was aspirated, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 10 min, washed three times with PBS, and the coverslips were placed in PBS for detection. Protein entry into cells was observed under a laser confocal microscope using a 63x oil immersion microscope. Figure 3 .

[0045] The results showed that the dual-targeting binding protein could effectively recognize cancer cells and was well taken up by the cells.

[0046] Example 3: Regulation of HER2 Receptor and Integrin Expression by the Dual-Targeting Binding Proteins of the Present Invention. Using breast cancer cells MCF-7 cells as an example, the regulatory effects of the dual-targeting binding proteins of the present invention on the expression of HER2 receptor and integrin were investigated by Western immunoblot assay. MCF-7 cells were seeded at a density of 100,000 cells per well in 6-well plates and grown overnight. Experimental groups: Single-targeting proteins RGD (SEQ ID No: 2), ELP-RGD (SEQ ID No: 9), nano-Her2 (SEQ ID No: 3), fusion proteins nano-Her2-RGD (SEQ ID No: 5), and ScFv-Her2-RGD (SEQ ID No: 6) were added to 6-well plates at a concentration of 500 μg / mL and co-cultured with cells for 24 h. The control group received the same volume of PBS buffer. Each group had 3 replicates. The culture medium was then aspirated, and the cells were washed three times with PBS. 200 μL of RIPA tissue cell lysis buffer was added to each well, and the cells were lysed at room temperature for 15 minutes. Thoroughly mix the lysis buffer and cell debris using a pipette, and collect the sample in a 1.5 mL centrifuge tube for later use. Dissolve the sample in 2× loading buffer, boil in water for 5 min, cool the sample on ice, and centrifuge at 10000 rpm for 1 min, collecting the supernatant. Separate the sample using a 10% protein gel electrophoresis gel. Transfer the sample from the separating gel to a PVDF membrane, remove the PVDF membrane, and immerse it in 5% skim milk for 1.5 h at room temperature. Then use EGFR, HER2, HER3, HER4, α... v The target band was incubated with primary antibodies against β3 and GAPDH at 37°C for 1 hour. The target band was then washed with TBST solution 3-5 times, every 5 minutes. The target band was then incubated with HRP-conjugated secondary antibodies at 37°C for 1 hour. The target band was again washed with TBST solution 3-5 times, every 5 minutes. Finally, ECL chemiluminescence buffer was used for imaging with an ECL chemiluminescence analyzer. Results are as follows: Figure 4 and Figure 5 As shown.

[0047] The results showed that nano-Her2-RGD and ScFv-Her2-RGD, which have dual targeting capabilities, can reduce HER2 and integrinα. vIt can reduce the expression level of β3 and simultaneously decrease the expression levels of other HER receptors, including EGFR, HER3, and HER4. However, targeting nano-Her2 alone, along with RGD and ELP-RGD, has virtually no effect on the expression of other receptors. Furthermore, different antibody forms, such as anti-HER2 nanobodies (nano-Her2) and anti-HER2 single-chain antibodies (ScFv-Her2), exhibit functional differences. However, nano-Her2-RGD and ScFv-Her2-RGD all demonstrate varying degrees of inhibitory activity against all four members of the HER family: EGFR, HER2, HER3, and HER4.

[0048] Example 4: Regulation of HER Receptor Gene Expression by the Dual-Targeting Binding Protein of the Present Invention. Using breast cancer cells MCF-7 cells as an example, the regulatory effect of the dual-targeting binding protein of the present invention on HER receptor expression was investigated by RT-PCR. MCF-7 cells were seeded at a density of 100,000 cells per well in 6-well plates and grown overnight. Experimental groups: Single-targeting proteins ELP-RGD (SEQ ID No: 9), nano-Her2 (SEQ ID No: 3), and the fusion protein nano-Her2-RGD (SEQ ID No: 5) were added to 6-well plates at a concentration of 500 μg / mL and co-cultured with cells for 24 h. The control group received the same volume of PBS buffer. Each group had 3 replicates. The culture medium was then aspirated, and the cells were washed three times with PBS. 500 μL of TRIzol (Invitrogen) reagent was added to each well. RNA was extracted according to the manufacturer's instructions, and then reverse transcribed into cDNA using the PrimeScript™ RT (Takara) kit for RT-PCR detection. Results are as follows: Figure 6 As shown.

[0049] The results showed that nano-Her2-RGD, which has dual targeting capabilities, can not only reduce the expression level of ERBB2, but also reduce the expression levels of other HER receptors, including ERBB1, ERBB3, and ERBB4. In contrast, single targeting of nano-Her2 and ELP-RGD has virtually no effect on the gene expression of other receptors.

[0050] Example 5: The regulatory effect of the dual-targeting binding protein on HER receptor expression described in this invention is illustrated using human breast cancer cells MCF-7 cells as an example. Cells were seeded at a density of 10,000 cells per well in six-well plates, with one coverslip added to each well, and allowed to grow overnight. Nano-Her2-RGD was added to the six-well plates at a concentration of 500 μg / mL and co-cultured with the cells for 24 h. The culture medium was then aspirated, and the cells were washed three times with PBS solution, fixed with 4% paraformaldehyde at room temperature for 10 min, and then washed three times with PBS. The cells were blocked with 5% BSA solution at room temperature for 1 h, followed by incubation with primary antibodies against EGFR, HER2, HER3, and HER4 at 4°C overnight. The cells were washed three times with PBST, co-incubated with the corresponding fluorescently labeled secondary antibodies, and then washed three times with PBST. DAPI was used for nuclear labeling; it was stored away from light, and the coverslips were placed in PBS for detection. The expression of the HER receptor after nano-Her2-RGD treatment was observed under a laser confocal microscope using a 63x oil immersion lens. Figure 7 .

[0051] Immunofluorescence results showed that the dual-targeting binding protein could simultaneously reduce the expression levels of HER receptors EGFR, HER2, HER3, and HER4 on the surface of tumor cells.

[0052] Example 6: Antitumor activity of the chemotherapeutic drug doxorubicin conjugated with the dual-targeting binding protein of the present invention as a drug delivery carrier.

[0053] The dual-targeting binding protein nano-Her2-RGD was used as a drug delivery carrier, and the chemotherapeutic drug doxorubicin (DOX) was conjugated to it according to the method in CN108187064A to synthesize the protein drug derivative nano-Her2-RGD-DOX. Positive control groups were set up using the single-targeting drug nano-HER2-DOX and ELP-RGD-DOX. Taking the dual-targeting binding protein nano-Her2-RGD as an example, it was first replaced by dialysis in 0.1M PB buffer solution at pH 6.5, and reacted with NHS-PEG1000-MAL at 4℃ for 12 h. Unreacted NHS-PEG1000-MAL was then removed by ultrafiltration, and the modified DOX drug was added to the reaction system, reacting at 4℃ for 12 h. The reaction product was separated by size gel chromatography. The cytotoxicity of the targeted protein drug derivative nano-Her2-RGD-DOX and the single-target drug derivatives nano-Her2-DOX and ELP-RGD-DOX against breast cancer cells MCF-7 cells was detected by the MTT assay. The concentration of the derivatives was calculated based on the DOX concentration. Results are as follows: Figure 8 As shown.

[0054] The results showed that, compared with single-target drugs, the dual-targeting nano-Her2-RGD-DOX significantly improved cytotoxicity. This indicates that the dual-targeting effect can enhance drug accumulation in cancer cells on the one hand, and on the other hand, the synergistic interaction between the targets further improves the therapeutic effect. sequence list <110> Nanjing University <120> A dual-targeting binding protein of human epidermal growth factor receptor 2 and integrin and its applications <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3 <212> PRT <213> Artificial Sequence <400> 1 Arg Gly Asp 1 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Cys Arg Gly Asp Lys Gly Pro Asp Cys 1 5 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <400> 3 Gly Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Pro 20 25 30 Asn Val Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Arg Arg Glu Trp 35 40 45 Val Ala Ala Ala Asn Lys Tyr Gly Thr Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Ala Thr Asn Trp Asp Tyr His Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 4 <211> 248 <212> PRT <213> Artificial Sequence <400> 4 Leu Glu Val Leu Phe Gln Gly Pro Glu Val Gln Leu Val Glu Ser Gly 1 5 10 15 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 20 25 30 Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala 35 40 45 Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly 50 55 60 Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala 65 70 75 80 Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 85 90 95 Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe 100 105 110 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135 140 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 180 185 190 Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220 Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly 225 230 235 240 Gln Gly Thr Lys Val Glu Ile Lys 245 <210> 5 <211> 143 <212> PRT <213> Artificial Sequence <400> 5 Gly Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Pro 20 25 30 Asn Val Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Arg Arg Glu Trp 35 40 45 Val Ala Ala Ala Asn Lys Tyr Gly Thr Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Ala Thr Asn Trp Asp Tyr His Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Cys Arg Gly Asp Lys Gly Pro Asp Cys 130 135 140 <210> 6 <211> 272 <212> PRT <213> Artificial Sequence <400> 6 Leu Glu Val Leu Phe Gln Gly Pro Glu Val Gln Leu Val Glu Ser Gly 1 5 10 15 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 20 25 30 Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala 35 40 45 Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly 50 55 60 Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala 65 70 75 80 Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 85 90 95 Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe 100 105 110 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135 140 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 180 185 190 Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220 Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly 225 230 235 240 Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255 Gly Ser Gly Gly Gly Gly Ser Cys Arg Gly Asp Lys Gly Pro Asp Cys 260 265 270 <210> 7 <211> 273 <212> PRT <213> Artificial Sequence <400> 7 Gly Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Ser Pro 20 25 30 Asn Val Met Gly Trp Tyr Arg Gln Thr Pro Gly Asn Arg Arg Glu Trp 35 40 45 Val Ala Ala Ala Asn Lys Tyr Gly Thr Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Ala Thr Asn Trp Asp Tyr His Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Val Pro Gly Lys Gly Val Pro Gly Val Gly 130 135 140 Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val 145 150 155 160 Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro 165 170 175 Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly 180 185 190 Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val 225 230 235 240 Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Gly Gly 245 250 255 Gly Gly Ser Gly Gly Gly Gly Ser Cys Arg Gly Asp Lys Gly Pro Asp 260 265 270 Cys <210> 8 <211> 387 <212> PRT <213> Artificial Sequence <400> 8 Leu Glu Val Leu Phe Gln Gly Pro Glu Val Gln Leu Val Glu Ser Gly 1 5 10 15 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 20 25 30 Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala 35 40 45 Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly 50 55 60 Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala 65 70 75 80 Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala 85 90 95 Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe 100 105 110 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135 140 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp 165 170 175 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 180 185 190 Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 195 200 205 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 210 215 220 Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly 225 230 235 240 Gln Gly Thr Lys Val Glu Ile Lys Val Pro Gly Lys Gly Val Pro Gly 245 250 255 Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys 260 265 270 Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly 275 280 285 Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val 290 295 300 Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro 305 310 315 320 Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly 325 330 335 Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys 340 345 350 Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly 355 360 365 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Arg Gly Asp Lys Gly 370 375 380 Pro Asp Cys 385 <210> 9 <211> 139 <212> PRT <213> Artificial Sequence <400> 9 Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val 1 5 10 15 Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro 20 25 30 Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly 35 40 45 Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys 50 55 60 Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly 65 70 75 80 Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val 85 90 95 Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro 100 105 110 Gly Lys Gly Val Pro Gly Val Gly Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Cys Arg Gly Asp Lys Gly Pro Asp Cys 130 135

Claims

1. A dual targeting binding protein of human epidermal growth factor receptor 2 and integrin, characterized in that An antibody of human epidermal growth factor receptor 2 and a polypeptide containing RGD motif are connected by a connecting peptide, the amino acid sequence of the polypeptide containing RGD motif is shown as SEQ ID No: 2, the amino acid sequence of the antibody of human epidermal growth factor receptor 2 is shown as SEQ ID No: 3 or 4, and the connecting peptide is (GGGGS) 3.

2. The dual targeting binding protein of claim 1, wherein The amino acid sequence is shown as SEQ ID No: 5 or 6.

3. A nucleic acid, characterized in that The double-targeting binding protein according to claim 1 or 2.

4. The double-targeting binding protein according to claim 1 or 2 is used for preparing a drug for preventing or treating cancer, the cancer is HER2 positive or HER2 drug-resistant cancer, and the cancer is breast cancer or gastric cancer.

5. Use according to claim 4, characterized in that The double-targeting binding protein inhibits the expression of cell surface HER family including EGFR, HER2, HER3 and HER4.

6. Use according to claim 4, characterized in that The double-targeting binding protein is used as a carrier for coupling drugs.

7. Use according to claim 6, characterized in that The drugs include one or more of paclitaxel, doxorubicin, maytansine, auristatin, calicheamicin or duocarmycins.

8. The double-targeting binding protein according to claim 1 or 2 is used for preparing a tumor cell imaging probe.

9. Use according to claim 8, characterized in that The probe includes a fluorescent imaging probe, a nuclear magnetic resonance imaging probe or a radioactive imaging material.

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