Protein targeting and binding to mesothelin, and encoding nucleic acid and use thereof

Abstract

A protein targeting and binding to mesothelin, which has an amino acid sequence as shown in one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No 4. The protein targeting and binding to mesothelin can specifically bind to a tumor cell expressing mesothelin. The protein targeting and binding to mesothelin is used in the preparation of a nuclide probe, a fluorescent probe and a tumor diagnostic kit, and in the preparation of a drug for tumor-targeted therapy or a carrier of a tumor-targeted drug delivery system, comprising a protein-drug conjugate and a multispecific fusion protein, and is used for immunocytokine-targeted therapy, etc. The protein targeting and binding to mesothelin plays a role in the prevention, imaging and pathological diagnosis, and treatment of mesothelin-positive tumors.

Description

Mesothelin targeting binding protein, its encoding nucleic acid and use Technical Field

[0001] The present invention relates to the field of biological targeted drugs, and in particular to a mesothelin targeted binding protein, its encoding nucleic acid and use. Background Art

[0002] Mesothelin (MSLN) is a cell-surface tumor differentiation antigen. It is expressed at low levels in mesothelial cells of a few non-critical organs and tissues, such as the pleura, pericardium, peritoneum, tonsils, thymus, seminal vesicles, fallopian tubes, uterus, and placenta. However, it is highly expressed in a variety of solid tumors, including ovarian cancer, pancreatic cancer, endometrial cancer, malignant mesothelioma, lung adenocarcinoma, triple-negative breast cancer, gastric cancer, cervical cancer, and bile duct cancer. Given the specific distribution and differential expression of mesothelin in tumor tissues, tumor immunotherapy strategies targeting mesothelin can reduce the risk of nonspecific toxicity, making it an ideal target for tumor immunotherapy.

[0003] The human mesothelin gene encodes a 71 kDa precursor protein composed of 628 amino acids. Furin cleaves this precursor protein at amino acid position Arg 295 to release two protein products: shed megakaryocyte potentiating factor (MPF) and mature mesothelin, which is anchored to the cell membrane. MPF is a 31 kDa soluble protein released into the serum and has the activity of stimulating bone marrow megakaryocyte colony formation. Mesothelin is a membrane-bound protein anchored to the cell membrane by glycosylated phosphatidylinositol (GPI). It is produced at the C-terminus of the precursor protein, is 40 kDa in size, and contains a CA125 / MUC16 binding site. CA125 / MUC16 is a member of the mucin family and is expressed in ovarian cancer and malignant mesothelioma.

[0004] When mesothelin is overexpressed, intracellular signaling pathways such as NF-κB, MAPK, and PI3K are activated, contributing to tumor cell adhesion, proliferation, anti-apoptosis, and chemoresistance. Mesothelin regulates cellular function through two primary pathways: First, it activates downstream signaling pathways by binding to its receptor, CA125 / MUC16. Second, aberrantly overexpressed mesothelin can activate intracellular pathways through its GPI domain. The interaction between CA125 / MUC16 and mesothelin mediates heterotypic cell adhesion in vitro and is therefore considered a potential mechanism for peritoneal metastasis of ovarian tumors. Studies have reported that CA125 / MUC16 binding to mesothelin downregulates DKK1 (Dickkopf-1, a WNT signaling pathway inhibitor) through the SGK3 / FOXO3 signaling pathway, thereby promoting migration. Blocking the binding of CA125 / MUC16 and mesothelin can restore DKK1 levels and prevent ovarian cancer metastasis.

[0005] Mesothelin is expressed in limited amounts in normal tissues but is overexpressed in a variety of malignant tumors, making it an ideal candidate for targeted therapy. Anti-tumor strategies targeting mesothelin are being developed in multiple directions and evaluated in preclinical and clinical trials, including monoclonal antibodies, antibody-drug conjugates, immunotoxins, tumor vaccines, chimeric antigen receptor T cell therapy, etc. The results of a phase I clinical trial (NCT02414269) applied mesothelin CAR-T cells in combination with PD-1 inhibitors to malignant pleural mesothelioma, metastatic lung cancer, and breast cancer. CAR-T cells targeting mesothelin were well tolerated when administered intrathoracically, had no significant toxicity to normal tissues expressing mesothelin, and showed potential for the treatment of solid tumors. The mesothelin-targeting ADC drug (BAY 94-9343) developed by the National Cancer Institute of the United States consists of the mesothelin antibody Anetumab ravtansine and the payload DM4. It has shown good tolerability, controllable adverse reactions, and good pharmacokinetics in Phase I clinical studies, and has demonstrated encouraging preliminary clinical activity in patients with advanced solid tumors.

[0006] For a long time, screening and preparing monoclonal antibodies against specific antigens or targets has been the most commonly used and successful strategy for preparing targeted binding proteins. However, current antibody drugs targeting mesothelin have limitations such as large molecular weight, poor tumor penetration, strong immunogenicity, complex preparation processes, and high production costs. Therefore, further research and development of mesothelin-targeting binding proteins with low molecular weight, high tumor penetration, and good tumor targeting, while maintaining high affinity, has extremely important clinical value. Summary of the Invention

[0007] The purpose of the present invention is to provide a mesothelin targeted binding protein, its encoding nucleic acid and use. The mesothelin targeted binding protein provided by the present invention can specifically bind to mesothelin protein, mesothelin-positive tumor cells or tumor tissues at the molecular protein level, in vitro cell and tissue level and in vivo experimental animal level, providing a tumor-targeted delivery vector with high affinity and good specificity for tumor targeted treatment strategies targeting mesothelin.

[0008] To achieve the above objectives, the present invention provides a mesothelin targeting binding protein, whose amino acid sequence is shown in one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4. The mesothelin targeting binding protein can specifically bind to the tumor molecular marker mesothelin.

[0009] The present invention also provides a nucleic acid molecule encoding the above-mentioned mesothelin targeting binding protein.

[0010] The present invention also provides a radionuclide probe, which is a radionuclide-labeled probe prepared by using the mesothelin targeting binding protein provided by the present invention.

[0011] The present invention also provides a fluorescent probe, which is obtained by covalently coupling the mesothelin-targeting binding protein provided by the present invention with a fluorescent dye.

[0012] The present invention also provides a protein-drug conjugate, which is composed of a fusion protein consisting of the mesothelin-targeted binding protein provided by the present invention and an albumin-binding domain capable of binding to human serum albumin, and is site-specifically coupled to four MMAE toxin molecules via cysteine ​​residues to form a mesothelin-targeted protein-drug conjugate; the amino acid sequence of the conjugate is shown as one of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10.

[0013] Wherein, the albumin binding domain is a natural protein from the N-terminal region of streptococcal protein G.

[0014] The present invention also provides a kit, which is a tumor diagnosis kit containing the mesothelin targeting binding protein provided by the present invention.

[0015] The present invention also provides the use of the above-mentioned mesothelin-targeted binding protein in the preparation of tumor-targeted drugs.

[0016] The tumor-targeted drug is an immune cell therapy drug.

[0017] The tumor-targeted drug is a tumor-targeted cytokine therapeutic drug.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The mesothelin targeted binding protein provided by the present invention can specifically bind to mesothelin protein, mesothelin-positive tumor cells or tumor tissues at the molecular protein level, in vitro cell and tissue level, and in vivo experimental animal level; the targeted binding protein is labeled with a fluorescent tracer to construct a mesothelin-specific fluorescent probe, which can efficiently target mesothelin-positive gastric cancer cells in in vitro and in vivo experiments; the mesothelin targeted protein-drug conjugate prepared based on the targeted binding protein can effectively kill mesothelin-positive cells.

[0020] Compared with existing anti-human mesothelin antibodies, the mesothelin-targeting binding protein provided by the present invention has the following characteristics: a small molecular weight of only about 18 kDa, which is about one-tenth of the antibody molecule; a small size and strong tissue penetration; low immunogenicity; better regulated pharmacokinetic performance; a simple production process, high yield and low production cost.

[0021] The targeted binding protein of the present invention has good targeting properties for mesothelin-positive tumor cells and can be connected or used in combination with existing anti-tumor drugs, drug delivery systems, and molecular imaging agents for tumor targeted therapy, tumor targeted drug delivery, and in vivo tumor imaging and tracing, providing new ideas and methods for tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows the binding of mesothelin targeted binding proteins to mesothelin molecules; targeted binding proteins 1, 2, and 3 have significant binding to mesothelin, among which targeted binding proteins 1 and 3 have the strongest binding to mesothelin and are the dominant mesothelin targeted binding proteins, while the non-targeted targeted binding protein 4 in the control group has no significant binding.

[0023] Figure 2 shows the SDS-PAGE and Western Bolt assay results of the superior mesothelin targeting protein (mesothelin targeting protein 1) after prokaryotic expression and protein purification. The molecular weight of the mesothelin targeting protein is approximately 18 kDa, and the purity of the purified targeting protein is greater than 90%.

[0024] Figure 3 shows the binding of the dominant mesothelin targeting protein (mesothelin targeting protein 1) to the surfaces of N87 and Huh7 cells. N87 is a mesothelin-positive cell line, while Huh7 is a mesothelin-negative cell line. MBP refers to the dominant mesothelin targeting protein. MBP clearly binds to mesothelin-positive N87 cells but not to mesothelin-negative Huh7 cells.

[0025] Figure 4 shows the biodistribution of the mesothelin-specific fluorescent probe in mice. The mesothelin-specific fluorescent probe was injected into the tail vein, and near-infrared imaging was performed on the tumor-bearing mice 8 hours and 24 hours after administration. The mesothelin-specific fluorescent probe showed obvious accumulation in the tumor tissue 8 hours after administration, and the fluorescent probe was completely targeted to the tumor site after 24 hours.

[0026] FIG5 shows the SPR results of mesothelin-targeted fusion proteins SEQ ID No. 5, SEQ ID No. 7, and SEQ ID No. 9.

[0027] FIG6 is the cell immunofluorescence results of mesothelin-targeted fusion proteins SEQ ID No. 5 and SEQ ID No. 7.

[0028] FIG. 7 shows the in vivo targeting results of the mesothelin-targeted binding protein SEQ ID No. 3 and the mesothelin-targeted fusion proteins SEQ ID No. 5 and SEQ ID No. 7.

[0029] FIG8 shows the cell killing experiment results of mesothelin-targeted protein-drug conjugates SEQ ID No. 5 and SEQ ID No. 7.

[0030] FIG9 shows the results of tumor inhibition experiments of mesothelin-targeted protein-drug conjugates SEQ ID No. 5 and SEQ ID No. 7 in the MKN45 gastric cancer-bearing mouse model.

[0031] FIG10 shows the results of tumor inhibition experiments of mesothelin-targeted protein-drug conjugates SEQ ID No. 5 and SEQ ID No. 7 in a PANC1 pancreatic cancer-bearing mouse model. DETAILED DESCRIPTION

[0032] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0033] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0034] The present invention provides a mesothelin targeting binding protein, the amino acid sequence of which is shown in one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. The mesothelin targeting binding protein can specifically bind to the tumor molecular marker mesothelin.

[0035] The mesothelin-targeted binding protein provided by the present invention is obtained through multiple rounds of panning and screening using phage display library technology. Multiple phage monoclones with strong binding ability are further screened by ELISA, and the nucleic acid sequences of the monoclones are determined. The mesothelin-targeted binding protein of the present invention is produced by prokaryotic expression and further identified by ELISA, flow cytometry, cell immunofluorescence, etc.

[0036] In an embodiment of the present invention, in the amino acid sequence of the targeted binding protein, the amino acid residue Xaa at position 31, 33, 34, 35, 36, 37, 38, 44, 45, 64, 66, 67, 69, 77, 78, 97, 99, 100, 102, 110 and 111 are respectively any amino acids, that is, the backbone sequences of the four mesothelin targeted binding protein sequences are the same, and only the amino acids at these positions marked above are different.

[0037] The mesothelin-targeted binding protein disclosed in the present invention can specifically bind to tumor cells expressing mesothelin. The mesothelin-targeted binding protein of the present invention is used to prepare radionuclide probes, fluorescent probes, and tumor diagnostic kits; to prepare tumor-targeted therapeutic drugs or tumor-targeted drug delivery system carriers, including but not limited to protein-drug conjugates, multi-specific fusion proteins, immune cell therapy, and tumor-targeted cytokine therapy, etc., to play a preventive, imaging and pathological diagnostic, and therapeutic role in mesothelin-positive tumors.

[0038] In an embodiment of the present invention, a mesothelin-specific fluorescent probe is used to track mesothelin-positive tumors, such as gastric cancer, in vivo.

[0039] The mesothelin-targeted binding protein of the present invention specifically binds to gastric cancer tissues that express positive mesothelin, but is not limited to gastric cancer. The mesothelin-targeted binding protein and its fluorescent probe can also specifically bind to other tumor tissues that highly express mesothelin, such as ovarian cancer, breast cancer, lung cancer, pancreatic cancer, cervical cancer, mesothelioma, etc.

[0040] Furthermore, the present invention discloses a red non-invasive tracing fluorescent probe, in which the mesothelin-targeting binding protein of the present invention is covalently coupled with the fluorescent dye CY5-NHS (Cyanine5-NHS) to form a specific fluorescent probe. In in vitro cell immunofluorescence and in vivo biodistribution experiments, the fluorescently labeled antigen targeting protein showed good targeting to mesothelin-positive tumors and tissues, and was enriched in tumor sites rather than normal organs in vivo.

[0041] The invention discloses applications of a mesothelin-specific fluorescent probe in the diagnosis and therapeutic effect evaluation of mesothelin-highly expressed tumors.

[0042] In an embodiment of the present invention, a mesothelin-targeted binding protein is fused with an albumin-binding domain (ABD), and four MMAE toxins are site-specifically coupled via cysteine ​​residues to prepare a mesothelin-targeted protein-drug conjugate with a uniform DAR value (Drug-to-Antibody Ratio).

[0043] ABD refers to a natural protein from the N-terminal region of streptococcal protein G. It is a protein composed of 46 amino acids that can bind to human and mouse serum albumin, thereby prolonging the serum circulation half-life of mesothelin-specific protein drug conjugates.

[0044] In an embodiment of the present invention, the amino acid sequence of the targeting module and the cysteine ​​linker of the mesothelin-targeted protein drug conjugate is shown as one of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10.

[0045] The mesothelin-targeted binding protein involved in the embodiments of the present invention is synthesized through an E. coli expression system, and has a simple synthesis process, high yield, and low cost.

[0046] The present invention is further described in detail below with reference to specific embodiments:

[0047] Example 1: Detection of affinity of target binding protein for mesothelin by enzyme-linked immunosorbent assay (ELISA)

[0048] 1. Synthesis and purification of targeted binding proteins:

[0049] Targeted binding proteins #1, 2, and 3 were synthesized using the Escherichia coli prokaryotic expression system, and negative control targeted binding protein #4 was expressed, and the proteins were purified using a nickel column.

[0050] Target binding protein #1 (SEQ ID No. 1 containing a histidine tag at the N-terminus);

[0051] Targeted binding protein #2 (SEQ ID No. 2 containing a histidine tag at the N-terminus);

[0052] Target binding protein #3 (SEQ ID No. 3 containing a histidine tag at the N-terminus);

[0053] Targeted binding protein #4 (SEQ ID No. 4 containing a histidine tag at the N-terminus).

[0054] 2. Co-incubation with mesothelin protein:

[0055] Mesothelin protein was coated onto a 96-well flat-bottom plate and blocked with 1% BSA. The plate was then incubated with target binding proteins #1, 2, 3, and 4 for 2 h. Free target binding proteins in each group were washed away with PBS containing 0.5% Tween 20 (0.5% PBST). Diluted anti-His secondary antibody was then added and incubated for another 1 h. The anti-His secondary antibody was washed away with 0.5% PBST, and TMB solution was added. The plate was incubated at room temperature in the dark for 30-120 s, until the solution turned from colorless to blue. The reaction was terminated by adding 1 M dilute hydrochloric acid, which caused the solution to turn from blue to yellow.

[0056] 3. Use a microplate reader to measure the absorbance at OD450:

[0057] As shown in FIG1 , compared with the negative control, targeted binding proteins #1, 2, and 3 all had significant binding to mesothelin protein, among which targeted binding proteins #1 and 3 had the strongest binding.

[0058] Example 2: Using polyacrylamide gel electrophoresis (SDS-PAGE) and protein immunoblotting (Western Blot) to detect the purity and molecular weight of the target binding protein

[0059] 1. Electrophoresis:

[0060] Prepare 10% separation gel first, and add the upper layer of concentration gel after the lower separation gel solidifies. Add 10ul of target binding protein #1 sample or protein marker expressed and purified in the prokaryotic system to each well, run at 70V for 30min, and adjust to 120V for 60min after the marker band runs out.

[0061] 2. Dyeing:

[0062] Carefully pry open the splint, cut off the edge according to the lowest edge of the marker band, cut the upper edge along the gap between the stacking gel and the separation gel, carefully remove the film and put it into the staining solution, and stain with Coomassie Brilliant Blue for half an hour.

[0063] 3. Determination:

[0064] Take out the film and put it in water for decolorization, then take a photo and analyze the protein purity and molecular weight;

[0065] 4. Protein Immunoblotting:

[0066] As in step 1, transfer the proteins on the SDS-PAGE gel to a polyvinylidene fluoride membrane (PVDF membrane) using the wet transfer method. The electrotransfer conditions were 350 mA for 80 min. Block with 5% skim milk powder for 30 min and wash with TBST three times for 5 min each. Incubate with a 1:2000 dilution of anti-His antibody at 4°C overnight. After washing with TBST, develop the color using BeyoECL Star and capture the bands using a chemiluminescence image analysis system.

[0067] As shown in FIG2 , the molecular weight of the mesothelin targeted binding protein 1 is about 18 kDa, and the purity of the obtained targeted binding protein after purification is greater than 90%.

[0068] Example 3: Binding of the dominant mesothelin-targeting binding protein on the surface of N87 and Huh7 cells

[0069] 1. In combination with N87 and Huh7 cells:

[0070] The mesothelin-positive gastric cancer cell line N87 and the mesothelin-negative liver cancer cell line Huh7 were plated at an appropriate density in a cell culture confocal microplate; blocked with 1% BSA and fixed with 4% paraformaldehyde, and incubated with the dominant mesothelin target binding protein #1 (MBP) at 4°C for 12 hours. The unbound protein was washed away, and anti-His fluorescent secondary antibody was added and incubated for another 1-2 hours. After washing away the secondary antibody, DAPI dye was used to stain the cell nuclei.

[0071] 2. Observe the fluorescence of cells under a confocal microscope:

[0072] As shown in FIG3 , the dominant mesothelin targeting binding protein #1 (MBP) significantly binds to N87 cells expressing positive mesothelin, but does not bind to Huh7 cells expressing negative mesothelin.

[0073] Example 4: Biodistribution of mesothelin-specific fluorescent probe in mice

[0074] 1. Preparation of fluorescent probes for binding proteins:

[0075] The target binding protein #1 was obtained by prokaryotic expression and reacted with CY5-NHS cyanine dye at room temperature for 12 hours to obtain the target binding protein fluorescent probe;

[0076] 2. Preparation of tumor-bearing mice:

[0077] N87 cells were cultured until adherent, and 4-week-old nude mice were selected and inoculated subcutaneously in the groin of the nude mice for 10 6 The cells were grown and observed for several days until the tumor grew into a mass.

[0078] 3. Injection of mesothelin-targeted binding protein fluorescent probe into tumor-bearing mice:

[0079] The mesothelin-targeting binding protein fluorescent probe was injected into tumor-bearing mice via tail vein injection.

[0080] 4. Optical imaging measurement:

[0081] Mice were anesthetized 8 and 24 hours after injection and placed in an IVIS small animal in vivo optical imaging system for imaging.

[0082] As shown in the imaging in Figure 4, it can be seen that the targeted binding protein fluorescent probe can be effectively enriched specifically in the tumor site, demonstrating its excellent potential as a clinical diagnostic and therapeutic drug.

[0083] Example 5: Mesothelin Therapeutic Experiment

[0084] By preparing mesothelin-targeted fusion protein SEQ ID No. 5 and SEQ ID No. 7 drug conjugates, the targeting binding ability and cell killing ability of the protein-drug conjugates were fully verified in vitro and in vivo experiments on mesothelin-positive tumor cells.

[0085] Among them, SEQ ID No. 5 is a mesothelin-targeted fusion protein composed of SEQ ID No. 3 at the N-terminus, an albumin-binding domain at the C-terminus, and a linker containing four cysteine ​​residues; SEQ ID No. 6 is a mesothelin-targeted fusion protein composed of SEQ ID No. 1 at the N-terminus, an albumin-binding domain at the C-terminus, and a linker containing four cysteine ​​residues; SEQ ID No. 7 is a mesothelin-targeted fusion protein composed of SEQ ID No. 3 and a G7 (GNNNQNY or GNNQQNY) heptapeptide sequence at the N-terminus, an albumin-binding domain at the C-terminus, and a linker containing four cysteine ​​residues; SEQ ID No. 8 is a mesothelin-targeted fusion protein composed of SEQ ID No. 1 and G7 at the N-terminus, an albumin-binding domain at the C-terminus, and a linker containing four cysteine ​​residues; SEQ ID No. 9 is a mesothelin-targeted fusion protein composed of SEQ ID No. 3 and an albumin-binding domain at the N-terminus, G7 at the C-terminus, and a linker containing four cysteine ​​residues; SEQ ID No. 10 is a mesothelin-targeted fusion protein composed of SEQ ID No. No.1, a mesothelin-targeting fusion protein consisting of an albumin-binding domain, G7 at the C-terminus, and a linker containing four cysteine ​​residues.

[0086] Taking SEQ ID No. 3 as an example, three mesothelin-targeting fusion proteins, SEQ ID No. 5, SEQ ID No. 7, and SEQ ID No. 9, constructed based on SEQ ID No. 3, were subjected to in vitro molecular and cellular binding validation and in vivo targeting validation prior to conjugation with MMAE toxin molecules. Surface plasmon resonance (SPR) experiments demonstrated that SEQ ID No. 5, SEQ ID No. 7, and SEQ ID No. 9 bind to mesothelin with high affinity, reaching nanomolar levels, indicating that the introduction of ABD and G7 does not negatively impact the mesothelin binding ability of SEQ ID No. 3. Furthermore, we demonstrated that the introduction of G7 further enhances the binding affinity of the mesothelin-targeting fusion proteins to mesothelin molecules, as demonstrated by higher KD values ​​for SEQ ID No. 7 and SEQ ID No. 9 than for SEQ ID No. 5, with the best affinity observed when G7 is placed between SEQ ID No. 3 and the albumin binding domain (Figure 5). We then selected SEQ ID No. 5 and SEQ ID No. 7 containing G7 for further cell binding experiments. Through cell immunofluorescence experiments, we demonstrated that both SEQ ID No. 5 and SEQ ID No. 7 could bind to mesothelin-positive tumor cells ( FIG. 6 ).

[0087] Biodistribution of mesothelin-targeted fusion protein in mice

[0088] SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 were obtained by prokaryotic expression and reacted with CY5-NHS cyanine dye at room temperature for 12 h to obtain a targeted binding protein fluorescent probe. The specific experimental method is shown in Example 4.

[0089] The experimental results demonstrated that the mesothelin-targeting binding protein could bind to mesothelin in MSLN-positive tumor-bearing mice and that the corresponding probe could be used for imaging the mesothelin-positive gastric cancer cell line N87 ( FIG. 7 ).

[0090] Cancer cell inhibition experiment

[0091] After co-incubation of different concentrations of mesothelin-targeted protein-drug conjugates with mesothelin-positive gastric cancer cells MKN45, the mesothelin-targeted protein-drug conjugates showed significant killing effects on MKN45 cells within the nanomolar concentration range ( FIG. 8 ).

[0092] In vivo tumor inhibition experiments in animals

[0093] 3×10 6MKN45 tumor cells were inoculated into the left groin of BALB / c nude mice and the tumor-bearing mice were randomly divided into 6 groups: NS group, isotype control group Isotype-MMAE, naked protein group SEQ ID No.5 and SEQ ID No.7, and protein-coupled drug group SEQ ID No.5-MMAE and SEQ ID No.7-MMAE. When the average size of the tumor reached 100-150 mm 3 Treatment was started at 14 days after tumor inoculation by injecting the above drugs into the tumor-bearing mice through the tail vein at a dose of 2.5 mg / kg, and the drugs were administered twice, respectively, on the 10th and 14th days after tumor inoculation.

[0094] 5×10 6 PANC1 tumor cells were inoculated into the left groin of BALB / c nude mice and the tumor-bearing mice were randomly divided into 4 groups: NS group, isotype control group (Isotype-MMAE), and protein-coupled drug group (SEQ ID No.5-MMAE and SEQ ID No.7-MMAE). When the average tumor size reached 100-150 mm 3 Treatment was started at 14:00 pm. The above drugs were injected into tumor-bearing mice via the tail vein at a dose of 2.5 mg / kg, and were administered twice on the 7th and 11th days after tumor inoculation.

[0095] The efficacy of the mesothelin-targeted protein-drug conjugates was further evaluated in MKN45 gastric cancer-bearing mice and PANC1 pancreatic cancer-bearing mice ( Figures 9 and 10 ). The results showed that the mesothelin-targeted protein-drug conjugates based on SEQ ID No. 5 and SEQ ID No. 7 could significantly inhibit the growth of mesothelin-positive tumors.

[0096] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent replacement and improvement made by any technician familiar with the profession to the above embodiment without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

[0097]

[0098]

[0099]

Claims

1. A mesothelin targeting binding protein, characterized in that: Its amino acid sequence is shown in one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.

4. The mesothelin targeting binding protein can specifically bind to the tumor molecular marker mesothelin.

2. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the mesothelin targeting binding protein according to claim 1.

3. A nuclear probe, characterized in that: A radionuclide-labeled probe prepared using the mesothelin targeting binding protein described in claim 1.

4. A fluorescent probe, characterized in that: A fluorescent probe is obtained by covalently coupling the mesothelin targeting binding protein according to claim 1 with a fluorescent dye.

5. A protein-drug conjugate, characterized in that: A fusion protein is formed by the mesothelin targeting binding protein according to claim 1 and an albumin binding domain capable of binding to human serum albumin, and four MMAE toxin molecules are site-specifically coupled via cysteine ​​residues to form a mesothelin targeting protein drug conjugate; its amino acid sequence is shown in one of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, and SEQ ID No.

10.

6. A kit, characterized in that: A tumor diagnostic kit containing the mesothelin targeting binding protein according to claim 1.

7. Use of the mesothelin targeted binding protein according to claim 1 in the preparation of tumor targeted drugs.

8. The use according to claim 7, characterized in that: The tumor includes solid cancer; preferably, the solid cancer includes gastric cancer and pancreatic cancer.

9. The use according to claim 7, characterized in that: The tumor targeting drug is an immune cell therapy drug.

10. The use according to claim 7, characterized in that: The tumor-targeted drug is a tumor-targeted cytokine therapeutic drug.

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