A biomarker for cancer diagnosis and its application

Abstract

The present invention discloses a monoclonal antibody, which can specifically bind to both KPNA4 protein and RAB10 protein; the monoclonal antibody includes monoclonal antibody 1 and monoclonal antibody 2, wherein the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4, and the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6. The present invention also discloses a biomarker combination for diagnosis and treatment of pancreatic cancer, the biomarker combination includes KPNA4 and RAB10, and the monoclonal antibody 1 and the monoclonal antibody 2 that specifically bind to KPNA4 protein and RAB10 protein. The monoclonal antibody of the present invention has good specificity and sensitivity, and can be used for diagnosis and treatment of pancreatic cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biomarker for cancer diagnosis and treatment and an application thereof. Background Art

[0002] Pancreatic cancer is a common pancreatic tumor with a high degree of malignancy, which is very difficult to diagnose and treat. About 90% of pancreatic cancers originate from ductal adenocarcinoma of the glandular duct epithelium. The incidence and mortality of pancreatic cancer have increased significantly in recent years, and the 5-year survival rate is less than 1%, making it one of the malignant tumors with the worst prognosis. Symptoms of pancreatic cancer include upper abdominal pain that can radiate to the back and worsen after eating, weight loss, diarrhea, yellowing of the skin and sclera, etc.

[0003] Currently, pancreatic cancer is mainly diagnosed through imaging examinations, such as CT scans, MRI scans, endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasound (EUS). These examination methods are slow, costly and harmful to the human body. Most pancreatic cancer patients are already in the advanced stage when diagnosed, so only 15% to 20% of pancreatic cancer patients can undergo surgery. After surgery, further treatment is mainly through chemotherapy. There is currently no drug that actively targets pancreatic cancer.

[0004] Therefore, exploring the pathogenesis of pancreatic cancer and finding biomarkers for diagnosis are of great significance for optimizing clinical treatment and improving patient prognosis. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, one of the purposes of the present invention is to provide a biomarker for the diagnosis and treatment of pancreatic cancer; a second purpose of the present invention is to provide an application of the biomarker for the diagnosis and treatment of pancreatic cancer in the diagnosis and treatment of pancreatic cancer.

[0006] Therefore, on one hand, the present invention discloses a monoclonal antibody, which can specifically bind to both KPNA4 protein and RAB10 protein; the monoclonal antibody that can specifically bind to both KPNA4 protein and RAB10 protein includes monoclonal antibody 1 and monoclonal antibody 2, wherein the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4, and the sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0007] Preferably, the monoclonal antibody 1 of the present invention specifically binds to the KPNA4 protein at aa371 to aa388, and its amino acid sequence is MIIHLLDKGDFGTQKEAA.

[0008] Preferably, the monoclonal antibody 1 of the present invention specifically binds to the RAB10 protein at aa8 to aa25, and its amino acid sequence is LLFKLLLIGDSGVGKTCV.

[0009] Preferably, the monoclonal antibody 2 of the present invention specifically binds to the KPNA4 protein at aa461-aa476, and its amino acid sequence is IEQLQNHENEDIYKLA.

[0010] Preferably, the monoclonal antibody 2 of the present invention specifically binds to the RAB10 protein at aa104 to aa119, and its amino acid sequence is LRNIDEHANEDVERML.

[0011] In another aspect, the present invention further discloses a biomarker combination for diagnosis and treatment of pancreatic cancer, wherein the biomarker combination comprises KPNA4 and RAB10, and monoclonal antibody 1 and monoclonal antibody 2 that can specifically bind to both KPNA4 protein and RAB10 protein.

[0012] In another aspect, the present invention also discloses the use of the monoclonal antibody 1 and the monoclonal antibody 2 in the preparation of drugs for treating pancreatic cancer.

[0013] In another aspect, the present invention also discloses the use of the monoclonal antibody 1 and the monoclonal antibody 2 in the preparation of diagnostic reagents for pancreatic cancer.

[0014] Based on the previous work, the inventors screened two good biomarkers for the diagnosis and treatment of pancreatic cancer from a large number of genes and proteins related to pancreatic cancer through molecular biology technology and bioinformatics technology, namely, the markers KPNA4 and RAB10. At the same time, the inventors compared and studied the sequences of KPNA4 and RAB10 in depth, and screened two monoclonal antibodies that can specifically bind to both KPNA4 protein and RAB10 protein; and on this basis, the inventors preliminarily studied the targeted therapeutic effect of the monoclonal antibodies on pancreatic cancer, and the results showed that the two monoclonal antibodies prepared by the present invention have good targeted therapeutic effects on pancreatic cancer.

[0015] It should be noted that the KPNA4 and RAB10 screened by the present invention can not only be used for the diagnosis of pancreatic cancer (such as using the PCR method to detect the expression of KPNA4 and RAB10, or using specific antibodies to perform western blot detection on the expression of KPNA4 and RAB10 proteins), but can also be used for targeted therapy of pancreatic cancer (as shown in Example 3 of the present invention).

[0016] It should be noted that the monoclonal antibody 1 and monoclonal antibody 2 of the present invention can also be used for the diagnosis of pancreatic cancer, such as using a monoclonal antibody that specifically binds to the KPNA4 protein as a capture antibody and the monoclonal antibody 1 or monoclonal antibody 2 of the present invention as a detection antibody to establish a double antibody sandwich method, thereby achieving quantitative or qualitative detection of the KPNA4 protein. This methodology is also applicable to the quantitative and qualitative detection of the RAB10 protein by monoclonal antibody 1 or monoclonal antibody 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Detection results of mRNA expression levels of KPNA4 and RAB10 in pancreatic cancer.

[0018] Figure 2 Amino acid sequence alignment results of KPNA4 protein and RAB10 protein.

[0019] Figure 3 Western blot detection results of monoclonal antibody 1 and monoclonal antibody 2 for KPNA4 protein and RAB10 protein, respectively.

[0020] Figure 4 Pancreatic cancer treatment outcomes in mice. DETAILED DESCRIPTION

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0022] "Biomarker" and "marker" are equivalent and interchangeable, referring to molecular indicators with specific biological properties, biochemical characteristics or aspects, which can be used to determine the presence or absence of a specific disease or condition and / or the severity of a specific disease or condition. In the present invention, "marker" refers to a parameter associated with one or more biological molecules (i.e., "biomarker"), such as a nucleic acid or protein produced naturally or artificially. "Marker" in the present invention also includes a single parameter that can be calculated or otherwise obtained by considering expression data from two or more different markers. In the present invention, the term "biomarker" refers to a gene, a fragment of a gene, or a protein, a protein fragment associated with pancreatic cancer.

[0023] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0024] Example 1: Analysis and screening of biomarkers in pancreatic cancer

[0025] Through literature search, it was found that there are many biomarkers related to pancreatic cancer, as many as hundreds, and it is difficult to screen out the best pancreatic cancer-related biomarkers.

[0026] Through years of basic research and continuous experimental verification, the researchers have detected the expression levels of pancreatic cancer-related genes and miRNAs through bioinformatics and molecular biology techniques, discovered genes or fragments with significant expression differences, and explored their relationship with the occurrence and development of pancreatic cancer, thereby finding better ways and methods for the detection and targeted treatment of pancreatic cancer. Through screening, the present invention found that KPNA4 (Karyopherin alpha) and RAB10 (Ras-related protein Rab-10) were significantly upregulated in pancreatic cancer (such as Figure 1 As shown), and a large number of experiments have proved that the use of this gene and protein has a high positive detection rate; further cell experiments have shown that changing the expression levels of KPNA4 and RAB10 in pancreatic cancer cells can affect the growth of pancreatic cancer cells, suggesting that KPNA4 and RAB10 can be used as biomarkers for pancreatic cancer for targeted detection and treatment.

[0027] Example 2: Testing of the preparation of KPNA4 and RAB10 monoclonal antibodies

[0028] First, we analyzed the amino acid sequences of KPNA4 (as shown in SEQ ID NO.1) and RAB10 (as shown in SEQ ID NO.2), and the sequence alignment results are shown in FIG. Figure 2 As shown, antigen epitope polypeptides are selected from them for the preparation of monoclonal antibodies, and the antigen epitope polypeptides are shown in Table 1.

[0029] Table 1 Antigenic epitope peptides

[0030]

[0031] The two antigen epitope polypeptides in Table 1 were synthesized respectively, and mice were immunized after emulsification respectively, and monoclonal antibodies were prepared respectively by conventional hybridoma cell technology.

[0032] After preparation, detection and analysis, the sequences of the heavy chain variable region and the light chain variable region of a monoclonal antibody (monoclonal antibody 1) screened after immunization with antigenic epitope polypeptide 1 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively; the sequences of the heavy chain variable region and the light chain variable region of a monoclonal antibody (monoclonal antibody 2) screened after immunization with antigenic epitope polypeptide 2 are shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0033] The two prepared monoclonal antibodies were used to perform western blot detection on recombinant KPNA4 protein (purchased from Huamei Biotechnology, CSB-YP012486HU) and recombinant RAB10 protein (purchased from Huamei Biotechnology, CSB-YP019152HU), and the results showed that ( Figure 3 ), these two monoclonal antibodies have specific binding to recombinant KPNA4 protein and recombinant RAB10 protein (although the two monoclonal antibodies have different binding abilities to the two proteins, it does not hinder the specific binding of the two monoclonal antibodies to the two proteins), indicating that these two monoclonal antibodies can be used as monoclonal antibodies for diagnosis or targeted treatment of KPNA4 protein and RAB10 protein.

[0034] It should be emphasized that the present invention directly discloses the two antigen epitope polypeptides and the monoclonal antibodies 1 and 2 prepared therefrom from the perspective of practical application. However, in the actual research process, the inventors used a large amount of experimental design and experimental verification as well as intellectual and creative labor to screen out the two relatively good antigen epitope polypeptides and the monoclonal antibodies prepared therefrom. Therefore, those skilled in the art cannot obtain these results based solely on existing technologies.

[0035] Example 3: Treatment of pancreatic cancer

[0036] The monoclonal antibody 1 and monoclonal antibody 2 prepared above are used to treat KPNA4 protein and RAB10 protein. The therapeutic drugs of conventional ADC drugs such as bleomycin are chemically coupled to monoclonal antibody 1 and monoclonal antibody 2 to prepare ADC drugs targeting KPNA4 protein and RAB10 protein.

[0037] Quickly take out Panc02 cells from the liquid nitrogen tank, wipe the bottle, quickly place the cell cryopreservation tube in a 37℃ water bath, shake the cryopreservation tube constantly to melt it as soon as possible, and observe the dissolution of the cell fluid. When the cells in the cryopreservation tube are observed to be completely dissolved, quickly take out the cryopreservation tube, wipe the outside of the cryopreservation tube with 75% alcohol, move the cryopreservation tube to the ultra-clean cell workbench, gently unscrew the cryopreservation tube cap, use a sterile pipette to blow and mix the cell suspension, use a pipette to transfer the cell suspension in the cryopreservation tube into a 15ml centrifuge tube, add PBS buffer to 10ml, and gently mix the cell suspension again with a pipette. After tightening the centrifuge tube cap, place the centrifuge tube in a centrifuge, balance it, and centrifuge it at 1000rpm / min for 5 minutes, discard the supernatant, and retain the cell pellet. Spray alcohol on the bottle, move it to the ultra-clean cell station, gently unscrew the centrifuge tube cap, carefully aspirate the cell supernatant with a sterile pipette to avoid dispersing the cell pellet, and finally add DMEM culture medium to the cell pellet to remix the cells. Use a pipette to transfer the cell suspension into the culture bottle, mark the name and date, and place the bottle in a cell culture incubator for continued culture after alcohol disinfection. Continue to expand the culture for several generations before use.

[0038] Ten SPF male C57BL / 6 mice, 10 weeks old, weighing 24-26g, were selected after 3 days of adaptive feeding. After disinfecting the abdominal skin of the mice with 75% ethanol, a 1ml sterile syringe was used to draw the prepared Panc02 cell suspension, the gas in the syringe was emptied, the skin on the back of the mice was pinched, the mice were fixed in a supine position, the left lower abdomen of the mice was taken, the needle was inserted obliquely, and 0.2ml of Panc02 cell suspension was injected into the abdominal cavity of each mouse after a breakthrough feeling (the number of injected cells was about 5×10 6 After the inoculation, the activity of the mice was observed and sufficient water and feed were given. The mice were used for this experiment after tumors were formed.

[0039] 40 mice with pancreatic tumors (all tumors of the same size) were selected. The mice were randomly divided into 4 groups, 10 in each group. During the feeding process, monoclonal antibody 1-bleomycin, monoclonal antibody 2-bleomycin, and bleomycin were injected into the mice at 1 mg / kg by tail vein injection. One group of injections was used as the control group. The mice were observed for 4 weeks after injection. After 4 weeks, the mice were euthanized and the tumor weight was measured.

[0040] The results show that Figure 4 ), monoclonal antibody 1-bleomycin, monoclonal antibody 2-bleomycin, and bleomycin groups were better than the control group, and monoclonal antibody 1-bleomycin and monoclonal antibody 2-bleomycin groups were better than the bleomycin group. This shows that the two monoclonal antibodies prepared by the present invention can be used for targeted treatment of pancreatic cancer and have good therapeutic effects.

[0041] In addition, it should be noted that the present invention conducted the same treatment study on the monoclonal antibodies of KPNA4 and RAB10 on the market and found that their treatment effects were not as good as those of monoclonal antibody 1 and monoclonal antibody 2 prepared by the present invention. The reason for this may be that the monoclonal antibodies on the market can only target one protein.

[0042] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody can specifically bind to both KPNA4 protein and RAB10 protein; the monoclonal antibody is selected from monoclonal antibody 1 or monoclonal antibody 2; the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody 1 specifically binds to the KPNA4 protein at aa371-aa388, and its amino acid sequence is MIIHLLDKGDFGTQKEAA.

3. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody 1 specifically binds to the RAB10 protein at aa8-aa25, and its amino acid sequence is LLFKLLLIGDSGVGKTCV.

4. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody 2 specifically binds to the KPNA4 protein at aa461-aa476, and its amino acid sequence is IEQLQNHENEDIYKLA.

5. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody 2 specifically binds to the RAB10 protein at aa104-aa119, and its amino acid sequence is LRNIDEHANEDVERML.

6. Use of the monoclonal antibody 1 and the monoclonal antibody 2 according to claim 1 in the preparation of a drug for treating pancreatic cancer.

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