Use of polypeptides in the diagnosis or prognosis of lung cancer

Abstract

The application relates to the technical field of biological medicine, and particularly provides application of a polypeptide in diagnosis or prognosis evaluation of lung cancer, and experiment proves that the polypeptide fragment selected in the application can be used for specific diagnosis of lung cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of peptides in the diagnosis or prognostic assessment of lung cancer. Background Technology

[0002] P53 (Ensembl:ENSG00000141510 MIM:191170) is encoded by the TP53 gene on chromosome 17 and is widely expressed in the spleen (RPKM 13.1), lymph nodes (RPKM 13.0), and 25 other tissues. P53 protein is a tumor suppressor and transcription factor, participating in cell cycle regulation and negatively regulating cell division to suppress tumors. In human cancers, p53 is the most frequently mutated gene, present in approximately 50% of human cancers. Mutated p53 protein loses its tumor-suppressive function. Highly expressed mutant p53 protein triggers an immune response, producing p53 autoantibodies. Serum p53 autoantibodies can serve as potential biomarkers for tumor diagnosis. Currently, antigen proteins are commonly used to detect autoantibodies, but these are generally difficult to obtain, and the positive rate of autoantibodies for each type of tumor is low, limiting their clinical value. Therefore, this application uses peptides as antigens. Peptide-mimicking protein antigens can bind to autoantibodies and are used for the detection of autoantibodies. They have the advantages of high throughput, multiple indicators, and miniaturization, and have high specificity and sensitivity in tumor detection. Summary of the Invention

[0003] In a first aspect, the present invention provides the use of a polypeptide in the preparation of products for diagnosing or assisting in the diagnosis or prognostic assessment of lung cancer, said polypeptide comprising SEQ ID NO: 1 or 2.

[0004] A second aspect of the invention provides the use of an autoantibody bound to a polypeptide as a biomarker in the preparation of products for the diagnosis or auxiliary diagnosis or prognostic assessment of lung cancer, said polypeptide comprising SEQ ID NO: 1 or 2.

[0005] Preferably, the length of the polypeptide is 15-30 aa, more preferably 15-20 aa. For example, it can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 aa.

[0006] Preferably, the polypeptide is SEQ ID NO: 1 or 2. Preferably, the polypeptide may contain modifications or labels, such as biotin.

[0007] Preferably, the diagnosis or auxiliary diagnosis or prognostic assessment of lung cancer includes detecting autoantibodies using peptides. More preferably, it involves detecting the presence or level of autoantibodies. More preferably, it involves detecting autoantibodies in serum or plasma.

[0008] Preferably, the autoantibody is IgG and / or IgA.

[0009] Preferably, the subject of the diagnosis, auxiliary diagnosis, or prognostic assessment can be a human or a non-human animal.

[0010] Preferably, the diagnosis or auxiliary diagnosis or prognostic assessment of lung cancer includes contacting the peptide with the sample to be tested and detecting the amount of autoantibodies binding to the peptide.

[0011] Specifically, the peptide is coated onto a solid-phase support, then the sample to be tested and a label (preferably a secondary antibody labeled with anti-human IgA cyanine dye (Cy5) or anti-human IgG cyanine dye (Cy3)) are added, and the signal molecule intensity of the label is detected. The detection methods for the signal molecule intensity include visible light colorimetry, chemiluminescence, and fluorescence luminescence. For example, detection can be performed using a microarray scanner. The signal strength is positively correlated with the amount of autoantibody bound to the peptide.

[0012] Preferably, the sample to be tested is serum or plasma.

[0013] Preferably, the product comprises a polypeptide and a solid-phase carrier;

[0014] Preferably, the solid support is selected from one or more combinations of enzyme-labeled microplates, microparticles, microspheres, affinity membranes, liquid phase chips, glass slides, test strips and plastic balls;

[0015] More preferably, the product is a reagent kit or a chip.

[0016] Preferably, the product further comprises reagents, such as labeled secondary antibodies, blocking buffers, and / or washing buffers.

[0017] Preferably, the lung cancer includes one or more of the following: lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, or small cell lung cancer.

[0018] In a third aspect, the present invention provides a polypeptide comprising SEQ ID NO: 1 or 2.

[0019] Preferably, the polypeptide is SEQ ID NO: 1 or 2.

[0020] Preferably, the polypeptide may contain modifications or labels, such as biotin.

[0021] Preferably, the polypeptide can bind to autoantibodies. More preferably, it binds to autoantibodies found in the serum or plasma of lung cancer patients.

[0022] Preferably, the polypeptide can be used to detect autoantibodies.

[0023] Preferably, the polypeptide can be used for the diagnosis or auxiliary diagnosis or prognostic assessment of lung cancer.

[0024] Preferably, the peptide or its binding autoantibody can serve as a biomarker for lung cancer diagnosis, auxiliary diagnosis, or prognostic assessment. For example, detecting the presence or level of binding autoantibodies can evaluate lung cancer. This evaluation may include, for example, diagnosis, auxiliary diagnosis, prognostic assessment, or clinical classification.

[0025] The polypeptide can be prepared using conventional methods available in the art, such as chemical synthesis or prokaryotic or eukaryotic expression.

[0026] In a fourth aspect, the present invention provides a nucleic acid encoding the aforementioned polypeptide.

[0027] In a fifth aspect, the present invention provides a vector comprising the above-described nucleic acid.

[0028] In a sixth aspect, the present invention provides a cell comprising the above-described nucleic acid or the above-described carrier.

[0029] In a seventh aspect, the present invention provides a reagent kit or chip comprising the aforementioned polypeptide, nucleic acid, carrier, or cell.

[0030] Preferably, the polypeptide contains a modification or label, such as biotin.

[0031] Preferably, the kit or chip further comprises a solid-phase support.

[0032] Preferably, the solid-phase carrier is selected from one or more combinations of enzyme-labeled microplates, microparticles, microspheres, affinity membranes, liquid phase chips, glass slides, test strips, and plastic balls.

[0033] Preferably, the kit or chip further includes reagents, such as labeled secondary antibodies, blocking buffers, and / or washing buffers.

[0034] An eighth aspect of the present invention provides a method for preparing a reagent kit or chip, the method comprising coating the above-mentioned polypeptide onto a solid support.

[0035] Preferably, the coating can be a direct coating method or an indirect coating method. The direct coating method involves directly immobilizing the polypeptide onto a solid support, while the indirect coating method involves first coupling the polypeptide with an unrelated protein such as bovine serum albumin (BSA), and then indirectly binding it to the surface of the solid support through the adsorption of the conjugate.

[0036] In a ninth aspect, the present invention provides a biomarker for lung cancer, wherein the biomarker for lung cancer is a polypeptide or an autoantibody that binds to the aforementioned polypeptide.

[0037] Preferably, the autoantibody is IgG and / or IgA.

[0038] Preferably, the autoantibody is derived from serum or plasma.

[0039] In a tenth aspect, the present invention provides a method for detecting autoantibodies, the method comprising using the above-described polypeptide or the above-described kit or chip.

[0040] Preferably, the method involves contacting the peptide with the sample to be tested and detecting the amount of autoantibodies bound to the peptide.

[0041] Specifically, the peptide is coated onto a solid-phase support, then the sample to be tested and a label (preferably a secondary antibody labeled with anti-human IgA cyanine dye (Cy5) or anti-human IgG cyanine dye (Cy3)) are added, and the signal molecule intensity of the label is detected. The detection methods for the signal molecule intensity include visible light colorimetry, chemiluminescence, and fluorescence luminescence. For example, detection can be performed using a microarray scanner. The signal strength is positively correlated with the amount of autoantibody bound to the peptide.

[0042] Preferably, the sample to be tested is serum or plasma.

[0043] In an eleventh aspect, the present invention provides a method for diagnosing lung cancer, the method comprising using the aforementioned polypeptide or the aforementioned reagent kit or chip.

[0044] Preferably, the method involves contacting the peptide with the sample to be tested and detecting the amount of autoantibodies bound to the peptide.

[0045] Specifically, the peptide is coated onto a solid-phase support, then the sample to be tested and a label (preferably a secondary antibody labeled with anti-human IgA cyanine dye (Cy5) or anti-human IgG cyanine dye (Cy3)) are added, and the signal molecule intensity of the label is detected. The detection methods for the signal molecule intensity include visible light colorimetry, chemiluminescence, and fluorescence luminescence. For example, detection can be performed using a microarray scanner. The signal strength is positively correlated with the amount of autoantibody bound to the peptide.

[0046] Preferably, the sample to be tested is serum or plasma.

[0047] The "diagnosis" described in this invention refers to determining whether a patient has had a disease or symptom in the past, at the time of diagnosis, or in the future, or determining the progression or potential future progression of a disease. The object of the diagnosis can be a human or a non-human animal, preferably a non-human mammal. More preferably, the non-human mammal can be a wild animal, zoo animal, commercially available animal, pet, laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys, etc.

[0048] The "assisted diagnosis" mentioned in this invention refers to the methods of this application as well as conventional methods or steps in the prior art.

[0049] The "prognostic assessment" described in this invention refers to assessing a patient's response to treatment and the risk of developing the disease in the future.

[0050] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence. Attached Figure Description

[0051] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0052] Figure 1 The expression level of IgG type autoantibodies binding to peptide P073 in plasma, where SNR is the signal-to-noise ratio, N is healthy individuals, LC is lung cancer patients, EC is esophageal cancer patients, GC is gastric cancer patients, and DC is patients with the other 7 types of cancer.

[0053] Figure 2 The expression level of IgA type autoantibodies binding to peptide P073 in plasma, where SNR is the signal-to-noise ratio, N is healthy individuals, LC is lung cancer patients, EC is esophageal cancer patients, GC is gastric cancer patients, and DC is patients with the other 7 types of cancer.

[0054] Figure 3 The expression level of IgG type autoantibodies binding to peptide P076 in plasma, where SNR is the signal-to-noise ratio, N is healthy individuals, LC is lung cancer patients, EC is esophageal cancer patients, GC is gastric cancer patients, and DC is patients with the other 7 types of cancer.

[0055] Figure 4The expression level of IgA type autoantibodies binding to peptide P076 in plasma, where SNR is the signal-to-noise ratio, N is healthy individuals, LC is lung cancer patients, EC is esophageal cancer patients, GC is gastric cancer patients, and DC is patients with the other 7 types of cancer.

[0056] Figure 5 ROC curves of IgG autoantibodies against peptide P076 in lung cancer patients and healthy groups.

[0057] Figure 6 ROC curves of IgA autoantibodies against peptide P076 in lung cancer patients and healthy groups. Detailed Implementation

[0058] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] The reagent formulations used in the examples are as follows:

[0060] Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4.

[0061] Washing buffer (PBST): 1×PBST at pH 7.4.

[0062] Blocking buffer: 1×PBST containing 5% BSA.

[0063] Example 1

[0064] 1. The designed polypeptide sequences are shown in Table 1.

[0065] Table 1: Peptide Sequences

[0066] serial number SEQ ID NO: x P53 polypeptide sequence Location information P073 1 GSRAHSSHLKSKKGQ 361-375 p076 2 STSRHKKLMFKTEGP 376-390

[0067] 2. Clinical samples are shown in Table 2.

[0068] Table 2: Clinical Samples

[0069]

[0070] Table 3: Patient information for lung adenocarcinoma samples

[0071]

[0072]

[0073] Table 4: Patient information for lung squamous cell carcinoma samples

[0074]

[0075]

[0076] Table 5: Patient information for large cell lung cancer samples

[0077]

[0078] Table 6: Patient information for small cell lung cancer samples

[0079]

[0080]

[0081] Table 7: Patient Information from Normal Physical Examination Samples

[0082]

[0083]

[0084] 3. Testing Steps

[0085] 1) P53 peptide array printing: Dilute the P53 peptides in Table 1 with PBS, and then use the Arrayjet microarray spotter to print the P53 peptides onto the surface of the glass slide four times. The prepared P53 peptide microarrays are stored at -20℃ until ready for use.

[0086] 2) Rewarming: Remove the P53 polypeptide slide from the -20℃ freezer and allow it to equilibrate to room temperature;

[0087] 3) Blocking: Assemble the P53 peptide slide into the incubation tray, add 400 μL of blocking buffer, incubate at room temperature for 1 h, and gently shake.

[0088] 4) Serum sample preparation: Thaw serum samples at 4℃ and centrifuge at 14,000×g for 10 min. Add 4 μL of serum to 400 μL of blocking buffer at a ratio of 1:100 to prepare serum samples;

[0089] 5) Add serum sample: Remove blocking buffer, add 400 μL of diluted serum, and incubate at room temperature for 2.5 h, gently shaking; after incubation, wash the slide three times with 400 μL PBST, 10 minutes each time;

[0090] 6) Add fluorescently labeled antibody: Dilute the secondary antibody (working concentration 4 μg / mL) with blocking buffer. Add 400 μL of diluted secondary antibody and incubate at room temperature for 1 hour, gently shaking. Wash the slide three times with 400 μL PBST for 10 minutes each time, then wash the slide twice with 400 μL distilled water for 2 minutes each time.

[0091] 7) Dry the slides: Remove any remaining liquid, empty the incubation tray using a vacuum pump or pipette, and then dry the slides;

[0092] 8) Reading data: Scan all arrays at 532nm or 635nm wavelength using a GenePix 4300A microarray scanner. Extract the median fluorescence signal intensity using GenePix Pro7 software and save the data.

[0093] 4. Test Results

[0094] The results of autoantibody expression levels of P073 and P076 peptides in Table 1 are shown below. Figure 1-4 See Table 8. Specifically, autoantibodies binding to peptides showed statistically significant differences between lung cancer patients and healthy controls, indicating that they can serve as specific biomarkers for lung cancer.

[0095] Table 8: Significance of expression levels of several exemplary peptides in different cancers

[0096]

[0097] In addition, the results of autoantibody ROC analysis of the binding peptides in lung cancer patients and healthy groups are shown below. Figure 5-6 Specifically:

[0098] For IgG autoantibodies, the AUC of peptide P076 is 0.7177. When the cutoff value of peptide P076, determined by the Youden index, is 1.211, the specificity is 73.68% and the sensitivity is 63.64%.

[0099] For IgA-type autoantibodies, the AUC of peptide P076 is 0.6525. When the cutoff value of peptide P076, determined by the Youden index, is 1.714, the specificity is 57.89% and the sensitivity is 76.14%.

[0100] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0101] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The application of peptides in the preparation of products for the diagnosis or prognostic assessment of lung cancer, characterized in that, The polypeptide is SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The lung cancer mentioned includes one or more of the following: lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, or small cell lung cancer.

3. The application according to claim 1, characterized in that, The diagnosis or prognostic assessment of lung cancer includes the use of peptides to detect autoantibodies.

4. The application according to claim 3, characterized in that, The detection of autoantibodies refers to the detection of the presence or content of autoantibodies.

5. The application according to claim 3, characterized in that, The detection of autoantibodies refers to the detection of autoantibodies in serum or plasma.

6. The application according to claim 3, characterized in that, The autoantibodies mentioned are IgG and / or IgA.

7. The application according to claim 1, characterized in that, The products include peptides and solid-phase carriers.

8. The application according to claim 7, characterized in that, The solid-phase carrier is selected from one or more combinations of enzyme-labeled microplates, microparticles, microspheres, affinity membranes, liquid phase chips, glass slides, test strips, and plastic balls.

9. The application according to claim 1, characterized in that, The product in question is a reagent kit or a chip.

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