Methods and kits for diagnosis and treatment of RDAA-positive diseases

Abstract

The present disclosure provides a method for diagnosing and / or treating a disease, which comprises at least the following steps: detecting the anaplastic lymphoma kinase (ALK) gene rearrangement, the anaplastic lymphoma kinase phosphorylation level and the RNase1 expression level of a subject; if the detection result of the ALK gene rearrangement is negative, and the ALK phosphorylation level and the RNase1 expression level are higher than the levels of a reference population, defining the disease as a disease caused by RNase1-driven ALK activation (RDAA-positive disease); and optionally administering an ALK inhibitor to the subject, wherein the RDAA-positive disease is RDAA-positive non-small cell lung cancer, and the ALK inhibitor does not include ensartinib.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and specifically to the diagnosis and treatment of diseases, and in particular to the specific application of ALK inhibitors in the treatment of RDAA-positive diseases. Background Art

[0002] ALK (anaplastic lymphoma kinase) is a type of receptor tyrosine kinase, and its mutation is associated with the occurrence of many cancers. ALK mutations include Alk gene rearrangement; activating mutations of the Alk gene; gene copy number variation, etc. ALK gene rearrangement can lead to the occurrence of many cancers such as lymphoma, lung cancer, neuroblastoma, myofibroblastoma, etc., among which the most common are anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC), but ALK gene rearrangement-positive only accounts for 6.7% of NSCLC patients and 50% of adult ALCL patients.

[0003] For different types of cancer, treatment options and prognostic assessments are not the same. For example, patients with tumors that are positive for ALK gene rearrangement can be treated with ALK inhibitors. However, for patients with wild-type ALK tumors (i.e., negative ALK gene rearrangement), no relevant cancer-promoting mechanisms have been found, and there is no effective targeted therapy. Summary of the invention

[0004] In view of the urgent problem that currently only effective treatment methods exist for ALK gene rearrangement-positive patients (i.e., administration of ALK inhibitors) in clinical practice, while no effective treatment methods have been found for ALK gene rearrangement-negative patients, the inventors of the present disclosure have identified RDAA-positive disease in the ALK gene rearrangement-negative patient population through the clever selection of biomarkers, and further found that the administration of ALK inhibitors effectively killed RDAA-positive cancer cells and significantly inhibited the tumor growth of RDAA-positive patients, thereby being able to effectively treat RDAA-positive disease, bringing good news to the majority of patients. In particular, the present disclosure provides a method for diagnosing and / or treating RDAA-positive disease; an anti-RNase1 antibody or its antigen-binding portion, an anti-ALK phosphorylation antibody or its antigen-binding portion used therein; a kit and equipment for diagnosing RDAA-positive disease; and the use of a combination of a reagent for detecting ALK gene rearrangement, a reagent for detecting ALK phosphorylation level, and a reagent for detecting RNase1 expression level in the preparation of a kit for detecting RDAA-positive disease.

[0005] In a first aspect of the present disclosure, a method for diagnosing and / or treating a disease may be provided, comprising at least the following steps:

[0006] The subject is tested for anaplastic lymphoma kinase (ALK) gene rearrangement, ALK phosphorylation level, and RNase1 expression level. If the test result of ALK gene rearrangement is negative, and both the ALK phosphorylation level and the RNase1 expression level are higher than the levels of the reference population, the disease is defined as a disease caused by RNase1-driven ALK activation (RDAA-positive disease), and an ALK inhibitor is optionally administered to the subject.

[0007] In a second aspect of the present disclosure, a method for diagnosing and / or treating a disease may be provided, comprising at least the following steps:

[0008] 1) Detect the ALK gene rearrangement of the subject. If the test result of ALK gene rearrangement is negative, perform the following steps:

[0009] 2) Detecting the ALK phosphorylation level and RNase1 expression level of the subjects; and

[0010] 3) comparing the detected ALK phosphorylation level and RNase1 expression level with the ALK phosphorylation level and RNase1 expression level of the reference population, respectively; and

[0011] 4) Optionally, when the detected ALK phosphorylation level and RNase1 expression level are respectively higher than the ALK phosphorylation level and RNase1 expression level of a reference population and reach or exceed a set threshold, determining that the subject suffers from RDAA-positive disease; and

[0012] 5) Optionally, administering an ALK inhibitor to a subject with RDAA-positive disease.

[0013] In a third aspect of the present disclosure, use of an ALK inhibitor in the preparation of a medicament for treating non-small cell lung cancer and / or pancreatic ductal adenocarcinoma may be provided.

[0014] In a fourth aspect of the present disclosure, an anti-RNase1 antibody or an antigen-binding portion thereof may be provided, comprising:

[0015] a light chain CDR1 having the amino acid sequence of SEQ ID NO:1, a light chain CDR2 having the amino acid sequence of SEQ ID NO:2, a light chain CDR3 having the amino acid sequence of SEQ ID NO:3, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6;

[0016] a light chain CDR1 having the amino acid sequence of SEQ ID NO:9, a light chain CDR2 having the amino acid sequence of SEQ ID NO:10, a light chain CDR3 having the amino acid sequence of SEQ ID NO:11, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:12, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:13, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:14;

[0017] a light chain CDR1 having the amino acid sequence of SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 18, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22;

[0018] a light chain CDR1 having the amino acid sequence of SEQ ID NO:25, a light chain CDR2 having the amino acid sequence of SEQ ID NO:26, a light chain CDR3 having the amino acid sequence of SEQ ID NO:27, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:28, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:30; or

[0019] A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:33, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:34, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:35, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:37, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:38.

[0020] In a fifth aspect of the present disclosure, an anti-ALK phosphorylation antibody or an antigen-binding portion thereof may be provided, comprising:

[0021] a light chain CDR1 having the amino acid sequence of SEQ ID NO:41, a light chain CDR2 having the amino acid sequence of SEQ ID NO:42, a light chain CDR3 having the amino acid sequence of SEQ ID NO:43, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:44, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:45, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:46;

[0022] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:49, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:50, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:51, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:52, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:53, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:54;

[0023] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:57, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:58, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:59, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:60, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:62;

[0024] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:65, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:66, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:67, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:68, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:69, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:70;

[0025] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:73, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:74, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:75, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:76, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:77, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:78;

[0026] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:81, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:82, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:83, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:84, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:85, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:86;

[0027] a light chain CDR1 having the amino acid sequence of SEQ ID NO:89, a light chain CDR2 having the amino acid sequence of SEQ ID NO:90, a light chain CDR3 having the amino acid sequence of SEQ ID NO:91, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94;

[0028] a light chain CDR1 having the amino acid sequence of SEQ ID NO:97, a light chain CDR2 having the amino acid sequence of SEQ ID NO:98, a light chain CDR3 having the amino acid sequence of SEQ ID NO:99, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:100, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:101, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:102; or

[0029] A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:105, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:106, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:107, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:108, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:109, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:110.

[0030] In a sixth aspect of the present disclosure, a kit may be provided, comprising:

[0031] 1) a reagent for detecting the phosphorylation level of ALK; and

[0032] 2) Reagents for detecting the expression level of RNase1.

[0033] In the seventh aspect of the present disclosure, there can be provided a combination of a reagent for detecting ALK phosphorylation level and a reagent for detecting RNase1 expression level for use in preparing a kit for detecting RDAA-positive disease, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0034] In the eighth aspect of the present disclosure, there can be provided a combination of a reagent for detecting the expression level of ALK gene rearrangement, a reagent for detecting the phosphorylation level of ALK, and a reagent for detecting the expression level of RNase1 for use in preparing a kit for detecting RDAA-positive diseases, wherein patients with the RDAA-positive diseases have a negative ALK gene rearrangement test result, an Rnase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0035] In the ninth aspect of the present disclosure, there can be provided the use of an ALK inhibitor in the preparation of a medicament for treating an RDAA-positive disease, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement detection result, an Rnase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0036] In a tenth aspect of the present disclosure, there may be provided an apparatus for diagnosing a RDAA-positive disease, comprising:

[0037] 1) Detection section: comprising a reagent section in which a reagent capable of detecting the expression level of ALK gene rearrangement, a reagent capable of detecting the phosphorylation level of ALK, and a reagent capable of detecting the expression level of RNase1 are respectively arranged, and a determination section for performing detection;

[0038] 2) Data collection unit: collects the results output by the detection unit;

[0039] 3) Data Analysis Department: Analyze the data collected by the Data Collection Department; and

[0040] 4) Result output section. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The results of Western immunoblotting hybridization experiments of five RNase1 monoclonal antibodies in Example 1 are shown.

[0042] Figure 2 The results of Western immunoblotting hybridization experiments of nine ALK protein phosphorylation monoclonal antibodies in Example 2 are shown.

[0043] Figure 3 The results of the experiment on the killing effect of ALK inhibitor on RDAA-positive lung cancer cells are shown.

[0044] Figure 4 The inhibitory effect of ALK inhibitor on tumor growth in RDAA-positive tumor-bearing mice is shown.

[0045] Figure 5 Results show the effect of ALK inhibitor on the survival of mice bearing RDAA-positive tumors.

[0046] Figure 6 The inhibitory effect of ALK inhibitors on tumor growth in RDAA-positive patients is shown.

[0047] Figure 7 The sensitivity of cells from RDAA-positive PDAC patients to ALK inhibitors is shown. DETAILED DESCRIPTION

[0048] Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. The terms used in the description of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0049] As used herein, the term "ALK" refers to anaplastic lymphoma kinase, for which detailed information can be found at https: / / www.ncbi.nlm.nih.gov / gene / 238 (ALK ALK receptor tyrosine kinase [Homo sapiens (human)] Gene ID: 238), and its homologs.

[0050] As used herein, the term "ALK gene rearrangement" includes, but is not limited to, those described in U.S. Patent No. 9,651,555 and Du et al. (Thoracic Cancer. 9:423-430, 2018), which are incorporated herein by reference in their entirety. ALK gene rearrangement can be the rearrangement of ALK with a gene selected from the group consisting of EML4, KIF5B, KLC1, TFG, TPR, HIP1, STRN, DCTN1, SQSTM1, NPM1, BCL11A, BIRC6, RANBP2, ATIC, CLTC, TMP4, and MSN, resulting in the formation of a fusion oncogene.

[0051] As used herein, the term "ALK gene rearrangement negative" refers to the presence of a negative ALK gene rearrangement detected by FISH (Fluorescence in situ hybridization), NGS (Next-generation sequencing), IHC

[0052] When the ALK gene rearrangement is detected by methods such as immunohistochemistry, the result is negative. For example, the Vysis ALK Break Apart FISH probe kit (Abbott Molecular, Inc.) is used to diagnose negative ALK rearrangement in the surgical or puncture pathological tissue sample of the patient; the second-generation high-throughput gene detection technology (Wuhan Ankeyi Technology Consulting Co., Ltd., Yishan Biotechnology Co., Ltd., Shenzhen BGI Co., Ltd., etc.) is used to diagnose that there is no ALK gene-related mutation and the ALK molecular rearrangement is negative; the anti-ALK (D5F3) rabbit monoclonal antibody reagent (immunohistochemistry) (VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody) is used to diagnose negative ALK molecular rearrangement in the surgical or puncture pathological tissue sample of the patient.

[0053] As used in this article, the term "Rnase1" refers to ribonuclease 1, which belongs to the human secretory ribonuclease family. For more information, please see https: / / www.ncbi.nlm.nih.gov / gene / 6035

[0054] (RNASE1 ribonuclease A family member 1, pancreatic [Homo sapiens (human)]), and its homologs.

[0055] As used herein, "RDAA-positive disease" refers to a disease associated with RNAse1-driven ALK activation.

[0056] As used herein, "a patient with RDAA-positive disease" refers to a patient with a negative ALK gene rearrangement test result, an ALK phosphorylation level that is higher than the ALK phosphorylation level of a reference population, and an RNase1 expression level that is higher than the RNase1 expression level of a reference population; in particular, the patient has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma ≥418 ng / ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0057] As used herein, "reference population" refers to healthy people or patients with RDAA-negative disease.

[0058] The inventors of the present disclosure have found for the first time that, for patients with RDAA-positive diseases, administration of ALK inhibitors can achieve effective therapeutic effects. Therefore, ALK inhibitors have good application prospects in the preparation of drugs for treating RDAA-positive diseases.

[0059] In a first aspect of the present disclosure, a method for diagnosing and / or treating a disease may be provided, comprising at least the following steps:

[0060] The subject's lymphoma kinase (ALK) gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level are detected. If the test result of ALK gene rearrangement is negative, and the ALK phosphorylation level and RNase1 expression level are higher than the reference population levels, the disease is defined as a disease caused by RNase1-driven ALK activation (RDAA-positive disease), and an ALK inhibitor is optionally administered to the subject.

[0061] In some embodiments, the subject may be a healthy person, or a patient suspected of a disease (e.g., cancer) or a patient suffering from a disease (e.g., cancer). In some embodiments, the subject may be a patient suspected of cancer or a cancer patient. In some embodiments, the cancer may be lung cancer or pancreatic cancer. In some embodiments, the cancer may be non-small cell lung cancer or pancreatic ductal adenocarcinoma.

[0062] In some embodiments, the detection is performed on a sample to be tested from a subject. In some embodiments, the sample to be tested may be from a tissue, cell and / or body fluid of a subject. In some embodiments, the sample to be tested may be from a tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a tumor tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a lung cancer or pancreatic cancer tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue section and / or blood of a subject.

[0063] In some embodiments, the ALK phosphorylation level of the reference population is a negative result of immunohistochemical staining for ALK phosphorylation. In some embodiments, the ALK phosphorylation level of the reference population is a score of <4 points of immunohistochemical staining for ALK phosphorylation.

[0064] In some embodiments, the RNase1 expression level of the reference population is the RNase1 expression level in plasma <418 ng / ml or the result of immunohistochemical staining for RNase1 is negative. In some embodiments, the RNase1 expression level of the reference population is the RNase1 expression level in plasma <418 ng / ml or the result of immunohistochemical staining for RNase1 is negative.

[0065] In some embodiments, the set threshold of ALK phosphorylation level is a positive result of immunohistochemical staining for ALK phosphorylation. In some embodiments, the set threshold of ALK phosphorylation level is a score of ≥4 points of immunohistochemical staining for ALK phosphorylation.

[0066] In some embodiments, the threshold value of RNase1 expression level is ≥418 ng / ml in plasma or the result of immunohistochemical staining for RNase1 is positive. In some embodiments, the threshold value of RNase1 expression level is ≥418 ng / ml in plasma or the result of immunohistochemical staining for RNase1 is ≥4 points.

[0067] In some embodiments, immunohistochemical staining is performed using diseased tissue. In some embodiments, immunohistochemical staining is performed using tumor tissue. In some embodiments, immunohistochemical staining is performed using lung cancer or pancreatic cancer tissue. In some embodiments, immunohistochemical staining is performed using non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue.

[0068] The detection of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level can be accomplished by any method known in the art, such as using commercial kits, sequencing, etc. In some embodiments, the detection of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level is independently selected from one or more of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. In some embodiments, the detection of ALK gene rearrangement expression level, ALK phosphorylation level and / or RNase1 expression level is performed using antibodies.

[0069] In some embodiments, the ALK inhibitor comprises an antibody or an antigen binding portion thereof capable of inhibiting ALK phosphorylation. In some embodiments, the ALK inhibitor comprises any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensartinib, or a combination thereof.

[0070] In a second aspect of the present disclosure, a method for diagnosing and / or treating a disease may be provided, comprising at least the following steps:

[0071] 1) Detect the ALK gene rearrangement expression level of the subject. If the ALK gene rearrangement test result is negative, perform the following steps:

[0072] 2) Detecting the ALK phosphorylation level and RNase1 expression level of the subjects; and

[0073] 3) comparing the detected ALK phosphorylation level and RNase1 expression level with the ALK phosphorylation level and RNase1 expression level of the reference population, respectively; and

[0074] 4) Optionally, when the detected ALK phosphorylation level and RNase1 expression level are respectively higher than the ALK phosphorylation level and RNase1 expression level of a reference population and reach or exceed a set threshold, determining that the subject suffers from RDAA-positive disease; and

[0075] 5) Optionally, administering an ALK inhibitor to a subject with RDAA-positive disease.

[0076] In some embodiments, the subject may be a healthy person, or a patient suspected of a disease (e.g., cancer) or a patient suffering from a disease (e.g., cancer). In some embodiments, the subject may be a patient suspected of cancer or a cancer patient. In some embodiments, the cancer may be lung cancer or pancreatic cancer. In some embodiments, the cancer may be non-small cell lung cancer or pancreatic ductal adenocarcinoma.

[0077] In some embodiments, the detection is performed on a sample to be tested from a subject. In some embodiments, the sample to be tested may be from a tissue, cell and / or body fluid of a subject. In some embodiments, the sample to be tested may be from a tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a tumor tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a lung cancer or pancreatic cancer tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue section and / or blood of a subject.

[0078] In some embodiments, the ALK phosphorylation level of the reference population is a negative result of immunohistochemical staining for ALK phosphorylation. In some embodiments, the ALK phosphorylation level of the reference population is a score of <4 points of immunohistochemical staining for ALK phosphorylation.

[0079] In some embodiments, the RNase1 expression level of the reference population is the RNase1 expression level in plasma <418 ng / ml or the result of immunohistochemical staining for RNase1 is negative. In some embodiments, the RNase1 expression level of the reference population is the RNase1 expression level in plasma <418 ng / ml or the result of immunohistochemical staining for RNase1 is negative.

[0080] In some embodiments, the set threshold of ALK phosphorylation level is a positive result of immunohistochemical staining for ALK phosphorylation. In some embodiments, the set threshold of ALK phosphorylation level is a score of ≥4 points of immunohistochemical staining for ALK phosphorylation.

[0081] In some embodiments, the threshold value of RNase1 expression level is ≥418 ng / ml in plasma or the result of immunohistochemical staining for RNase1 is positive. In some embodiments, the threshold value of RNase1 expression level is ≥418 ng / ml in plasma or the result of immunohistochemical staining for RNase1 is ≥4 points.

[0082] In some embodiments, immunohistochemical staining is performed using diseased tissue. In some embodiments, immunohistochemical staining is performed using tumor tissue. In some embodiments, immunohistochemical staining is performed using lung cancer or pancreatic cancer tissue. In some embodiments, immunohistochemical staining is performed using non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue.

[0083] The detection of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level can be accomplished by any method known in the art, such as using commercial kits, sequencing, etc. In some embodiments, the detection of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level is independently selected from one or more of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. In some embodiments, the detection of ALK gene rearrangement expression level, ALK phosphorylation level and / or RNase1 expression level is performed using antibodies.

[0084] In some embodiments, the ALK inhibitor comprises an antibody or an antigen binding portion thereof capable of inhibiting ALK phosphorylation. In some embodiments, the ALK inhibitor comprises any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensartinib, or a combination thereof.

[0085] In a third aspect of the present disclosure, use of an ALK inhibitor in the preparation of a medicament for treating non-small cell lung cancer and / or pancreatic ductal adenocarcinoma may be provided.

[0086] In a fourth aspect of the present disclosure, an anti-RNase1 antibody or an antigen-binding portion thereof may be provided, comprising:

[0087] a light chain CDR1 having the amino acid sequence of SEQ ID NO:1, a light chain CDR2 having the amino acid sequence of SEQ ID NO:2, a light chain CDR3 having the amino acid sequence of SEQ ID NO:3, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6;

[0088] a light chain CDR1 having the amino acid sequence of SEQ ID NO:9, a light chain CDR2 having the amino acid sequence of SEQ ID NO:10, a light chain CDR3 having the amino acid sequence of SEQ ID NO:11, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:12, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:13, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:14;

[0089] a light chain CDR1 having the amino acid sequence of SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 18, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22;

[0090] a light chain CDR1 having the amino acid sequence of SEQ ID NO:25, a light chain CDR2 having the amino acid sequence of SEQ ID NO:26, a light chain CDR3 having the amino acid sequence of SEQ ID NO:27, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:28, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:30; or

[0091] A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:33, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:34, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:35, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:37, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:38.

[0092] In some embodiments, the anti-RNase1 antibody or antigen-binding portion thereof comprises:

[0093] a light chain variable region having the amino acid sequence shown in SEQ ID NO:7, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8;

[0094] a light chain variable region having the amino acid sequence shown in SEQ ID NO: 15, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 16;

[0095] a light chain variable region having the amino acid sequence shown in SEQ ID NO:23, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24;

[0096] A light chain variable region having the amino acid sequence shown in SEQ ID NO:31, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:32; or

[0097] A light chain variable region having the amino acid sequence shown in SEQ ID NO:39, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:40.

[0098] In a fifth aspect of the present disclosure, an anti-ALK phosphorylation antibody or an antigen-binding portion thereof may be provided, comprising:

[0099] a light chain CDR1 having the amino acid sequence of SEQ ID NO:41, a light chain CDR2 having the amino acid sequence of SEQ ID NO:42, a light chain CDR3 having the amino acid sequence of SEQ ID NO:43, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:44, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:45, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:46;

[0100] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:49, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:50, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:51, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:52, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:53, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:54;

[0101] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:57, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:58, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:59, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:60, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:62;

[0102] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:65, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:66, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:67, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:68, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:69, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:70;

[0103] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:73, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:74, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:75, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:76, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:77, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:78;

[0104] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:81, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:82, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:83, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:84, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:85, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:86;

[0105] a light chain CDR1 having the amino acid sequence of SEQ ID NO:89, a light chain CDR2 having the amino acid sequence of SEQ ID NO:90, a light chain CDR3 having the amino acid sequence of SEQ ID NO:91, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94;

[0106] a light chain CDR1 having the amino acid sequence of SEQ ID NO:97, a light chain CDR2 having the amino acid sequence of SEQ ID NO:98, a light chain CDR3 having the amino acid sequence of SEQ ID NO:99, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:100, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:101, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:102; or

[0107] A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:105, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:106, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:107, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:108, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:109, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:110.

[0108] In some embodiments, the anti-ALK phosphorylation antibody, or antigen binding portion thereof, comprises:

[0109] a light chain variable region having the amino acid sequence shown in SEQ ID NO:47, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:48;

[0110] a light chain variable region having the amino acid sequence shown in SEQ ID NO:55, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:56;

[0111] a light chain variable region having the amino acid sequence shown in SEQ ID NO:63, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:64;

[0112] a light chain variable region having the amino acid sequence shown in SEQ ID NO:71, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:72;

[0113] a light chain variable region having the amino acid sequence shown in SEQ ID NO:79, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:80;

[0114] a light chain variable region having the amino acid sequence shown in SEQ ID NO:87, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:88;

[0115] a light chain variable region having the amino acid sequence shown in SEQ ID NO:95, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:96;

[0116] A light chain variable region having the amino acid sequence shown in SEQ ID NO: 103, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 104; or

[0117] A light chain variable region having the amino acid sequence shown in SEQ ID NO:111, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:112.

[0118] In a sixth aspect of the present disclosure, a kit may be provided, comprising:

[0119] 1) a reagent for detecting the phosphorylation level of ALK; and

[0120] 2) Reagents for detecting the expression level of RNase1.

[0121] In some embodiments, the reagent for detecting the phosphorylation level of ALK, the reagent for detecting the expression level of RNase1, and the reagent for detecting the expression level of ALK gene rearrangement can be any reagent known in the art that can achieve this function. In some embodiments, the reagent for detecting the phosphorylation level of ALK includes the anti-ALK phosphorylation antibody or its antigen binding portion described in the present disclosure. In some embodiments, the reagent for detecting the expression level of RNase1 includes the anti-RNase1 antibody or its antigen binding portion described in the present disclosure. In some embodiments, the kit also contains a reagent for detecting the expression level of ALK gene rearrangement.

[0122] In the seventh aspect of the present disclosure, there can be provided a combination of a reagent for detecting ALK phosphorylation level and a reagent for detecting RNase1 expression level for use in preparing a kit for detecting RDAA-positive disease, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0123] In some embodiments, a positive immunohistochemical staining result for RNase1 refers to an immunohistochemical staining score for RNase1 of ≥4 points.

[0124] In some embodiments, a positive immunohistochemical staining result for ALK phosphorylation refers to an immunohistochemical staining score for ALK phosphorylation of ≥4 points.

[0125] In the eighth aspect of the present disclosure, there can be provided a combination of a reagent for detecting the expression level of ALK gene rearrangement, a reagent for detecting the phosphorylation level of ALK, and a reagent for detecting the expression level of RNase1 for use in preparing a kit for detecting RDAA-positive diseases, wherein patients with the RDAA-positive diseases have a negative ALK gene rearrangement test result, an Rnase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0126] In some embodiments, a positive immunohistochemical staining result for RNase1 refers to an immunohistochemical staining score for RNase1 of ≥4 points.

[0127] In some embodiments, a positive immunohistochemical staining result for ALK phosphorylation refers to an immunohistochemical staining score for ALK phosphorylation of ≥4 points.

[0128] In the ninth aspect of the present disclosure, there can be provided the use of an ALK inhibitor in the preparation of a medicament for treating an RDAA-positive disease, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement detection result, an Rnase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0129] In some embodiments, a positive immunohistochemical staining result for RNase1 refers to an immunohistochemical staining score for RNase1 of ≥4 points.

[0130] In some embodiments, a positive immunohistochemical staining result for ALK phosphorylation refers to an immunohistochemical staining score for ALK phosphorylation of ≥4 points.

[0131] ALK inhibitors can be any substance known in the art that can effectively inhibit the activity and / or expression of ALK. In some embodiments, ALK inhibitors include but are not limited to any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensartinib or a combination thereof. In some embodiments, ALK inhibitors include antibodies or antigen binding portions thereof that can inhibit ALK phosphorylation. In some embodiments, ALK inhibitors include anti-ALK phosphorylation antibodies or antigen binding portions thereof described in the present disclosure.

[0132] In a tenth aspect of the present disclosure, there may be provided an apparatus for diagnosing a RDAA-positive disease, comprising:

[0133] 1) Detection section: comprising a reagent section in which a reagent capable of detecting ALK gene rearrangement, a reagent capable of detecting ALK phosphorylation level, and a reagent capable of detecting RNase1 expression level are respectively arranged, and a determination section for performing detection;

[0134] 2) Data collection unit: collects the results output by the detection unit;

[0135] 3) Data Analysis Department: Analyze the data collected by the Data Collection Department; and

[0136] 4) Result output section.

[0137] In some embodiments, the subject may be a healthy person, or a patient suspected of a disease (e.g., cancer) or a patient suffering from a disease (e.g., cancer). In some embodiments, the subject may be a patient suspected of cancer or a cancer patient. In some embodiments, the cancer may be lung cancer or pancreatic cancer. In some embodiments, the cancer may be non-small cell lung cancer or pancreatic ductal adenocarcinoma.

[0138] In some embodiments, the detection is performed on a sample to be tested from a subject. In some embodiments, the sample to be tested may be from a tissue, cell and / or body fluid of a subject. In some embodiments, the sample to be tested may be from a tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a tumor tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a lung cancer or pancreatic cancer tissue section and / or blood of a subject. In some embodiments, the sample to be tested may be from a non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue section and / or blood of a subject.

[0139] In some embodiments, the detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level is independently selected from one or more of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. In some embodiments, the detection of ALK gene rearrangement expression level, ALK phosphorylation level and / or RNase1 expression level is performed using antibodies.

[0140] In some embodiments, the data analysis unit compares the collected data of ALK gene rearrangement expression level, ALK phosphorylation level, and RNase1 expression level with a set threshold.

[0141] In some embodiments, the set threshold for ALK phosphorylation level is a positive immunohistochemical staining result for ALK phosphorylation.

[0142] In some embodiments, the set threshold of ALK phosphorylation level is an immunohistochemical staining score for ALK phosphorylation of ≥ 4 points.

[0143] In some embodiments, the set threshold of RNase1 expression level is that the RNase1 expression level in plasma is ≥418 ng / ml or the result of immunohistochemical staining for RNase1 is positive.

[0144] In some embodiments, the set threshold of RNase1 expression level is that the RNase1 expression level in plasma is ≥418 ng / ml or the immunohistochemical staining score for RNase1 is ≥4 points.

[0145] In some embodiments, when the analysis by the data analysis unit shows that the ALK gene rearrangement expression level is negative, the immunohistochemical staining result for ALK phosphorylation is positive, and the Rnase1 expression level in plasma is ≥418 ng / ml or the immunohistochemical staining result for Rnase1 is positive, it is determined to be a RDAA-positive disease.

[0146] In some embodiments, when the analysis by the data analysis unit shows that the ALK gene rearrangement expression level is negative, the immunohistochemical staining score for ALK phosphorylation is ≥4 points, and the Rnase1 expression level in plasma is ≥418 ng / ml or the immunohistochemical staining score for Rnase1 is ≥4 points, it is determined to be RDAA-positive disease.

[0147] In some embodiments, the detection section, data collection section, data analysis section, and result output section can be separated into separate device units or form an integrated device.

[0148] The various embodiments and preferences described above for the method of the present invention can be combined with each other (as long as they are not inherently contradictory to each other), and are also applicable to the anti-RNase1 antibodies or their antigen-binding portions, anti-ALK phosphorylated antibodies or their antigen-binding portions, kits, uses and equipment of the present invention, and vice versa. The various embodiments formed by such combinations are considered to be part of the disclosure of this application.

[0149] In particular, the present disclosure provides the following embodiments:

[0150] 1. A method for diagnosing a disease, comprising at least the following steps:

[0151] 1) Detecting the anaplastic lymphoma kinase (ALK) gene rearrangement, anaplastic lymphoma kinase phosphorylation level and RNase1 expression level of the subjects; and

[0152] 2) If the test result of anaplastic lymphoma kinase gene rearrangement is negative, and the ALK phosphorylation level and RNase1 expression level are higher than the reference population level, the disease is defined as a disease caused by RNase1-driven ALK activation (RDAA-positive disease).

[0153] 2. A method for diagnosing a disease, comprising at least the following steps:

[0154] 1) Detect the anaplastic lymphoma kinase (ALK) gene rearrangement of the subject. If the ALK gene rearrangement test result is negative, perform the following steps:

[0155] 2) Detecting the ALK phosphorylation level and RNase1 expression level of the subjects; and

[0156] 3) comparing the detected ALK phosphorylation level and RNase1 expression level with the ALK phosphorylation level and RNase1 expression level of the reference population, respectively; and

[0157] 4) Optionally, when the detected ALK phosphorylation level and RNase1 expression level are respectively higher than the ALK phosphorylation level and RNase1 expression level of a reference population and reach or exceed a set threshold, the subject is determined to have RDAA-positive disease.

[0158] 3. The method according to any one of the preceding embodiments, wherein the set threshold value of ALK phosphorylation level is a positive result of immunohistochemical staining for ALK phosphorylation.

[0159] 4. The method according to any one of the preceding embodiments, wherein the set threshold value of RNase1 expression level is that the RNase1 expression level in plasma is ≥418 ng / ml or the immunohistochemical staining result for RNase1 is positive.

[0160] 5. The method according to any one of the preceding embodiments, wherein the detection is performed on a sample to be tested from a subject, and the sample to be tested is from a tissue, cell and / or body fluid of the subject;

[0161] Preferably, tissue sections and / or blood from a subject;

[0162] Preferably, tumor tissue sections and / or blood from a subject;

[0163] Preferably, lung cancer or pancreatic cancer tissue sections and / or blood from a subject;

[0164] Preferably, non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue sections and / or blood from a subject.

[0165] 6. The method according to any one of the preceding embodiments, wherein the detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level is independently selected from one or more of protein blotting, immunohistochemistry and enzyme-linked immunosorbent assay.

[0166] 7. The method of any one of the preceding embodiments, wherein the detection of ALK gene rearrangement, ALK phosphorylation level and / or RNase1 expression level is performed by using an antibody or an antigen-binding portion thereof.

[0167] 8. The method according to any one of the preceding embodiments, wherein the subject is a patient suspected of having cancer, and preferably the cancer is non-small cell lung cancer or pancreatic ductal adenocarcinoma.

[0168] 9. A method for treating a disease, comprising at least the following steps:

[0169] Anaplastic lymphoma kinase (ALK) inhibitors are administered to patients with RDAA-positive disease, defined as disease caused by RNase1-driven ALK activation.

[0170] wherein the sample from the patient with RDAA-positive disease has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation, and

[0171] wherein the RDAA-positive disease is RDAA-positive non-small cell lung cancer, and

[0172] The ALK inhibitor does not include ensartinib.

[0173] 10. The method of embodiment 9, wherein the sample is from a patient's tissue, cell and / or body fluid;

[0174] Preferably, tissue sections and / or blood from a patient;

[0175] Preferably, tumor tissue sections and / or blood from the patient;

[0176] Preferably, non-small cell lung cancer tissue sections and / or blood from a patient.

[0177] 11. The method of embodiment 9 or 10, wherein the ALK inhibitor comprises an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.

[0178] 12. The method according to any one of embodiments 9-11, wherein the ALK inhibitor comprises any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib or a combination thereof.

[0179] 13. An anti-RNase1 antibody or an antigen-binding portion thereof, comprising:

[0180] a light chain CDR1 having the amino acid sequence of SEQ ID NO:1, a light chain CDR2 having the amino acid sequence of SEQ ID NO:2, a light chain CDR3 having the amino acid sequence of SEQ ID NO:3, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6;

[0181] a light chain CDR1 having the amino acid sequence of SEQ ID NO:9, a light chain CDR2 having the amino acid sequence of SEQ ID NO:10, a light chain CDR3 having the amino acid sequence of SEQ ID NO:11, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:12, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:13, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:14;

[0182] a light chain CDR1 having the amino acid sequence of SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 18, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22;

[0183] a light chain CDR1 having the amino acid sequence of SEQ ID NO:25, a light chain CDR2 having the amino acid sequence of SEQ ID NO:26, a light chain CDR3 having the amino acid sequence of SEQ ID NO:27, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:28, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:30; or

[0184] A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:33, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:34, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:35, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:37, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:38.

[0185] 14. The anti-RNase1 antibody or antigen-binding portion thereof of embodiment 13, comprising:

[0186] a light chain variable region having the amino acid sequence shown in SEQ ID NO:7, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8;

[0187] a light chain variable region having the amino acid sequence shown in SEQ ID NO: 15, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 16;

[0188] a light chain variable region having the amino acid sequence shown in SEQ ID NO:23, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24;

[0189] A light chain variable region having the amino acid sequence shown in SEQ ID NO:31, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:32; or

[0190] A light chain variable region having the amino acid sequence shown in SEQ ID NO:39, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:40.

[0191] 15. An anti-ALK phosphorylation antibody or an antigen-binding portion thereof, comprising:

[0192] a light chain CDR1 having the amino acid sequence of SEQ ID NO:41, a light chain CDR2 having the amino acid sequence of SEQ ID NO:42, a light chain CDR3 having the amino acid sequence of SEQ ID NO:43, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:44, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:45, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:46;

[0193] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:49, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:50, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:51, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:52, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:53, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:54;

[0194] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:57, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:58, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:59, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:60, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:62;

[0195] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:65, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:66, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:67, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:68, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:69, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:70;

[0196] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:73, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:74, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:75, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:76, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:77, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:78;

[0197] a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:81, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:82, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:83, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:84, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:85, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:86;

[0198] a light chain CDR1 having the amino acid sequence of SEQ ID NO:89, a light chain CDR2 having the amino acid sequence of SEQ ID NO:90, a light chain CDR3 having the amino acid sequence of SEQ ID NO:91, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94;

[0199] a light chain CDR1 having the amino acid sequence of SEQ ID NO:97, a light chain CDR2 having the amino acid sequence of SEQ ID NO:98, a light chain CDR3 having the amino acid sequence of SEQ ID NO:99, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:100, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:101, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:102; or

[0200] A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:105, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:106, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:107, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:108, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:109, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:110.

[0201] 16. The anti-ALK phosphorylated antibody or antigen binding portion thereof of embodiment 15, comprising:

[0202] a light chain variable region having the amino acid sequence shown in SEQ ID NO:47, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:48;

[0203] a light chain variable region having the amino acid sequence shown in SEQ ID NO:55, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:56;

[0204] a light chain variable region having the amino acid sequence shown in SEQ ID NO:63, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:64;

[0205] a light chain variable region having the amino acid sequence shown in SEQ ID NO:71, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:72;

[0206] a light chain variable region having the amino acid sequence shown in SEQ ID NO:79, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:80;

[0207] a light chain variable region having the amino acid sequence shown in SEQ ID NO:87, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:88;

[0208] a light chain variable region having the amino acid sequence shown in SEQ ID NO:95, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:96;

[0209] A light chain variable region having the amino acid sequence shown in SEQ ID NO: 103, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 104; or

[0210] A light chain variable region having the amino acid sequence shown in SEQ ID NO:111, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:112.

[0211] 17. A kit comprising:

[0212] 3) a reagent for detecting the phosphorylation level of ALK; and

[0213] 4) Reagents for detecting the expression level of RNase1.

[0214] 18. The kit of embodiment 17, wherein

[0215] The reagent for detecting the ALK phosphorylation level comprises the anti-ALK phosphorylation antibody or antigen binding portion thereof according to any one of embodiments 15-16.

[0216] 19. The kit of embodiment 17, wherein

[0217] The reagent for detecting the expression level of RNase1 includes the anti-RNase1 antibody or antigen-binding portion thereof according to any one of embodiments 13-14.

[0218] 20. The kit according to any one of embodiments 17-19, further comprising a reagent for detecting ALK gene rearrangement.

[0219] 21. Use of a combination of a reagent for detecting ALK phosphorylation levels and a reagent for detecting RNase1 expression levels in the preparation of a kit for detecting RDAA-positive diseases, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma ≥418 ng / ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0220] 22. Use of a combination of a reagent for detecting ALK gene rearrangement, a reagent for detecting ALK phosphorylation level, and a reagent for detecting RNase1 expression level in the preparation of a kit for detecting RDAA-positive disease, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma ≥418 ng / ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation.

[0221] 23. Use of an ALK inhibitor in the preparation of a drug for treating an RDAA-positive disease, wherein a sample from a patient suffering from the RDAA-positive disease has a negative ALK gene rearrangement detection result, an Rnase1 expression level in plasma of ≥418 ng / ml or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation, and wherein the RDAA-positive disease is RDAA-positive non-small cell lung cancer, and the ALK inhibitor does not include ensartinib.

[0222] 24. The use according to embodiment 23, wherein the sample is from a patient's tissue, cell and / or body fluid;

[0223] Preferably, tissue sections and / or blood from a patient;

[0224] Preferably, tumor tissue sections and / or blood from the patient;

[0225] Preferably, non-small cell lung cancer tissue sections and / or blood from a patient.

[0226] 25. The use of embodiment 23 or 24, wherein the ALK inhibitor comprises any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib or a combination thereof.

[0227] 26. The use of any one of embodiments 23-25, wherein the ALK inhibitor comprises an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.

[0228] 27. The use of any one of embodiments 23-26, wherein the ALK inhibitor comprises the anti-ALK phosphorylated antibody or antigen-binding portion thereof of any one of embodiments 15-16.

[0229] 28. An apparatus for diagnosing a RDAA-positive disease, comprising:

[0230] 1) Detection section: comprising a reagent section in which a reagent capable of detecting the expression level of ALK gene rearrangement, a reagent capable of detecting the phosphorylation level of ALK, and a reagent capable of detecting the expression level of RNase1 are respectively arranged, and a determination section for performing detection;

[0231] 2) Data collection unit: collects the results output by the detection unit;

[0232] 3) Data Analysis Department: Analyze the data collected by the Data Collection Department; and

[0233] 4) Result output section.

[0234] 29. The device according to embodiment 28, wherein the detection part is performed on a sample to be tested from a subject, and the sample to be tested is from tissue, cells and / or body fluid of the subject;

[0235] Preferably, tissue sections and / or blood from a subject;

[0236] Preferably, tumor tissue sections and / or blood from a subject; preferably, lung cancer or pancreatic cancer tissue sections and / or blood from a subject;

[0237] Preferably, non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue sections and / or blood from a subject;

[0238] The detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level is independently selected from one or more of Western blotting, immunohistochemistry and enzyme-linked immunosorbent assay.

[0239] 30. The apparatus according to embodiment 28, wherein the data analysis unit compares the collected data of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level with a set threshold value.

[0240] 31. The device of embodiment 28, wherein when the analysis of the data analysis unit indicates

[0241] 4) ALK gene rearrangement is negative,

[0242] 5) Immunohistochemical staining for ALK phosphorylation was positive, and

[0243] 6) When the expression level of RNase1 in plasma is ≥418 ng / ml or the immunohistochemical staining result for RNase1 is positive,

[0244] It is judged as RDAA-positive disease.

[0245] 32. The device of embodiment 28, wherein the detection part, data acquisition part, data analysis part and result output part can be separated into individual device units or form an integrated device.

[0246] The technical solution of the present disclosure will be more clearly and explicitly described in the following examples in an illustrative manner. It should be understood that these examples are only for illustrative purposes and are not intended to limit the scope of protection of the present disclosure. The scope of protection of the present disclosure is limited only by the claims.

[0247] Example

[0248] Example 1: Preparation and binding properties of anti-RNase1 antibodies

[0249] Preparation of RNase1 antibody

[0250] 1. Animal selection: purebred BALB / C mice

[0251] 2. Selection of RNase1 Antibody Immunization Scheme

[0252] The RNase1 protein purified from prokaryotic cells was used as an antigen (5 mg in total) in combination with an adjuvant. Commonly used adjuvants: Freund's complete adjuvant and Freund's incomplete adjuvant.

[0253] a: Primary immunization antigen 50 μg plus Freund's complete adjuvant was injected subcutaneously at multiple points or intrasplenicly (1 ml in total, 0.2 ml / point);

[0254] b: 3 weeks later, the second immunization dose was the same as above, with the addition of Freund's incomplete adjuvant, with a dose of 0.3 ml injected intraperitoneally;

[0255] c: 3 weeks later, the third immunization dose was the same as above, without adjuvant, and injected intraperitoneally;

[0256] d: 3 weeks later, boost immunization with a dose of 0.5 mg, injected intraperitoneally;

[0257] e: After 3 days, the spleen was harvested for fusion.

[0258] 3. Cell Fusion

[0259] 1. Preparation before cell fusion

[0260] (1) Selection of myeloma cell line: P3 / X63-Ag8 (X63) (derived from BALB / C myeloma MOPC-21).

[0261] Myeloma cells were cultured in DMEM medium. The concentration of calf serum was 10% and the cell density was 1×10 5 / ml is appropriate. When the cells are in the middle of logarithmic growth, subculture at a ratio of 1:3. Subculture once every 3 days. (Regularly treat with 8-azaguanine to make the surviving cells uniformly sensitive to HAT.)

[0262] (2) Feeder cells:

[0263] In this study, mouse peritoneal macrophages were selected and the amount was 10 5 cells / well.

[0264] 2. Steps of cell fusion

[0265] (1) Preparation of feeder cell layer:

[0266]

[0267] (2) Preparation of immune spleen cells

[0268]

[0269] (3) Preparation of myeloma cells

[0270]

[0271] (4) Fusion

[0272] ① Mix myeloma cells and spleen cells at a ratio of 1:10 or 1:5, wash once with serum-free incomplete culture medium in a 50ml centrifuge tube, centrifuge at 1200rpm for 8min; discard the supernatant and use a pipette to suck out the remaining liquid to avoid affecting the concentration of polyethylene glycol (PEG). Gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly.

[0273] ② Add 1 ml of 45% PEG solution pre-warmed at 37℃ within 90 seconds, shaking gently while adding. Incubate in a 37℃ water bath for 90 seconds.

[0274] ③ Add incomplete culture medium pre-warmed at 37°C to terminate the action of PEG, and add 1ml, 2ml, 3ml, 4ml, 5ml and 6ml respectively every 2 minutes.

[0275] ④ Centrifugation, 800rpm, 6min.

[0276] ⑤ Fill up the supernatant and resuspend in HAT selection culture medium containing 20% ​​calf serum.

[0277] ⑥ Add the above cells to a 96-well plate with a feeder cell layer, adding 100 μl to each well. One immune spleen can be inoculated into 4 96-well plates.

[0278] ⑦ Place the culture plate in a 37°C, 5% CO2 incubator.

[0279] 3. Selection of hybridoma cells and antibody detection

[0280] 1) HAT selection of hybridoma cells. Fusion cells are cultured in HAT selection medium. A large number of tumor cells die within 1 to 2 days of culture. After 3 to 4 days, the tumor cells disappear and hybrid cells form small colonies. After 7 to 10 days of HAT selection medium, HT medium is used. After another 2 weeks, general medium is used. During the selection culture period, when hybridoma cells cover 1 / 10 of the bottom of the well, specific antibodies can be detected to screen out the desired hybridoma cell lines. During the selection culture period, half of the culture medium is generally replaced every 2 to 3 days.

[0281] IV. Cloning of RNase1 Antibody-Producing Hybridomas

[0282] 1) Prepare feeder cell layer 1 day before cloning (for cell fusion).

[0283] 2) Gently blow dry the hybridoma cells to be cloned from the culture wells and count them.

[0284] 3) Adjust the cell density to 10 cells / ml.

[0285] 4) Take the cell culture plate with the feeder cell layer prepared the day before, add 100 μl of diluted cells to each well, and incubate in a 37°C, 5% CO2 incubator.

[0286] 5) Change the medium on the 7th day, and every 2 days thereafter.

[0287] 6) Cell clone formation can be seen after 8 days, and antibody activity can be detected in time.

[0288] 7) Move the cells from the positive wells to a 24-well plate for expansion culture.

[0289] 8) Cryopreservation of cell clones.

[0290] 5. Mass production of RNase1 monoclonal antibodies

[0291] Hybridoma cells were inoculated in vivo and ascites was prepared.

[0292] Preparation of ascites: BALB / C mice were routinely injected with 0.5 ml pristane or liquid paraffin intraperitoneally. Two weeks later, 1×10 6 Hybridoma cells can produce ascites 10 days after inoculation. The mice are killed and the ascites is sucked into a test tube with a dropper. Generally, 10 ml of ascites can be obtained from one mouse.

[0293] The monoclonal antibody content in ascites can reach 5 mg / ml.

[0294] Five anti-RNase1 antibodies were obtained, and their specific amino acid sequences are shown in the following table.

[0295]

[0296]

[0297] The five antibodies were used for western blot hybridization experiments. The experimental steps are as follows:

[0298] 1. Preparation of protein lysis buffer

[0299] 1. Protein lysis solution 2×sample buffer 40ml system configuration: add the following ingredients: 80% glycerol 10ml, sterile water 1ml, 10% SDS 24ml, 1M Tris-HCL (pH6.8) 5ml, mix thoroughly and store at room temperature for later use.

[0300] 2. Preparation of protein loading buffer: Mix β-mercaptoethanol and appropriate amount of bromophenol blue powder in a 1.5 ml EP tube and store in a -20 ° C refrigerator for future use

[0301] 2. Total Protein Extraction

[0302] 1) Take out the cell culture dish where protein extraction is required from the 37°C incubator and evaluate the cell density under a microscope;

[0303] 2) Place the culture dish on ice (the entire operation is performed on ice), remove the upper culture medium, and wash twice with cold PBS;

[0304] 3) Add 20-50 μl of PBS and protein lysis buffer to each well in a ratio of 1:1 according to the cell density. After the liquid covers the surface of the culture dish, use a cell scraper to mix the added solution and the cells attached to the surface of the culture dish to lyse them and collect them into an EP tube;

[0305] 4) Heat in a 100°C constant temperature metal bath for 10 minutes to denature the protein, centrifuge at 12,000 rpm, and store at 4°C for a short period of time for future use.

[0306] 3. Protein concentration quantification (BCA method)

[0307] 1) Before the experiment, quantitatively mix PBS and BSA stock solution to prepare a BSA standard with a concentration of 0.5 mg / ml and store it on ice or at 4°C for later use;

[0308] 2) Prepare BCA mixed solution: According to the number of test samples and corresponding gradient standards in the experimental setting, prepare a light green BCA mixed working solution at a ratio of BCA reagent:Cu reagent = 50:1, mix thoroughly and store at room temperature for short-term use;

[0309] 3) Starting from the first well, add the standard sample to the protein standard sample wells in decreasing volumes from 20 μl to 0 μl, and then add PBS to make up the liquid in each well to 20 μl, so that the concentration of the standard sample in each well forms a series of concentration gradients;

[0310] 4) The absorbance was detected by an ELISA instrument at a wavelength of 562 nm, the OD value was recorded, and a protein standard curve was drawn;

[0311] 5) Sample protein treatment: Take a 96-well plate and the prepared protein sample, add 18 μl PBS and 2 μl sample to each well, and add 200 μl BCA mixture, and react in a 37°C metal bath or incubator for 30 minutes;

[0312] 6) Take the sample after the reaction, detect the OD value, calculate the sample protein concentration according to the drawn protein standard curve, and calculate the sample volume during electrophoresis according to the protein loading amount. The specific calculation formula is: protein loading volume = protein loading amount / sample protein concentration;

[0313] 7) Mix the sample protein and the prepared protein loading buffer in a ratio of 20:1, centrifuge at 12000 rpm and store in a -20℃ refrigerator for later use.

[0314] IV. SDS-PAGE gel electrophoresis

[0315] 1) Preparation of separation gel: Select separation gels of different concentrations according to the molecular weight of the protein to be detected in the experiment. In this experiment, the separation gel concentrations required for detecting proteins RNase1, p-ALK (Y1604), and p-ALK (Y1282 / 1283) are 10%, 8%, and 8%. The separation gel system is shown in Table 1. Install the gel plate rack on a horizontal table, prepare the mixed solution (5 ml) according to the separation gel system, and quickly, carefully and steadily inject it into the reserved gaps between the gel plates. Then, slowly inject 100-200 μl of isopropanol or anhydrous ethanol horizontally from left to right along the edge of the gel plate wall to flatten the gel surface, slightly lift and shake one side of the gel rack to expel potential bubbles, and finally place it on a horizontal table at room temperature for 30-60 minutes to allow the separation gel to solidify;

[0316] Table 1 Separation gel preparation system (5ml)

[0317]

[0318]

[0319] 2) Preparation of concentrated gel: After the separation gel solidifies, carefully remove the isopropanol or anhydrous ethanol and dry it under natural ventilation. Then prepare the upper layer of gel (2 ml) according to the concentrated gel system (as shown in Table 2), and quickly and steadily inject it into the gap between the gel plates until the liquid overflows. Slowly and steadily insert the 15-hole comb. Let it stand on a horizontal table at room temperature for 30-60 minutes;

[0320] Table 2 Concentrated gel preparation system (2ml)

[0321]

[0322] 3) After the upper concentrated gel has solidified, carefully remove the gel plate from the gel preparation rack, fix it tightly in the electrophoresis tank, carefully and slowly pull out the comb vertically to leave the sample loading hole, and pour an appropriate amount of electrophoresis solution into the electrophoresis tank according to the number of gel plates;

[0323] 4) Protein loading: Take out the protein samples stored in the -20℃ refrigerator and wait for them to thaw and return to room temperature. Load the samples in equal amounts according to the calculated protein loading volume, and reserve 1-2 wells for 5μl of protein loading indicator (marker) according to experimental needs;

[0324] 5) Electrophoresis: After loading the sample, start electrophoresis at a constant voltage of 80V. When bead-like bubbles begin to form at the bottom of the gel plate, it indicates the start of electrophoresis. Then, the bromophenol blue in the protein sample is compressed to form a straight line. According to the marker, the protein reaches a transparent separation gel interface with a clear distinction. Adjust the voltage to a constant voltage of 120V. When the bromophenol blue indicator drops to the bottom edge of the gel plate, stop electrophoresis and start membrane transfer.

[0325] 6) Transfer: Prepare the filter paper required for transfer in advance, equipped with a methanol box for activation, transfer clip, PVDF membrane, etc., and pre-cool the transfer solution at 4°C before transfer. Place a piece of filter paper in a horizontal container and pour an appropriate amount of transfer solution so that its height slightly exceeds the thickness of the filter paper. Moisten and activate the PVDF membrane with methanol, and then place it on the filter paper. Take out the gel plate after electrophoresis, peel off the protein gel from the gel plate, trim the border appropriately and place it on the PVDF membrane, and then cover it with another layer of filter paper, and constantly remove the bubbles generated during the operation. Finally, follow the "black glue and white film" principle, use the transparent surface of the transfer clip as the bottom layer, cover it with a sponge pad and place the filter paper and PVDF membrane clamped in the previous step horizontally, and then cover it with a layer of sponge pad, align the black surface of the transfer clip, close and buckle the transfer clip buckle. Fix the buckled transfer clip in the transfer tank, add transfer solution until it submerges the transfer indicator line, place the transfer tank in a suitable container and start the transfer, and add ice water to the container to cool down. Use 300mA constant flow transfer and select appropriate transfer time according to the size of the target protein. The transfer time for R1 and p-ALK proteins is 1 and 2 hours, respectively.

[0326] 7) Blocking: Pre-prepare 5% skim milk prepared with PBST and an appropriate amount of PBST for washing. After the transfer is completed, open the transfer clamp and carefully remove the PVDF membrane that has completed the transfer. Wash the membrane in PBST for 2-3 times, 2 minutes each time. Then move it into 5% skim milk prepared with PBST, place it on a shaker, and block it for 1 hour;

[0327] 8) Incubate the primary antibody: Use the primary antibody diluent, 5% milk or 5% BSA to prepare the primary antibody in proportion. The concentration of the primary antibody in this experiment is 1:1000 (the source of the antibodies in this experiment is homemade monoclonal mouse antibody), and 4 ml is prepared for each antibody. Prepare the antibodies used before incubation, pour them into the grid of the corresponding strip in the incubation box and place them on ice to maintain the activity of the antibody at low temperature. Take out the PVDF membrane in the 5% skim milk and rinse it slightly with PBST to remove the residual milk. According to the experimental detection of protein band size, use the color marker as a scale to make the membrane into strips, and then put it into the grid of the corresponding antibody. After sealing, place it on a shaker at 4°C and shake slowly for 12 hours;

[0328] 9) Recover the primary antibody: Recover the primary antibody after 12 hours;

[0329] 10) Incubate with secondary antibody: Use horseradish enzyme-labeled goat anti-mouse IgG (H+L) secondary antibody (Zhongshan Jinqiao, catalog number: ZB-2305) and prepare the corresponding secondary antibody dilution solution in advance with 5% skim milk prepared in PBST at a ratio of 1:5000. Rinse the PVDF membrane with PBST at least 3 times, 5-10 minutes each time, and pour off the liquid after rinsing. Then pour the secondary antibody dilution solution into the PVDF membrane grid of the corresponding species and shake it slowly on a shaker for 1 hour;

[0330] 11) Recovering the secondary antibody: Recover the secondary antibody and rinse the PVDF membrane again with PBST for 3 times, each time for 5-10 minutes;

[0331] 12) ECL development: Prepare ultrasensitive ECL developer (UltraSignal ultrasensitive ECL chemiluminescent substrate, Tetrastellar, catalog number: 4AW011-100) in advance, mix equal volumes of ECL A solution and B solution in an EP tube, and store at low temperature away from light for later use. Before development, place the PVDF membrane on a foam board, slightly wipe off the residual PBST, and then drip the prepared ultrasensitive ECL developer on it according to the size of the membrane, and wait until both sides are completely infiltrated. Expose in a Bio-rad imaging instrument.

[0332] The experimental results are shown in Figure 1These five RNase1 monoclonal antibodies can specifically recognize endogenous RNase1 protein, the target band is clear and consistent with the protein size of RNase1, and there is no non-specific band. The results show that these five anti-RNase1 antibodies can specifically bind to RNase1 and can be effectively used to detect RNase1 in samples.

[0333] Example 2: Preparation of anti-ALK phosphorylation antibody

[0334] The preparation process of this antibody is the same as that of the above-mentioned RNase1 antibody, and the only difference is the antigen used. The antigen used to prepare the ALK phosphorylation antibody is a synthetic ALK protein phosphorylation peptide, with a total of 2 ALK protein phosphorylation peptides, each 5 mg.

[0335] Nine anti-ALK phosphorylation antibodies were obtained, and their specific amino acid sequences are shown in the following table.

[0336]

[0337]

[0338]

[0339] The 9 antibodies were used to perform a protein immunoblotting hybridization experiment, and the experimental method was the same as the western blot process of RNase1 in Example 1.

[0340] The experimental results are shown in Figure 2 These 9 ALK phosphorylation monoclonal antibodies can specifically recognize endogenous ALK phosphorylated protein, the target band is clear and consistent with the size of ALK phosphorylated protein, and there is no non-specific band. The results show that these 9 anti-ALK phosphorylation antibodies all bind specifically to phosphorylated ALK, and can be effectively used to detect the ALK phosphorylation level in the sample.

[0341] Example 3: Killing effect of ALK inhibitors on RDAA-positive disease cells

[0342] The second-generation sequencing method was used to detect the expression level of ALK gene rearrangement in tumor tissue section samples of 3 subjects: the patients' surgical or puncture pathological tissue samples were diagnosed with no ALK gene rearrangement and related mutations using the second-generation high-throughput gene detection technology (Shenzhen BGI Co., Ltd.), and were diagnosed as ALK gene rearrangement negative.

[0343] Subjects with negative ALK gene rearrangement will continue with the following testing.

[0344] The expression level of RNase1 in plasma samples from three ALK gene rearrangement-negative subjects was detected by enzyme-linked immunosorbent assay (ELISA). The detection method is as follows:

[0345] Elisa detection reagent and process use RNase1 Elisa kit (product name and item number: ELISA Kit for Ribonuclease1, human; Product Number: SEA297Hu)

[0346] 1. Preparation of reagents and items (items in the kit)

[0347] Reagents Quantity (dose) Reagents Quantity (dose) Pre-treated, ready-to-use 96-well plates 1 96-well plate seal 4 Standards 2 Standard diluent 1×20mL Detection Reagent A 1×120μL Detection diluent A 1×12mL Detection Reagent B 1×120μL Detection diluent B 1×12mL TMB reaction substrate solution 1×9mL Stop Solution 1×6mL Washing buffer (30× concentration) 1×20mL manual 1 serving

[0348] 2. Experimental steps

[0349] 1. Prepare all reagents, samples and standards;

[0350] 2. Add 100 μL of standard or sample to each well and incubate at 37°C for 1 hour;

[0351] 3. Draw out 100 μL / well of the prepared detection reagent A and incubate at 37°C for 1 hour;

[0352] 4. Aspirate and clean 3 times;

[0353] 5. Add 100 μL / well of the prepared detection reagent B and incubate at 37°C for 30 minutes;

[0354] 6. Aspirate and clean 5 times;

[0355] 7. Add 90 μL / well TMB reaction substrate solution. Incubate at 37°C for 120 minutes;

[0356] 8. Add 50 μL / well of stop solution and read on a microplate reader at 450 nm.

[0357] According to the absorbance value, the standard was used as a reference, and the dilution multiple was used to calculate the concentration of RNase1 in the sample. The concentration of 418ng / ml was used as the dividing point to distinguish high and low RNase1 expression. (The average concentration of RNase1 in the plasma of patients with non-small cell lung cancer is 418ng / ml).

[0358] Immunohistochemistry (IHC) was used to detect the expression level of RNase1 in tumor tissue samples from three ALK gene rearrangement-negative subjects. Immunohistochemistry experimental steps:

[0359] (1) Bake tissue sections in an oven at 65°C for 3 hours;

[0360] (2) Paraffin sections were dewaxed with xylene and hydrated with different concentrations of ethanol;

[0361] (3) 3% hydrogen peroxide inhibited endogenous peroxidase for 15 min;

[0362] (4) Wash with PBS three times, 5 minutes each time;

[0363] (5) Immerse the slices in citric acid buffer and repair them with microwaves at high temperature for 5 minutes and medium temperature for 5 minutes (or heat at high pressure at 120°C for 2 minutes);

[0364] (6) After cooling naturally to room temperature (about 60 minutes), rinse with PBS three times, 5 minutes each time;

[0365] (7) Remove excess liquid from the slide, add RNase1 monoclonal antibody at a dilution concentration of 1:100 to 1:1000, place in a wet box at 4°C overnight;

[0366] (8) On the second day, take out the samples and place them at room temperature for 1 hour, then wash them with PBS three times, 5 minutes each time.

[0367] (9) Add polymerase-labeled secondary antibody and place at room temperature for 30 minutes, then rinse with PBS three times, 5 minutes each time;

[0368] (10) DAB color developing solution was used for 3 minutes and then observed under a microscope;

[0369] (11) Flow water to stop color development and counterstain with hematoxylin for 1 minute;

[0370] (12) Rinse with tap water and differentiate with differentiation solution for 5 seconds;

[0371] (13) Rinse the sections, dehydrate with ethanol, make them transparent with xylene, and seal them with neutral gum.

[0372] Method for judging immunohistochemical results: All hematoxylin-stained sections were read by two pathologists separately. The tumor content of cancer specimens was greater than 70%. The RNase1 staining was evaluated by double-blind method to observe and determine the location of cells with positive RNase1 expression, which was manifested as brown or brown particles. The immunohistochemical staining score was obtained by multiplying the number of positively stained cells by the staining intensity. Tissue specimens were observed under an optical microscope and scored according to the percentage of positively stained cells: 0: negative expression; 1 point: the number of positive cells was <15%; 2 points: positive cells 15-50%; 3 points: positive cells ≥50%; the staining intensity was scored according to the depth of color: 0 points: no positive; 1 point: light yellow; 2 points: brown; 3 points: brown. The immunohistochemical staining score = 4 points was used as the dividing point for distinguishing high and low RNase1 expression levels.

[0373] The HIC method was used to detect the ALK phosphorylation level in tumor tissue samples from 3 subjects using an ALK phosphorylation monoclonal antibody (CDA04 among the 9 ALK phosphorylation monoclonal antibodies shown in the antibody preparation section). The immunohistochemical staining score = 4 points was used as the cutoff point for distinguishing high and low ALK phosphorylation levels.

[0374] The subjects were divided into RDAA-positive subjects and non-RDAA-positive (RDAA-negative) subjects according to the following criteria:

[0375] RDAA-positive subjects: RNase1 expression level in plasma ≥418 ng / ml or immunohistochemical staining score for RNase1 ≥4 points; and immunohistochemical staining score for ALK phosphorylation ≥4 points;

[0376] RDAA-negative subjects: the expression level of RNase1 in plasma is less than 418 ng / ml or the immunohistochemical staining score for RNase1 is less than 4 points; and the immunohistochemical staining score for ALK phosphorylation is less than 4 points.

[0377] Cancer cells from one RDAA-positive and one RDAA-negative non-small cell lung cancer subject were treated with six ALK inhibitors, including placebo (PBS buffer), crizotinib, ceritinib, loratinib, alectinib, brigatinib and ensartinib (the final concentration of the above drugs was 1 microgram per milliliter of culture medium, and the cell density was 50,000 cells per milliliter of culture medium). Cell survival was observed, the number of surviving cells was calculated every day, and growth curves were drawn.

[0378] The experimental results are as follows Figure 3 As shown, the horizontal axis is the time of medication, in days, and the vertical axis is the relative number of cells. The results showed that the placebo could not inhibit the growth of RDAA-positive lung cancer cells, but various ALK inhibitors effectively killed RDAA-positive lung cancer cells (P<0.01), but had no significant effect on RDAA-negative lung cancer cells.

[0379] Example 4: Therapeutic effect of ALK inhibitor on RDAA-positive lung cancer mice

[0380] Cancer cells from a RDAA-positive non-small cell lung cancer patient were inoculated into the subcutaneous tissue of the abdomen of nude mice. The number of inoculated cells was 500,000 cells per mouse. Ten days after the inoculation, a mass was observed growing under the subcutaneous tissue of the abdomen of the mice, thus constructing RDAA-positive tumor-bearing mice.

[0381] Tumor-bearing mice bearing RDAA-positive tumors were randomly divided into seven groups (5 mice in each group) and orally administered placebo (PBS buffer), crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, or ensartinib (the dose of ALK inhibitor was 25 mg per kg body weight per day, and the dose of placebo was 2 ul per mouse per day. The drug was started 2 weeks after tumor inoculation and stopped from the fourth week).

[0382] The experimental results are as follows Figure 4 , Figure 5 The results showed that compared with the placebo group, the ALK inhibitor significantly inhibited the tumor growth of RDAA-positive tumor-bearing mice (P<0.01); at the same time, the survival of the tumor-bearing mice was also significantly improved (P<0.01).

[0383] Example 5: Therapeutic effect of ALK inhibitors on RDAA-positive patients

[0384] 40 patients from West China Hospital of Sichuan University were determined to be ALK gene rearrangement negative according to the detection method in Example 3. Then, the RNase1 expression level in the patient's plasma sample and the RNase1 protein expression level and ALK phosphorylation level in the tumor tissue sample were measured using this kit, and the patients were divided into RDAA-positive patients and non-RDAA-positive patients according to the following criteria:

[0385] RDAA-positive patients: plasma RNase1 expression level ≥418 ng / ml or RNase1 immunohistochemical staining score ≥4 points; and ALK phosphorylation immunohistochemical staining score ≥4 points;

[0386] RDAA-negative patients: the expression level of RNase1 in plasma is less than 418 ng / ml or the immunohistochemical staining score for RNase1 is less than 4 points; and the immunohistochemical staining score for ALK phosphorylation is less than 4 points.

[0387] A total of 3 RDAA-positive patients were identified, all of whom were patients with non-small cell lung cancer. The CT results before and after 4 weeks of continuous use of crizotinib at a dose of 250 mg twice a day were as follows: Figure 6 As shown in Figure 2, it can be clearly seen that crizotinib significantly inhibited the tumor growth of RDAA-positive patients.

[0388] Example 6: Therapeutic effect of ALK inhibitors on RDAA-positive PDAC patients

[0389] Ten PDAC patients with negative ALK gene rearrangement were identified by the second-generation high-throughput gene sequencing (NGS) method. The tumor tissue sections of these patients were subjected to immunohistochemical detection of RNase1 and ALK phosphorylation mentioned in this kit. Among them, the immunohistochemical results of one tumor tissue section were RNase1 expression>4 points and ALK phosphorylation>4 points, and were identified as RDAA-positive PDAC patients. The patient's tumor cells were cultured and treated with six ALK inhibitors, including placebo control (control, PBS buffer), crizotinib, ceritinib, loratinib, alectinib, brigatinib and ensartinib (the above drugs were used at a final concentration of 1 microgram per milliliter of culture medium and a cell density of 50,000 cells per milliliter of culture medium), and cell survival was observed, the number of surviving cells was calculated every day, and the growth curve was drawn.

[0390] The experimental results are as follows Figure 7 As shown, the horizontal axis is the time of medication, in days, and the vertical axis is the relative number of cells. The results showed that the placebo could not inhibit the growth of RDAA-positive PDAC cells, but various ALK inhibitors effectively killed RDAA-positive PDAC cells (P<0.01).

Claims

1. An anti-RNase1 antibody or an antigen-binding portion thereof, comprising: The light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, having the amino acid sequence shown in SEQ ID NO: NO:2, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:3, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:4, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:5, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:6; The light chain CDR1 having the amino acid sequence shown in SEQ ID NO:9, having the amino acid sequence shown in SEQ ID NO: a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:10, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:11, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:12, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:13, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:14; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 18, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22; a light chain CDR1 having the amino acid sequence of SEQ ID NO:25, a light chain CDR2 having the amino acid sequence of SEQ ID NO:26, a light chain CDR3 having the amino acid sequence of SEQ ID NO:27, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:28, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:30; or A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:33, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:34, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:35, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:37, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:

38.

2. The anti-RNase1 antibody or antigen-binding portion thereof according to claim 1, comprising: a light chain variable region having the amino acid sequence shown in SEQ ID NO:7, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:8; a light chain variable region having the amino acid sequence shown in SEQ ID NO: 15, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 16; a light chain variable region having the amino acid sequence shown in SEQ ID NO:23, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:24; A light chain variable region having the amino acid sequence shown in SEQ ID NO:31, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:32; or A light chain variable region having the amino acid sequence shown in SEQ ID NO:39, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

40.

3. An anti-anaplastic lymphoma kinase ALK phosphorylation antibody or an antigen-binding portion thereof, comprising: a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:41, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:42, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:43, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:44, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:45, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:46; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:49, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:50, a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO:51, a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:52, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:53, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO:

54. Heavy chain CDR3 having the amino acid sequence shown in NO:54; a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:57, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:58, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:59, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:60, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:61, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:62; a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:65, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:66, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:67, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:68, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:69, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:70; a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:73, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:74, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:75, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:76, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:77, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:78; a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:81, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO:82, a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:83, a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:84, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:85, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:86; a light chain CDR1 having the amino acid sequence of SEQ ID NO:89, a light chain CDR2 having the amino acid sequence of SEQ ID NO:90, a light chain CDR3 having the amino acid sequence of SEQ ID NO:91, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 97, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 98, a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 99, a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 100, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO:

102. The heavy chain CDR3 of the amino acid sequence shown in NO:102; or A light chain CDR1 having the amino acid sequence shown in SEQ ID NO:105, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO:106, a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:107, a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:108, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:109, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:

110.

4. The anti-anaplastic lymphoma kinase ALK phosphorylated antibody or antigen binding portion thereof according to claim 3, comprising: a light chain variable region having the amino acid sequence shown in SEQ ID NO:47, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:48; a light chain variable region having the amino acid sequence shown in SEQ ID NO:55, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:56; a light chain variable region having the amino acid sequence shown in SEQ ID NO:63, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:64; a light chain variable region having the amino acid sequence shown in SEQ ID NO:71, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:72; a light chain variable region having the amino acid sequence shown in SEQ ID NO:79, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:80; a light chain variable region having the amino acid sequence shown in SEQ ID NO:87, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:88; a light chain variable region having the amino acid sequence shown in SEQ ID NO:95, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:96; A light chain variable region having the amino acid sequence shown in SEQ ID NO: 103, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 104; or A light chain variable region having the amino acid sequence shown in SEQ ID NO:111, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:

112.

5. A kit comprising: 1) a reagent for detecting the phosphorylation level of ALK; and 2) Reagents for detecting RNase1 expression levels; The reagent for detecting the phosphorylation level of ALK comprises the anti-anaplastic lymphoma kinase ALK phosphorylation antibody or the antigen binding portion thereof according to any one of claims 3 to 4. The kit according to claim 5 , further comprising a reagent for detecting ALK gene rearrangement.

7. A kit comprising: 1) a reagent for detecting the phosphorylation level of ALK; and 2) Reagents for detecting RNase1 expression levels; The reagent for detecting the expression level of RNase1 comprises the anti-RNase1 antibody or the antigen-binding portion thereof according to any one of claims 1 to 2.

8. The kit of claim 7, wherein the reagent for detecting the phosphorylation level of ALK comprises the anti-anaplastic lymphoma kinase ALK phosphorylation antibody or the antigen-binding portion thereof according to any one of claims 3-4.

9. The kit according to claim 7 or 8, further comprising a reagent for detecting ALK gene rearrangement.

10. Use of a combination of a reagent for detecting ALK phosphorylation levels and a reagent for detecting RNase1 expression levels in the preparation of a kit for detecting RDAA-positive disease caused by RNase1-driven ALK activation, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma of ≥418 ng / ml or an immunohistochemical staining result for RNase1 with a staining score of ≥4 points, and an immunohistochemical staining result for ALK phosphorylation with a staining score of ≥4 points.

11. The use according to claim 10, wherein the detection is performed on a sample to be tested from a subject, and the sample to be tested is from a tissue, cell and / or body fluid of the subject.

12. The use according to claim 11, wherein the sample to be tested is from a tissue section and / or blood of a subject.

13. The use according to claim 11, wherein the sample to be tested is from a tumor tissue section and / or blood of a subject.

14. The use according to claim 11, wherein the sample to be tested is from a lung cancer or pancreatic cancer tissue section and / or blood of a subject.

15. The use according to claim 11, wherein the sample to be tested is from a non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue section and / or blood of a subject.

16. The use according to claim 11, wherein the subject is a patient suspected of cancer.

17. The use of claim 16, wherein the cancer is non-small cell lung cancer or pancreatic ductal adenocarcinoma.

18. Use of a combination of a reagent for detecting ALK gene rearrangement, a reagent for detecting ALK phosphorylation level, and a reagent for detecting RNase1 expression level in the preparation of a kit for detecting RDAA-positive disease, wherein the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result, an RNase1 expression level in plasma of ≥418 ng / ml or an immunohistochemical staining result for RNase1 with a staining score of ≥4 points, and an immunohistochemical staining result for ALK phosphorylation with a staining score of ≥4 points.

19. The use according to claim 18, wherein the detection is performed on a test sample from a subject, and the test sample is from a tissue, cell and / or body fluid of the subject.

20. The use according to claim 19, wherein the sample to be tested is from a tissue section and / or blood of a subject.

21. The use according to claim 19, wherein the sample to be tested is from a tumor tissue section and / or blood of a subject.

22. The use of claim 19, wherein the sample to be tested is from a lung cancer or pancreatic cancer tissue section and / or blood of a subject.

23. The use of claim 19, wherein the sample to be tested is from a non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue section and / or blood of a subject.

24. The use of claim 19, wherein the subject is a patient suspected of cancer.

25. The use of claim 24, wherein the cancer is non-small cell lung cancer or pancreatic ductal adenocarcinoma.

26. Use of an ALK inhibitor in the preparation of a drug for treating an RDAA-positive disease, wherein a sample from a patient suffering from the RDAA-positive disease has a negative ALK gene rearrangement detection result, an RNase1 expression level in plasma of ≥418 ng / ml or an immunohistochemical staining result for RNase1 with a staining score of ≥4, and an immunohistochemical staining result for ALK phosphorylation with a staining score of ≥4, and wherein the RDAA-positive disease is RDAA-positive non-small cell lung cancer, and the ALK inhibitor does not include ensartinib.

27. The use according to claim 26, wherein the sample is from tissues, cells and / or body fluids of a patient.

28. The use according to claim 26, wherein the sample is from a tissue section and / or blood of a patient.

29. The use according to claim 26, wherein the sample is from a tumor tissue section and / or blood of a patient.

30. The use according to claim 26, wherein the sample is from a non-small cell lung cancer tissue section and / or blood of a patient.

31. The use of any one of claims 26-30, wherein the ALK inhibitor comprises any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, or a combination thereof.

32. The use of any one of claims 26-30, wherein the ALK inhibitor comprises an antibody or an antigen binding portion thereof that is capable of inhibiting ALK phosphorylation.

33. The use of any one of claims 26-30, wherein the ALK inhibitor comprises the anti-anaplastic lymphoma kinase ALK phosphorylated antibody or antigen binding portion thereof of any one of claims 3-4.

34. An apparatus for diagnosing a RDAA-positive disease, comprising: 1) Detection section: comprising a reagent section in which a reagent capable of detecting the expression level of ALK gene rearrangement, a reagent capable of detecting the phosphorylation level of ALK, and a reagent capable of detecting the expression level of RNase1 are respectively arranged, and a determination section for performing detection; 2) Data collection unit: collects the results output by the detection unit; 3) Data Analysis Department: Analyze the data collected by the Data Collection Department; and 4) Result output unit; Among them, when the analysis of the data analysis department shows that 1) ALK gene rearrangement is negative, 2) Immunohistochemical staining results for ALK phosphorylation with a staining score of ≥ 4 points, and 3) When the expression level of RNase1 in plasma is ≥418 ng / ml or the immunohistochemical staining result for RNase1 is ≥4 points, It is judged as RDAA-positive disease.

35. The device of claim 34, wherein the detection part is performed on a sample to be tested from a subject, and the sample to be tested is from tissue, cells and / or body fluid of the subject.

36. The device of claim 35, wherein the sample to be tested is from a tissue section and / or blood of a subject.

37. The device according to claim 35, wherein the sample to be tested is from a tumor tissue section and / or blood of a subject.

38. The device according to claim 35, wherein the sample to be tested is from a lung cancer or pancreatic cancer tissue section and / or blood of a subject.

39. The device according to claim 35, wherein the sample to be tested is from a non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue section and / or blood of a subject.

40. The device according to any one of claims 34-39, wherein the detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level is independently selected from one or more of Western blotting, immunohistochemistry and enzyme-linked immunosorbent assay.

41. The apparatus of claim 34, wherein the data analysis unit compares the collected data of ALK gene rearrangement expression level, ALK phosphorylation level, and RNase1 expression level with a set threshold value.

42. The device of claim 34, wherein the detection section, the data collection section, the data analysis section and the result output section can be separated into individual device units or form an integrated device.

Images