WO2024017338A1

WO2024017338A1 - Method for diagnosing and treating rdaa positive disease, and kit

Info

Publication number: WO2024017338A1
Application number: PCT/CN2023/108418
Authority: WO
Inventors: 刘伦旭; 查正宇
Original assignee: 贝达药业股份有限公司
Priority date: 2022-07-22
Filing date: 2023-07-20
Publication date: 2024-01-25
Also published as: CN115993453A

Abstract

A method for diagnosing and/or treating a disease is provided, which comprises at least the following steps: evaluating the anaplastic lymphoma kinase (ALK) gene rearrangement, anaplastic lymphoma kinase phosphorylation levels, and RNase1 expression levels of a subject; if the evaluation result for ALK gene rearrangement is negative, and the ALK phosphorylation levels and the RNase1 expression levels are both higher than levels of a reference population, then defining the disease as a disease caused by RNase1-driven ALK activation (an RDAA positive disease), and 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 comprise ensartinib.

Description

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

Technical field

The present disclosure relates to the field of biomedical technology, specifically to the diagnosis and treatment of diseases, especially the specific application of ALK inhibitors in the treatment of RDAA-positive diseases.

Background technique

ALK (anaplastic lymphoma kinase) is a type of receptor tyrosine kinase, and its mutations are associated with the occurrence of various cancers. Mutated forms of ALK include Alk gene rearrangements; activating mutations of the Alk gene; gene copy number variations, etc. ALK gene rearrangement can lead to the occurrence of lymphoma, lung cancer, neuroblastoma, myofibroblastoma and other cancers, the most common of which are anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC). However, ALK gene rearrangement positivity accounts for only 6.7% of NSCLC patients and 50% of adult patients with ALCL.

Treatment options and prognosis assessments vary for different types of cancer. For example, patients with tumors that are positive for ALK gene rearrangement can be treated with ALK inhibitors. However, for tumor patients with wild-type ALK (i.e., ALK gene rearrangement negative), no relevant cancer-promoting mechanism has been discovered, and there is no effective targeted therapy.

Contents of the invention

In view of the urgent problem that there are currently only effective treatments for patients with positive ALK gene rearrangements (i.e., the administration of ALK inhibitors) in clinical practice, but no effective treatment methods have been found for patients with negative ALK gene rearrangements, the inventors of the present disclosure Through clever selection of biomarkers, RDAA-positive diseases were identified in a group of ALK gene rearrangement-negative patients, and it was further found that administration of ALK inhibitors effectively killed RDAA-positive cancer cells and significantly inhibited tumor growth in RDAA-positive patients. This can effectively treat RDAA-positive diseases and bring good news to the majority of patients. In particular, the present disclosure provides methods for diagnosing and/or treating RDAA-positive diseases; anti-RNase1 antibodies or antigen-binding portions thereof, anti-ALK phosphorylated antibodies or antigen-binding portions thereof used therein; methods for diagnosing RDAA-positive diseases; Kit, equipment; 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 preparing a kit for detecting RDAA-positive diseases.

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

Detect the subject's anaplastic lymphoma kinase (ALK) gene rearrangement, ALK phosphorylation level and RNase1 expression level. If the detection result of ALK gene rearrangement is negative and the ALK phosphorylation level and RNase1 expression level are higher than the reference At the population level, the disease is defined as a disease caused by RNase1 driven ALK activation (RDAA positive disease), and the subjects are optionally administered an ALK inhibitor.

In a second aspect of the present disclosure, a method for diagnosing and/or treating diseases can be provided, which at least includes the following steps:

1) Detect ALK gene rearrangement in the subject. If the test result for ALK gene rearrangement is negative, proceed to the following steps:

2) Detect the ALK phosphorylation level and RNase1 expression level of the subject; and

3) Compare the detected ALK phosphorylation level and RNase1 expression level with the ALK phosphorylation level and RNase1 expression level of the reference population respectively; and

4) Optionally, in the case where the detected ALK phosphorylation level and RNase1 expression level are respectively higher than the ALK phosphorylation level and RNase1 expression level of the reference population and reach or exceed the set threshold, determine the said The subject has an RDAA-positive disease; and

5) Optionally, administer an ALK inhibitor to a subject with RDAA-positive disease.

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

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

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 6 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 13, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 14 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:20, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:21, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:22 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 25, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 26, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 27 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 28, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 29, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 30 chain CDR3; or

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:33, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:34, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:35 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 36, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 37, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 38 Chain CDR3.

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

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:41, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:42, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:43 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:44, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:45, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:46 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 49, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 50, light chain CDR3 having the amino acid sequence 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 CDR2 having an amino acid sequence as shown in SEQ ID NO:54 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:57, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:58, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:59 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 60, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 61, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 62 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 65, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 66, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 67 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:68, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:69, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:70 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:73, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:74, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:75 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:76, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:77, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:78 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:81, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:82, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:83 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:84, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:85, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:86 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:89, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:90, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:91 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 92, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 93, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 94 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:97, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:98, light chain CDR3 having the amino acid sequence 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 CDR2 having an amino acid sequence as shown in SEQ ID NO: 102 chain CDR3; or

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 105, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 106, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 107 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 108, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 109, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 110 Chain CDR3.

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

1) Reagents for detecting ALK phosphorylation levels; and

2) Reagents for detecting RNase1 expression levels.

In a seventh aspect of the present disclosure, the use of a combination of a reagent for detecting ALK phosphorylation level and a reagent for detecting RNase1 expression level in preparing a kit for detecting RDAA-positive disease in patients with negative RDAA-positive disease can be provided. ALK gene rearrangement test results, Rnase1 expression level in plasma ≥418ng/ml or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation.

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

In a ninth aspect of the present disclosure, the use of an ALK inhibitor in preparing a medicament for treating RDAA-positive disease can be provided, and the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result and a plasma concentration of ≥418ng/ml. Rnase1 expression levels in or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation.

In a tenth aspect of the present disclosure, a device for diagnosing RDAA-positive diseases may be provided, which includes:

1) Detection part: including a reagent part that is respectively provided with a reagent capable of detecting the expression level of ALK gene rearrangement, a reagent that detects the ALK phosphorylation level, and a reagent that detects the RNase1 expression level, and a measurement part that performs detection;

2) Data collection department: collects the results output by the detection department;

3) Data Analysis Department: analyze the data collected by the Data Collection Department; and

4) Result output department.

Description of drawings

Figure 1 shows the results of Western immunoblot hybridization experiments of five RNase1 monoclonal antibodies in Example 1.

Figure 2 shows the results of Western immunoblot hybridization experiments of nine ALK protein phosphorylated monoclonal antibodies in Example 2.

Figure 3 shows the experimental results of the killing effect of ALK inhibitors on RDAA-positive lung cancer cells.

Figure 4 shows the inhibitory effect of ALK inhibitor on tumor growth in RDAA-positive tumor-bearing mice.

Figure 5 shows the results of the survival effect of ALK inhibitors on RDAA-positive tumor-bearing mice.

Figure 6 shows the tumor growth inhibitory effect of ALK inhibitors on RDAA-positive patients.

Figure 7 shows the sensitivity of cells from RDAA-positive PDAC patients to ALK inhibitors.

Detailed ways

Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

As used herein, the term "ALK" refers to anaplastic lymphoma kinase, as detailed at https://www.ncbi.nlm.nih.gov/gene/238(ALK ALK receptor tyrosine kinase[Homo sapiens( human)]Gene ID: 238), and its homologues.

As used herein, the term "ALK gene rearrangements" 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 by reference in their entirety. Incorporated herein. The ALK gene rearrangement may be a 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, leading to the formation of fusion oncogenes.

When used in this article, the term "ALK gene rearrangement negative" refers to the result of ALK gene rearrangement when detected by FISH (Fluorescence in situ hybridization) method, NGS (Next-generation sequencing) method, IHC (Immunohistochemistry) method, etc. row negative. For example, the Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Inc.) was used to diagnose ALK rearrangement negative for the patient's surgical or puncture pathological tissue samples; the patient's surgical or puncture pathological tissue samples were diagnosed as negative for ALK rearrangement using the second-generation Qualcomm Quantitative genetic testing technology (Wuhan Angkeyi Technology Consulting Co., Ltd., Yishan Biotechnology Co., Ltd., Shenzhen BGI Co., Ltd., etc.) diagnosed no ALK gene-related mutations and was diagnosed as negative for ALK molecular rearrangement; the patient underwent surgery or puncture Pathological tissue samples were diagnosed as negative for ALK molecular rearrangement using anti-ALK(D5F3) Rabbit Monoclonal Antibody reagent (immunohistochemistry) (VENTANA anti-ALK(D5F3) Rabbit Monoclonal Primary Antibody).

When used in this article, the term "Rnase1" refers to ribonuclease 1 (ribonuclease1), which belongs to the human secreted ribonuclease family. For detailed information, please see https://www.ncbi.nlm.nih.gov/gene/ 6035 (RNASE1 ribonuclease A family member 1, pancreatic [Homo sapiens (human)]), and its homologs.

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

As used herein, a “patient with RDAA-positive disease” means that the patient has a negative ALK gene rearrangement test result, an ALK phosphorylation level that is higher than that of the reference population, and an RNase1 expression that is higher than that of the reference population. level of RNase1 expression; specifically, the patient had a negative ALK gene rearrangement test result, a plasma RNase1 expression level ≥418 ng/ml or a positive immunohistochemical stain for RNase1, and a positive ALK phospho immunohistochemical staining results.

As used herein, "reference population" refers to healthy individuals or patients with RDAA-negative disease.

The inventors of the present disclosure discovered for the first time that administration of an ALK inhibitor can achieve effective therapeutic effects in patients with RDAA-positive disease. Therefore, ALK inhibitors have good application prospects in the preparation of drugs for the treatment of RDAA-positive diseases.

Detect the subject's lymphoma kinase (ALK) gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level. If the detection result of ALK gene rearrangement is negative and the ALK phosphorylation level and RNase1 expression level are higher than the reference At the population level, the disease is defined as a disease caused by RNase1 driven ALK activation (RDAA positive disease), and the subjects are optionally administered an ALK inhibitor.

In some embodiments, the subject may be a healthy person, a patient suspected of a disease (eg, cancer), or a patient suffering from a disease (eg, cancer). In some embodiments, the subject may be a cancer suspect 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.

In some embodiments, the detection is performed on a sample from the subject to be tested. In some embodiments, the sample to be tested may be from tissues, cells and/or body fluids of a subject. In some embodiments, the sample to be tested may be from tissue sections and/or blood of the subject. In some embodiments, the sample to be tested may be from a subject's tumor tissue section and/or blood. In some embodiments, the sample to be tested may be from lung or pancreatic cancer tissue sections and/or blood of the 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 the subject.

In some embodiments, the reference population has ALK phosphorylation levels that are negative for immunohistochemical staining for ALK phosphorylation. In some embodiments, the reference population has an ALK phosphorylation level with an immunohistochemical staining score for ALK phosphorylation of <4 points.

In some embodiments, the RNase1 expression level in the reference population is an Rnasel expression level in plasma <418 ng/ml or a negative immunohistochemical staining result for Rnasel. In some embodiments, the RNase1 expression level in the reference population is an Rnasel expression level in plasma of <418 ng/ml or an immunohistochemical staining score for Rnasel of <4 points.

In some embodiments, the set threshold for ALK phosphorylation level is a positive immunohistochemical stain for ALK phosphorylation. In some embodiments, the set threshold for ALK phosphorylation level is an immunohistochemical staining score for ALK phosphorylation ≥4 points.

In some embodiments, the set threshold for RNase1 expression level is an Rnasel expression level in plasma ≥ 418 ng/ml or a positive immunohistochemical staining result for Rnasel. In some embodiments, the set threshold for RNase1 expression level is an Rnasel expression level in plasma ≥418 ng/ml or an immunohistochemical staining score for RNase1 ≥4 points.

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

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

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

1) Detect the expression level of ALK gene rearrangement in the subject. If the test result of ALK gene rearrangement is negative, proceed to the following steps:

In some embodiments, the detection is performed on a sample from the subject to be tested. In some embodiments, the sample to be tested may be from tissues, cells and/or body fluids of a subject. In some embodiments, the sample to be tested may be from tissue sections and/or blood of the subject. In some embodiments, the sample to be tested may be from a subject Tumor tissue sections and/or blood of the subject. In some embodiments, the sample to be tested may be from lung or pancreatic cancer tissue sections and/or blood of the 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 the subject.

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 20, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 21, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 22 chain CDR3;

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

A light chain variable region having an amino acid sequence as shown in SEQ ID NO:7, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:8;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16;

A light chain variable region having an amino acid sequence shown in SEQ ID NO: 23, and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 24;

A light chain variable region having an amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 32; or

A light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 39, and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 40.

In some embodiments, the anti-ALK phosphorylated antibody or antigen-binding portion thereof comprises:

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 47, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 48;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 55, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 56;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 63, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 64;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO:71, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:72;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO:79, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:80;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 87, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 88;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 95, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 96;

A light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 103, and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104; or

A light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 111, and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 112.

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

1) Reagents for detecting ALK phosphorylation levels; and

2) Reagents for detecting RNase1 expression levels.

In some embodiments, the reagent for detecting ALK phosphorylation level, the reagent for detecting RNase1 expression level, and the reagent for detecting ALK gene rearrangement expression level can be any reagent known in the art that can achieve this function. In some embodiments, a reagent for detecting ALK phosphorylation levels includes an anti-ALK phosphorylation antibody or an antigen-binding portion thereof as described in this disclosure. In some embodiments, reagents for detecting RNase1 expression levels include an anti-RNase1 antibody or an antigen-binding portion thereof as described in the present disclosure. In some embodiments, the kit further includes a reagent for detecting the expression level of ALK gene rearrangement.

In some embodiments, a positive immunohistochemical staining result for Rnase1 refers to an immunohistochemical staining score for Rnase1 of ≥4 points.

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

The ALK inhibitor 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 combinations thereof . In some embodiments, ALK inhibitors include antibodies or antigen-binding portions thereof capable of inhibiting ALK phosphorylation. In some embodiments, an ALK inhibitor includes an anti-ALK phosphorylated antibody of the present disclosure, or an antigen-binding portion thereof.

1) Detection part: including a reagent part that is respectively provided with a reagent capable of detecting ALK gene rearrangement, a reagent that detects ALK phosphorylation level, and a reagent that detects RNase1 expression level, and a measurement part that performs detection;

4) Result output department.

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

In some embodiments, the data analysis section compares the collected data on ALK gene rearrangement expression levels, ALK phosphorylation levels and RNase1 expression levels with set thresholds.

In some embodiments, the set threshold for ALK phosphorylation level is a positive immunohistochemical stain for ALK phosphorylation.

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

In some embodiments, the set threshold for RNase1 expression level is an Rnasel expression level in plasma ≥ 418 ng/ml or a positive immunohistochemical staining result for Rnasel.

In some embodiments, the set threshold for RNase1 expression level is an Rnasel expression level in plasma ≥418 ng/ml or an immunohistochemical staining score for RNase1 ≥4 points.

In some embodiments, when the analysis by the data analysis department 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 the plasma is ≥418ng/ml or the Rnase1 expression level is negative. When the immunohistochemical staining result is positive, it is determined to be RDAA-positive disease.

In some embodiments, when the analysis by the Data Analysis Department 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 the plasma is ≥418ng/ml or for Rnase1 When the immunohistochemical staining score is ≥4 points, it is determined to be RDAA-positive disease.

In some embodiments, the detection part, data acquisition part, data analysis part and result output part may be separated into separate equipment units or formed into an integrated equipment.

The various embodiments and preferences described above for the methods of the present disclosure may be combined with each other (so long as they are not inherently inconsistent with each other) and are equally applicable to the anti-RNase1 antibodies or antigen-binding portions thereof, anti-ALK Phosphorylated antibodies or antigen-binding portions thereof, kits, uses and devices, and vice versa, and various embodiments resulting from combinations thereof are considered part of the disclosure of this application.

In particular, the present disclosure provides the following embodiments:

1. A method for diagnosing disease, which at least includes the following steps:

1) Detect the subject's anaplastic lymphoma kinase (ALK) gene rearrangement, anaplastic lymphoma kinase phosphorylation level and RNase1 expression level; and

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

2. A method for diagnosing disease, which at least includes the following steps:

1) Test the subject for anaplastic lymphoma kinase (ALK) gene rearrangement. If the test result for ALK gene rearrangement is negative, proceed with the following steps:

4) Optionally, in the case where the detected ALK phosphorylation level and RNase1 expression level are respectively higher than the ALK phosphorylation level and RNase1 expression level of the reference population and reach or exceed the set threshold, determine the said The subject had RDAA-positive disease.

3. The method of any one of the preceding embodiments, wherein the set threshold for ALK phosphorylation level is a positive immunohistochemical stain for ALK phosphorylation.

4. The method of any one of the preceding embodiments, wherein the set threshold for RNase1 expression level is an Rnasel expression level in plasma ≥ 418 ng/ml or a positive immunohistochemical staining result for Rnase1.

5. The method of any one of the preceding embodiments, wherein the detection is performed on a sample to be tested from the subject's tissue, cells and/or body fluids;

Preferably, tissue sections and/or blood from the subject;

Preferably, tumor tissue sections and/or blood from the subject;

Preferably, lung or pancreatic cancer tissue sections and/or blood from the subject;

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

6. The method of any one of the preceding embodiments, wherein the detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level are independently selected from the group consisting of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. of one or more.

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

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

9. A method for treating diseases, which at least includes the following steps:

Administration of anaplastic lymphoma kinase (ALK) inhibitors to patients with RDAA-positive disease, defined as disease resulting from RNase1-driven ALK activation,

wherein samples from patients with RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression levels in plasma of ≥418ng/ml or positive immunohistochemical staining results for Rnase1, and positive for Immunohistochemical staining results of ALK phosphorylation, and

wherein said RDAA-positive disease is RDAA-positive non-small cell lung cancer, and

The ALK inhibitor does not include ensartinib.

10. The method of embodiment 9, wherein the sample is from tissue, cells and/or body fluids of a patient;

Preferably, tissue sections and/or blood from the patient;

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

Preferably, non-small cell lung cancer tissue sections and/or blood from the patient.

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.

12. The method of any one of embodiments 9-11, the ALK inhibitor includes any one of crizotinib, ceritinib, lorlatinib, alectinib, and brigatinib species or combination thereof.

13. An anti-RNase1 antibody or antigen-binding portion thereof, comprising:

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:20, having as SEQ ID NO:20 The heavy chain CDR2 of the amino acid sequence shown in NO:21, and the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:22;

14. The anti-RNase1 antibody or antigen-binding portion thereof according to embodiment 13, comprising:

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 24;

15. An anti-ALK phosphorylated antibody or antigen-binding portion thereof, comprising:

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:89, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:90, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:91 , a heavy chain CDR1 having an amino acid sequence shown in SEQ ID NO:92, a heavy chain CDR2 having an amino acid sequence shown in SEQ ID NO:93, and a heavy chain CDR2 having an amino acid sequence shown in SEQ ID NO:94 chain CDR3;

16. The anti-ALK phosphorylated antibody or antigen-binding portion thereof according to embodiment 15, comprising:

A light chain variable region having an amino acid sequence set forth in SEQ ID NO: 103, and a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 104; or

17. A test kit containing:

1) Reagents for detecting ALK phosphorylation levels; and

2) Reagents for detecting RNase1 expression levels.

18. The kit of embodiment 17, wherein

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

19. The kit of embodiment 17, wherein

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

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

21. Use of a combination of a reagent for detecting ALK phosphorylation levels and a reagent for detecting RNase1 expression levels in preparing a kit for detecting RDAA-positive diseases, where patients with RDAA-positive diseases have negative ALK gene rearrangement test results, Rnase1 expression level in plasma of ≥418ng/ml or positive immunohistochemical staining for Rnase1 and positive immunohistochemical staining for ALK phosphorylation.

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 preparing a kit for detecting RDAA-positive disease, in which patients with RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression level in plasma ≥418ng/ml or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation.

23. Use of an ALK inhibitor in the preparation of a medicament for the treatment of RDAA-positive disease, wherein samples from patients suffering from said RDAA-positive disease have negative ALK gene rearrangement detection results, ≥418ng/ml in plasma Rnase1 expression level or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation, and wherein the RDAA-positive disease is RDAA-positive non-small cell lung cancer, and the ALK inhibition Agents do not include ensartinib.

24. The use of embodiment 23, wherein the sample is from tissue, cells and/or body fluids of a patient;

Preferably, tissue sections and/or blood from the patient;

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

25. The use of embodiment 23 or 24, wherein the ALK inhibitor includes any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib or any thereof. combination.

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.

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

28. A device for diagnosing RDAA-positive diseases, comprising:

4) Result output department.

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

Preferably, tissue sections and/or blood from the subject;

Preferably, tumor tissue sections and/or blood from the subject; Preferably, lung cancer or pancreatic cancer tissue sections and/or blood from the subject;

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

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

30. The device of embodiment 28, wherein the data analysis section compares the collected data of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level with a set threshold.

31. The device of embodiment 28, wherein when analysis by the data analysis part indicates

1) ALK gene rearrangement is negative,

2) The immunohistochemical staining result for ALK phosphorylation is positive, and

3) When the expression level of Rnase1 in plasma is ≥418ng/ml or the immunohistochemical staining result for Rnase1 is positive, it is determined to be an RDAA-positive disease.

32. The device of embodiment 28, wherein the detection part, the data collection part, the data analysis part and the result output part can be separated into separate equipment units or form an integrated equipment.

The technical solution of the present disclosure will be explained more clearly and specifically by way of illustration in conjunction with the embodiments below. It should be understood that these examples are for illustrative purposes only and are in no way intended to limit the scope of the present disclosure. The scope of the present disclosure is limited only by the claims.

Example

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

Preparation of RNase1 antibodies

1. Animal selection: purebred BALB/C mice

2. Selection of RNase1 antibody immunization regimen

Prokaryotic cell purified RNase1 protein was used as antigen (5 mg in total) in combination with adjuvant. Commonly used adjuvants: Freund's complete adjuvant, Freund's incomplete adjuvant.

a: Primary immunization antigen 50 μg plus Freund's complete adjuvant subcutaneous injection at multiple points or intraspleen injection (total 1ml, 0.2ml/point);

b: After 3 weeks, the second immunization dose is the same as above, plus Freund's incomplete adjuvant for intraperitoneal injection, the dose is 0.3ml;

c: After 3 weeks, the third immunization dose is the same as above, without adjuvant, and injected intraperitoneally;

d: After 3 weeks, boost immunity, dose 0.5mg, intraperitoneal injection;

e: After 3 days, the spleen was removed and fused.

3. Cell fusion

1. Preparation before cell fusion

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

Myeloma cells were cultured in DMEM medium. The concentration of calf serum is 10% and the cell concentration is 1×10 ⁵ /ml. When cells are in the mid-phase of logarithmic growth, passage them at a ratio of 1:3. Passage every 3 days. (Regular treatment with 8-azaguanine makes surviving cells uniformly sensitive to HAT.)

(2) Feeder cells:

Mouse peritoneal macrophages were used in this study, and the amount was 10 ⁵ cells/well.

2. Steps of cell fusion

(1) Preparation of feeder cell layer:

(2) Preparation of immune spleen cells

(3) Preparation of myeloma cells

(4) Fusion

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

②Add 1ml of 45% PEG solution pre-warmed at 37°C within 90 seconds, and shake slightly while adding. 37°C water bath for 90 seconds.

③Add incomplete culture medium pre-warmed at 37°C to terminate the PEG effect. Add 1ml, 2ml, 3ml, 4ml, 5ml and 6ml every 2 minutes.

④Centrifuge, 800rpm, 6min.

⑤Add supernatant and resuspend in HAT selective culture medium containing 20% calf serum.

⑥ Add the above cells to the 96-well plate with the feeder cell layer, and add 100 μl to each well. One immune spleen can inoculate four 96-well plates.

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

3. Select hybridoma cells and antibody detection

1) HAT selects hybridoma cells and culture fusion cells in HAT selection culture medium. A large number of tumor cells will die within 1 to 2 days of culture. After 3 to 4 days, the tumor cells will disappear and the hybrid cells will form small colonies. The HAT selection culture medium will be maintained for 7 to 10 days. After 3 days, switch to HT culture medium, maintain it for another 2 weeks, and then switch to normal culture medium. During the selection culture period, when the hybridoma cells cover 1/10 of the bottom area of the well, specific antibodies can be detected and the required hybridoma cell lines can be screened out. During the selection culture period, half of the culture medium is generally changed every 2 to 3 days.

4. Cloning of hybridomas producing RNase1 antibodies

1) Prepare the feeder cell layer (same cell fusion) one day before cloning.

2) Gently dry the hybridoma cells to be cloned from the culture wells and count.

3) Adjust the cells to 10 cells/ml.

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

5) Change the medium on the 7th day, and then every 2 days.

6) Cell clone formation can be seen in 8 days, and antibody activity can be detected in a timely manner.

7) Move the cells in the positive wells to a 24-well plate for expansion and culture.

8) Cryopreservation of cell clones.

5. Mass production of RNase1 monoclonal antibodies

Hybridoma cells were inoculated in vivo to prepare ascites.

Preparation of ascites: Routinely intraperitoneally inject 0.5 ml of Pristane (phytane) or liquid paraffin into BALB/C mice. Two weeks later, 1×10 ⁶ hybridoma cells are intraperitoneally injected. Ascites can be produced 10 days after the cells are inoculated. The mice were killed, and the ascites was sucked into the test tube with a dropper. Generally, 10 ml of ascites could be obtained from one mouse.

The monoclonal antibody content in ascites fluid can reach 5mg/ml.

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

Use these 5 antibodies to perform Western blot hybridization experiments (western blot). The experimental steps are as follows:

1. Configuration of protein lysis solution

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

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

2. Total protein extraction

1) Take out the cell culture dish from which the protein needs to be extracted from the 37°C constant temperature incubator, and evaluate the cell density under a microscope;

2) Place the culture dish on ice (the entire operation steps are performed on ice), suck off the upper culture medium, and wash twice with cold PBS;

3) Add 20-50 μl of PBS and protein lysis solution to each well in a ratio of 1:1 according to the cell density. After the liquid covers the surface of the culture dish, immediately use a cell scraper to fully stir and lyse the added solution and cells attached to the surface of the culture dish. , and collected into EP tube;

4) Heating in a constant-temperature metal bath at 100°C for 10 minutes to denature the protein, immediately detached at 12,000rpm, and stored at 4°C for short-term use.

3. Quantification of protein concentration (BCA method)

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;

2) Prepare BCA mixed solution: According to the number of test samples and corresponding gradient standards set in the experiment, prepare a light green BCA mixed working solution at the ratio of BCA reagent:Cu reagent = 50:1. Mix thoroughly and store it at room temperature for short-term use. ;

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

4) The microplate reader uses a wavelength of 562nm to detect the absorbance, record the OD value, and draw a protein standard curve;

5) Sample protein treatment: Take the 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;

6) Take the post-reaction sample and detect the OD value. Calculate the sample protein concentration based on the drawn protein standard curve, and calculate the sample volume during electrophoresis based on the protein loading volume. The specific calculation formula is: protein loading volume = protein loading. Amount/sample protein concentration;

7) Thoroughly mix the sample protein and the prepared protein loading buffer at a ratio of 20:1, and flash it at 12000 rpm in a -20°C refrigerator for later use.

4. SDS-PAGE gel electrophoresis

1) Preparation of separation gel: Select separation gels of different concentrations according to the molecular weight of the proteins detected in the experiment. The concentration of separation gel required for the detection of proteins RNase1, p-ALK (Y1604), and p-ALK (Y1282/1283) in this experiment is 10 %, 8%, 8%. The separating gel system is shown in Table 1. Install the rubber plate holder on a horizontal table, prepare the mixed solution (5ml) according to the separation gel system, and quickly and carefully pour it into the reserved gap between the rubber plates. Then slowly pour 100-200μl volume of isopropyl alcohol or absolute ethanol horizontally from left to right along the edge of the glue making board wall to flatten the glue surface, slightly lift and shake one side of the glue making rack to expel potential air bubbles, and finally let it sit. Place on a horizontal table at room temperature for 30-60 minutes until the separation gel solidifies;

Table 1 Separating gel configuration system (5ml)

2) Preparation of stacking gel: After the separation gel solidifies, carefully absorb the isopropyl alcohol or absolute ethanol and dry it under natural ventilation conditions. Then configure the upper glue (2ml) according to the concentrated gel system (as shown in Table 2), and quickly and smoothly pour it into the gap between the glue making plates until the liquid overflows. Insert the 15-hole comb slowly and smoothly. Let it sit on a horizontal table at room temperature for 30-60 minutes;

Table 2 Stacking gel configuration system (2ml)

3) After the upper layer of concentrated gel has solidified, carefully remove the gel plate from the gel preparation rack and secure it tightly in the electrophoresis tank. Carefully, slowly and vertically pull out the comb to leave a loading hole, and place the gel plate in the electrophoresis tank according to the gel. Pour an appropriate amount of electrophoresis solution for the number of plates;

4) Protein loading: Take out the protein sample stored in the -20°C refrigerator and wait for it to thaw and return to room temperature. Load equal amounts of protein in sequence according to the calculated protein loading volume, and reserve 1-2 wells to add 5 μl of protein loading indicator (marker) according to experimental needs;

5) Electrophoresis: After loading the sample, start electrophoresis at a constant voltage of 80V. When you see bead-like bubbles starting to appear at the bottom of the gel plate, it marks the start of electrophoresis. Then you can see that the bromophenol blue in the protein sample is compressed to form a straight line. According to the marker After judging that the protein has reached a clearly differentiated transparent separation gel interface, 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 transfer;

6) Membrane transfer: Prepare the filter paper required for membrane transfer in advance, install the methanol box for activation, transfer clamp, PVDF membrane, etc., and pre-cool the transfer liquid 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 the height slightly exceeds the thickness of the filter paper. Methanol wets and activates the PVDF membrane, which is then placed on filter paper. Take out the gel plate after electrophoresis, peel off the protein gel from the gel plate, trim the borders appropriately and place it on the PVDF membrane, cover it with a layer of filter paper, and continuously remove the bubbles generated during the operation. Finally, follow the principle of "black glue and white film", use the transparent surface of the film transfer clamp as the bottom layer, cover it with a sponge pad, and place the filter paper and PVDF membrane clamped in the previous step horizontally, then cover it with a layer of sponge pad, and transfer Align the black surface of the film clip and close it to fasten the buckle of the transfer film clip. Fix the buckled transfer clamp in the transfer tank, add transfer liquid until it submerges the transfer indicator line, place the transfer tank in a suitable container, and start transfer. Add ice water to the container to prepare for cooling. Use 300mA constant flow to transfer the membrane, and select the appropriate transfer time according to the size of the target protein. The transfer times for R1 and p-ALK proteins are 1 and 2 hours respectively;

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

8) Incubate primary antibody: Use primary antibody diluent, 5% milk or 5% BSA according to the proportion to configure the primary antibody. The concentration of the primary antibody configuration in this experiment is 1:1000 (the sources of the antibodies in this experiment are self-made monoclonal mouse antibodies). Each antibody is configured with 4ml. Prepare the antibody before incubation, pour it into the corresponding compartment of the incubation box and place it on ice at low temperature to maintain antibody activity. Remove the PVDF membrane from the 5% skimmed milk and rinse slightly with PBST to remove residual milk. According to the size of the protein band detected in the experiment, use the color marker as a scale to form the membrane into a strip shape, and then place it into the grid corresponding to the antibody. After sealing, place it in a shaker at 4°C and shake slowly for 12 hours;

9) Recover the primary antibody: Recover the primary antibody after 12 hours;

10) Incubate the secondary antibody: use horseradish enzyme-labeled goat anti-mouse IgG (H+L) secondary antibody (Zhongshan Jinqiao, Cat. No.: ZB-2305) according to the ratio of 1:5000, use 5% skim milk prepared with PBST and prepare accordingly in advance. Secondary antibody diluent. Rinse the PVDF membrane with PBST at least 3 times for 5-10 minutes each time. Pour off the liquid after rinsing. Then pour the secondary antibody dilution into the PVDF membrane grid of the corresponding species and shake slowly on the shaker for 1 hour;

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

12) ECL development: Pre-configure the ultra-sensitive ECL developer (UltraSignal ultra-sensitive ECL chemiluminescent substrate, Sizhengbai, product number: 4AW011-100). Mix equal volumes of ECL A solution and B solution in an EP tube and protect from light. Reserve at low temperature. Before development, place the PVDF film on the foam board, dry the remaining PBST slightly, and then drop the prepared ultra-sensitive ECL developer on it according to the size of the film until both sides are completely infiltrated. Exposed in Bio-rad imaging instrument.

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

Example 2: Preparation of anti-ALK phosphorylated antibodies

The preparation process of this antibody is the same as that of the above-mentioned RNase1 antibody, except that the antigen is used. The antigen used to prepare ALK phosphorylated antibodies is a synthetic ALK protein phosphorylated peptide, a total of 2 ALK protein phosphorylated peptides, 5 mg each.

Nine anti-ALK phosphorylated antibodies were obtained, and their specific amino acid sequences are shown in the table below.

Use these 9 antibodies to perform Western blot hybridization experiments. The experimental method is the same as the western blot process of RNase1 in Example 1.

The experimental results are shown in Figure 2. These nine ALK phosphorylated monoclonal antibodies can specifically recognize endogenous ALK phosphorylated protein. The target band is clear and consistent in size with the ALK phosphorylated protein, and there are no non-specific bands. The results show that these nine anti-ALK phosphorylated antibodies specifically bind to phosphorylated ALK and can be effectively used to detect ALK phosphorylation levels in samples.

Example 3: Killing effect of ALK inhibitor on RDAA-positive disease cells

The second-generation sequencing method was used to detect the expression level of ALK gene rearrangement in the tumor tissue section samples of 3 subjects: the patient's surgical or puncture pathological tissue samples used second-generation high-throughput gene detection technology (Shenzhen BGI Co., Ltd. ) The diagnosis is that there is no ALK gene rearrangement and related mutations, and the diagnosis is negative for ALK gene rearrangement.

Subjects who are negative for ALK gene rearrangement continue to undergo the following tests.

Enzyme-linked immunosorbent (ELISA) method was used to detect the expression level of RNase1 in plasma samples from three ALK gene rearrangement-negative subjects. The detection method is as follows:

Elisa detection reagents and procedures use Rnase1Elisa kit (product name and item number: ELISA Kit for Ribonuclease1, human; Product Number: SEA297Hu)

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

2. Experimental steps

1. Prepare all reagents, samples and standards;

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

3. Add 100μL/well of the prepared detection reagent A and incubate at 37°C for 1 hour;

4. Aspirate and clean 3 times;

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

6. Aspirate and clean 5 times;

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

8. Add 50 μL/well of stop solution. Microplate reader reads at 450nm.

Calculate the concentration of RNase1 in the sample based on the absorbance value, using the standard as a reference, and the dilution factor. The concentration of 418ng/ml was used as the cut-off point to distinguish high and low expression of RNase1. (The average concentration of RNase1 in the plasma of patients with non-small cell lung cancer is 418ng/ml).

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:

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

(2) Paraffin sections were dewaxed with xylene and hydrated with ethanol at different concentrations;

(3) 3% hydrogen peroxide inhibits endogenous peroxidase for 15 minutes;

(4) Wash 3 times with PBS, 5 minutes each time;

(5) Immerse the slices in citric acid buffer and use microwave to repair them. Microwave on high heat for 5 minutes and on medium heat for 5 minutes (or heat at high pressure at 120°C for 2 minutes);

(6) After naturally cooling to room temperature (about 60 minutes), rinse with PBS 3 times, 5 minutes each time;

(7) Remove the excess liquid on the slide, add RNase1 monoclonal antibody dropwise, the antibody dilution concentration is 1:100 to 1:1000, place it in a humid box and keep it overnight at 4°C;

(8) On the second day, take it out and leave it at room temperature for 1 hour, then wash it 3 times with PBS for 5 minutes each time;

(9) Add polymerase-labeled secondary antibody dropwise and leave it at room temperature for 30 minutes, rinse with PBS 3 times, 5 minutes each time;

(10) DAB chromogenic solution develops color for 3 minutes and observes under the microscope;

(11) Run water to stop color development and counterstain with hematoxylin for 1 minute;

(12) Rinse with tap water and differentiate the differentiation solution for 5 seconds;

(13) Rinse the sections, dehydrate them with ethanol, clear them with xylene, and seal them with neutral gum.

Method for determining immunohistochemistry results: All hematoxylin-stained sections were reviewed independently by two pathologists. Cancer specimens contain greater than 70% tumor content. A double-blind method was used to evaluate RNase1 staining, and the location of cells with positive RNase1 expression was observed and determined, which showed brown-yellow or tan particles. The immunohistochemical staining score was calculated by multiplying the number of positively stained cells by the staining intensity. Observe the tissue specimen under a light microscope and score according to the percentage of positively stained cells: 0: negative expression; 1 point: the number of positive cells is <15%; 2 points: 15-50% of positive cells; 3 points: positive cells ≥ 50%; the staining intensity is scored according to the depth of color development: 0 points: no positive; 1 point: light yellow; 2 points: brown; 3 points: tan. The immunohistochemical staining score = 4 points was used as the cut-off point to distinguish high and low RNase1 expression levels.

Using the HIC method described above, ALK phosphorylated monoclonal antibodies (CDA04 among the 9 ALK phosphorylated monoclonal antibodies shown in the Antibody Preparation section) were used to detect ALK phosphorylation levels in tumor tissue samples from 3 subjects. The immunohistochemical staining score = 4 points was used as the cut-off point to distinguish high and low ALK phosphorylation levels.

According to the following criteria, they are divided into RDAA-positive subjects and non-RDAA-positive (RDAA-negative) subjects:

RDAA-positive subjects: The expression level of Rnase1 in plasma is ≥418ng/ml or the immunohistochemical staining score for Rnase1 is ≥4 points; and the immunohistochemical staining score for ALK phosphorylation is ≥4 points;

RDAA negative subjects: Rnase1 expression level in plasma is <418ng/ml or immunohistochemical staining score for Rnase1 is <4 points; and immunohistochemical staining score for ALK phosphorylation is <4 points.

Cancer cells from 1 RDAA-positive and 1 RDAA-negative non-small cell lung cancer subject were treated with placebo (PBS buffer), crizotinib, ceritininb, and loratinib, respectively. Cells were treated with six ALK inhibitors: Loratinib, Alectinib, Brigatinib and Ensartinib (the final concentration of the above drugs was 1 μg per ml of culture) Base, cell density is 50,000 cells per ml of culture medium), observe cell survival, count the number of surviving cells every day, and draw a growth curve.

The experimental results are shown in Figure 3. The abscissa axis is the medication time in (days), and the ordinate axis is the relative cell number. The results showed that 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.

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

Cancer cells from an RDAA-positive non-small cell lung cancer patient were inoculated into the abdominal subcutaneous tissue of nude mice. The number of cells inoculated was 500,000 cells per mouse. Mass growth under the abdominal skin of the mice could be observed 10 days after inoculation. , that is, RDAA-positive tumor-bearing mice were constructed.

Tumor-bearing mice bearing RDAA-positive tumors were randomly divided into seven groups (5 mice in each group), and placebo (PBS buffer), crizotinib, ceritinib, lorlatinib, and aletinib were orally administered. The dose of ALK inhibitor, brigatinib, or ensartinib (ALK inhibitor) was 25 mg per kilogram of body weight per day, and the dose of placebo was 2 μl per mouse per day, starting 2 weeks after tumor inoculation. , stop administration from the fourth week).

The experimental results are shown in Figures 4 and 5. The results showed that compared with mice in the placebo group, 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).

Example 5: Therapeutic effect of ALK inhibitor on RDAA positive patients

Forty patients from West China Hospital of Sichuan University have been determined to be negative for ALK gene rearrangement according to the detection method in Example 3. Then, use this kit to measure the RNase1 expression level in the patient's plasma samples and the RNase1 protein expression level and ALK phosphorylation level in the tumor tissue samples, and divide them into RDAA-positive patients and non-RDAA-positive patients according to the following standards:

RDAA-positive patients: The expression level of Rnase1 in plasma is ≥418ng/ml or the immunohistochemical staining score for Rnase1 is ≥4 points; and the immunohistochemical staining score for ALK phosphorylation is ≥4 points;

RDAA-negative patients: The expression level of Rnase1 in plasma is <418ng/ml or the immunohistochemical staining score for Rnase1 is <4 points; and the immunohistochemical staining score for ALK phosphorylation is <4 points.

A total of three RDAA-positive patients were identified, all of whom were patients with non-small cell lung cancer. These three patients were administered crizotinib at a dose of 250 mg twice a day. The CT results before and after 4 weeks of continuous use are shown in Figure 6. It can be clearly seen that crizotinib significantly inhibited tumor growth in RDAA-positive patients.

Example 6: Therapeutic effect of ALK inhibitor on patients with RDAA-positive PDAC

Through the second-generation high-throughput gene sequencing (NGS) method, 10 PDAC patients with negative ALK gene rearrangement were identified. Tumor tissue sections from these patients were subjected to immunohistochemical detection of RNase1 and ALK phosphorylation mentioned in this kit. Among them, the immunohistochemistry results of the tumor tissue section of one patient were RNase1 expression >4 points and ALK phosphorylation >4 points, and he was identified as an RDAA-positive PDAC patient. The patient's tumor cells were cultured and treated with placebo control (PBS buffer), Crizotinib, Ceritininb, Loratinib, Alectinib ( Cells were treated with six ALK inhibitors (Alectinib), Brigatinib and Ensartinib (the final concentration of the above drugs was 1 μg per ml of culture medium, and the cell density was 50,000 cells per ml). base), observe cell survival, count the number of surviving cells every day, and draw a growth curve.

The experimental results are shown in Figure 7. The abscissa axis is the medication time in (days), and the ordinate axis is the relative cell number. The results showed that 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

A method for diagnosing diseases, which at least includes the following steps:

1) Detect the subject's anaplastic lymphoma kinase (ALK) gene rearrangement, anaplastic lymphoma kinase phosphorylation level and RNase1 expression level; and

2) If the test result for anaplastic lymphoma kinase gene rearrangement is negative, and the ALK phosphorylation level and RNase1 expression level are both higher than the reference population level, then the disease is defined as a disease caused by RNase1-driven ALK activation (RDAA positive disease).
A method for diagnosing diseases, which at least includes the following steps:

1) Test the subject for anaplastic lymphoma kinase (ALK) gene rearrangement. If the test result for ALK gene rearrangement is negative, proceed with the following steps:

2) Detect the ALK phosphorylation level and RNase1 expression level of the subject; and

3) Compare the detected ALK phosphorylation level and RNase1 expression level with the ALK phosphorylation level and RNase1 expression level of the reference population respectively; and

4) Optionally, in the case where the detected ALK phosphorylation level and RNase1 expression level are respectively higher than the ALK phosphorylation level and RNase1 expression level of the reference population and reach or exceed the set threshold, determine the said The subject had RDAA-positive disease.
The method of any one of the preceding claims, wherein the set threshold for ALK phosphorylation levels is a positive immunohistochemical stain for ALK phosphorylation.
The method of any one of the preceding claims, wherein the set threshold for RNase1 expression level is an Rnasel expression level in plasma ≥ 418 ng/ml or a positive immunohistochemical staining result for Rnase1.
The method of any one of the preceding claims, wherein the detection is performed on a sample to be tested from a subject's tissues, cells and/or body fluids;

Preferably, tissue sections and/or blood from the subject;

Preferably, tumor tissue sections and/or blood from the subject;

Preferably, lung or pancreatic cancer tissue sections and/or blood from the subject;

Preferably, non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue sections and/or blood from the subject.
The method of any one of the preceding claims, wherein the detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level are independently selected from one of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. Kind or variety.
The method of any one of the preceding claims, wherein the detection of ALK gene rearrangements, ALK phosphorylation levels and/or RNase1 expression levels is performed by using an antibody or an antigen-binding portion thereof.
The method of any one of the preceding claims, wherein the subject is a suspected cancer patient, preferably the cancer is non-small cell lung cancer or pancreatic ductal adenocarcinoma.
A method for treating diseases, which at least includes the following steps:

Administration of anaplastic lymphoma kinase (ALK) inhibitors to patients with RDAA-positive disease, defined as disease resulting from RNase1-driven ALK activation,

wherein samples from patients with RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression levels in plasma of ≥418ng/ml or positive immunohistochemical staining results for Rnase1, and positive for Immunohistochemical staining results of ALK phosphorylation, and

wherein said RDAA-positive disease is RDAA-positive non-small cell lung cancer, and

The ALK inhibitor does not include ensartinib.
The method of claim 9, wherein the sample is from tissue, cells and/or body fluids of the patient;

Preferably, tissue sections and/or blood from the patient;

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

Preferably, non-small cell lung cancer tissue sections and/or blood from the patient.
The method of claim 9 or 10, wherein the ALK inhibitor includes an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.
The method of any one of claims 9-11, the ALK inhibitor includes any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib or its combination.
An anti-RNase1 antibody or antigen-binding portion thereof, comprising:

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 6 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:12, having as SEQ ID NO:12 The heavy chain CDR2 of the amino acid sequence shown in NO:13, and the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:14;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 20, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 21, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 22 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 25, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 26, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 27 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 28, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 29, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 30 chain CDR3; or

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:33, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:34, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:35 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 36, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 37, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 38 Chain CDR3.
The anti-RNase1 antibody or antigen-binding portion thereof according to claim 13, comprising:

A light chain variable region having an amino acid sequence as shown in SEQ ID NO:7, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:8;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 24;

A light chain variable region having an amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 32; or

A light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 39, and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 40.
An anti-ALK phosphorylated antibody or antigen-binding portion thereof, comprising:

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:41, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:42, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:43 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:44, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:45, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:46 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 49, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 50, light chain CDR3 having the amino acid sequence 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 CDR2 having an amino acid sequence as shown in SEQ ID NO:54 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:57, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:58, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:59 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 60, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 61, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 62 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 65, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 66, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 67 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:68, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:69, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:70 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:73, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:74, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:75 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:76, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:77, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:78 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:81, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:82, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:83 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:84, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:85, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:86 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:89, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:90, light chain CDR3 having the amino acid sequence shown in SEQ ID NO:91 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 92, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 93, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 94 chain CDR3;

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO:97, light chain CDR2 having the amino acid sequence shown in SEQ ID NO:98, light chain CDR3 having the amino acid sequence 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 CDR2 having an amino acid sequence as shown in SEQ ID NO: 102 chain CDR3; or

Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 105, light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 106, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 107 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 108, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 109, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 110 Chain CDR3.
The anti-ALK phosphorylated antibody or antigen-binding portion thereof according to claim 15, comprising:

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 47, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 48;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 55, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 56;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 63, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 64;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO:71, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:72;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO:79, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:80;

A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 87, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 88;

A light chain variable region having an amino acid sequence shown in SEQ ID NO: 95, and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 96;

A light chain variable region having an amino acid sequence set forth in SEQ ID NO: 103, and a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 104; or

A light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 111, and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 112.
A kit containing:

1) Reagents for detecting ALK phosphorylation levels; and

2) Reagents for detecting RNase1 expression levels.
The kit of claim 17, wherein

The reagent for detecting ALK phosphorylation level includes the anti-ALK phosphorylation antibody or the antigen-binding portion thereof according to any one of claims 15-16.
The kit of claim 17, wherein

The reagent for detecting RNase1 expression level includes the anti-RNase1 antibody or antigen-binding portion thereof according to any one of claims 13-14.
The kit according to any one of claims 17 to 19, further comprising a reagent for detecting ALK gene rearrangement.
Use of a combination of a reagent for detecting ALK phosphorylation level and a reagent for detecting RNase1 expression level in preparing a kit for detecting RDAA-positive disease, where patients with the RDAA-positive disease have negative ALK gene rearrangement test results, ≥418ng /ml plasma Rnase1 expression level or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation.
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 preparing a kit for detecting RDAA-positive disease in which patients with negative ALK Gene rearrangement test results, Rnase1 expression level in plasma ≥418ng/ml or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation.
Use of an ALK inhibitor in the preparation of a medicament for the treatment of an RDAA-positive disease, wherein a sample from a patient suffering from said RDAA-positive disease has a negative ALK gene rearrangement test result, Rnase1 expression in plasma of ≥418ng/ml level 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 including ensartinib.
The use of claim 23, wherein the sample is from tissue, cells and/or body fluids of a patient;

Preferably, tissue sections and/or blood from the patient;

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

Preferably, non-small cell lung cancer tissue sections and/or blood from the patient.
The use of claim 23 or 24, wherein the ALK inhibitor includes any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib or a combination thereof.
The use of any one of claims 23-25, wherein the ALK inhibitor comprises an antibody or antigen-binding portion thereof capable of inhibiting ALK phosphorylation.
The use of any one of claims 23-26, wherein the ALK inhibitor comprises the anti-ALK phosphorylated antibody or antigen-binding portion thereof of any one of claims 15-16.
A device for diagnosing RDAA-positive diseases, which includes:

1) Detection part: including a reagent part that is respectively provided with a reagent capable of detecting the expression level of ALK gene rearrangement, a reagent that detects the ALK phosphorylation level, and a reagent that detects the RNase1 expression level, and a measurement part that performs detection;

2) Data collection department: collects the results output by the detection department;

3) Data Analysis Department: analyze the data collected by the Data Collection Department; and

4) Result output department.
The device of claim 28, wherein the detection part is performed on a sample to be tested from the subject, and the sample to be tested is from the tissue, cells and/or body fluids of the subject;

Preferably, tissue sections and/or blood from the subject;

Preferably, tumor tissue sections and/or blood from the subject; Preferably, lung cancer or pancreatic cancer tissue sections and/or blood from the subject;

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

The detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level are independently selected from one or more of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay.
The device of claim 28, wherein the data analysis section compares the collected data of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level with a set threshold.
The apparatus of claim 28, wherein when analysis by the data analysis section indicates

1) ALK gene rearrangement is negative,

2) The immunohistochemical staining result for ALK phosphorylation is positive, and

3) When the expression level of Rnase1 in plasma is ≥418ng/ml or the immunohistochemical staining result for Rnase1 is positive,

It is determined to be an RDAA-positive disease.
The device of claim 28, wherein the detection part, the data collection part, the data analysis part and the result output part can be separated into separate equipment units or form an integrated equipment.