TW202404596A - Method and kit for diagnosis and treatment of RDAA-positive diseases - Google Patents

Abstract

Provided in the present disclosure is a method for treating diseases, which at least comprises the following step: administering an anaplastic lymphoma kinase (ALK) inhibitor to a patient suffering from an RDAA-positive disease, the RDAA-positive disease being defined as a disease caused by RNase1 driven ALK activation, wherein a specimen from the patient suffering from the RDAA positive disease has an ALK gene rearrangement detection result that is negative, has an Rnase1 expression level in plasma that is ≥ 418 ng/mL or has an Rnase1-targeted immunohistochemical staining result that is positive, and has an ALK-phosphorylation-targeted immunohistochemical staining result that is positive, and the patient suffering from the RDAA-positive disease is not a patient with non-small cell lung cancer.

Description

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

This disclosure relates to the field of biomedical technology, 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.

ALK (anaplastic lymphoma kinase, 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; Alk gene priming mutations; 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), but 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 (ie, ALK gene rearrangement negative), no relevant cancer-promoting mechanism has been discovered, and there are no effective targeted treatments.

In view of the urgent problem that currently there are only effective treatments for patients with positive ALK gene rearrangements (i.e., the administration of ALK inhibitors) in clinical practice, but no effective treatments have been found for patients with negative ALK gene rearrangements, the inventor 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 diseases can 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 the subject’s ALK gene rearrangement. 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, the subject is determined The subject has an RDAA-positive disease; and 5) Optionally, administer an ALK inhibitor to the subject with RDAA-positive disease.

In the third party of the present disclosure, the use of ALK inhibitors in the preparation of drugs for the treatment of non-small cell lung cancer and/or pancreatic ductal adenocarcinoma can be provided.

In a fourth aspect of the present disclosure, an anti-RNase1 antibody or an antigen-binding portion thereof may be provided, comprising: A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and an amine group as shown in SEQ ID NO: 3 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:4, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:5, and a heavy chain CDR2 with an amino acid sequence as SEQ ID NO:5 Heavy chain CDR3 of the amino acid sequence shown in NO:6; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:9, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:10, and an amine group as shown in SEQ ID NO:11 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:12, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:13, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:13 Heavy chain CDR3 of the amino acid sequence shown in NO:14; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 18, and an amine group as shown in SEQ ID NO: 19 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:20, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:21, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:21 Heavy chain CDR3 of the amino acid sequence shown in NO:22; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:25, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:26, and an amine group as shown in SEQ ID NO:27 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:28, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:29, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:28 Heavy chain CDR3 of the amino acid sequence shown in NO:30; or A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:33, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:34, and an amine group as shown in SEQ ID NO:35 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:36, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:37, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:37 Heavy chain CDR3 of the amino acid sequence shown in NO:38.

In a fifth aspect of the present disclosure, an anti-ALK phosphorylated antibody or an antigen-binding portion thereof can be provided, which includes: A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:41, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:42, and an amine group as shown in SEQ ID NO:43 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:44, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:45, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:44 Heavy chain CDR3 of the amino acid sequence shown in NO:46; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 49, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 50, having an amine group as shown in SEQ ID NO: 51 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 52, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 53, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 52 Heavy chain CDR3 of the amino acid sequence shown in NO:54; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:57, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:58, and an amine group as shown in SEQ ID NO:59 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:60, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:61, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:61 Heavy chain CDR3 of the amino acid sequence shown in NO: 62; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:65, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:66, and an amine group as shown in SEQ ID NO:67 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:68, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:69, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:69 Heavy chain CDR3 of the amino acid sequence shown in NO:70; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:73, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:74, and an amine group as shown in SEQ ID NO:75 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:76, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:77, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:77 Heavy chain CDR3 of the amino acid sequence shown in NO:78; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 81, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 82, having an amine group as shown in SEQ ID NO: 83 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 84, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 85, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 85 NO: Heavy chain CDR3 of the amino acid sequence shown in 86; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:89, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:90, having an amine group as shown in SEQ ID NO:91 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:92, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:93, and a heavy chain CDR2 with an amino acid sequence as SEQ ID NO:93 Heavy chain CDR3 of the amino acid sequence shown in NO: 94; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:97, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:98, having an amine group as shown in SEQ ID NO:99 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:100, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:101, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:101 Heavy chain CDR3 of the amino acid sequence shown in NO:102; or A light chain CDR1 having an amino acid sequence shown in SEQ ID NO: 105, a light chain CDR2 having an amino acid sequence shown in SEQ ID NO: 106, having an amine group shown in SEQ ID NO: 107 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:108, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:109, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:109 Heavy chain CDR3 of the amino acid sequence shown in NO:110.

In a sixth aspect of the present disclosure, a kit may be provided, which may include: Reagents for detecting ALK phosphorylation levels; and A reagent for detecting RNase1 expression levels.

In a seventh aspect of the present disclosure, there can be provided 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, wherein patients with RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression level in plasma ≥418 ng/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 ALK gene rearrangement expression level, a reagent for detecting ALK phosphorylation level, and a reagent for detecting RNase1 expression level in preparing a kit for detecting RDAA-positive diseases can be provided. , patients with RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression levels in plasma of ≥418 ng/ml or positive immunohistochemical staining results for Rnase1, and positive ALK phosphorylation. Immunohistochemical 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, ≥418 ng/ml. Rnase1 expression levels in plasma 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 can be provided, which includes: 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; Data acquisition department: collects the results output by the testing department; Data Analysis Department: analyzes the data collected by the Data Acquisition Department; and Result output section.

Detailed implementation

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 invention belongs. The terminology used in the description of the disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

When used in this article, 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 homologs.

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 VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody reagent (immunohistochemistry).

When used in this article, the term "Rnase1" refers to ribonuclease 1 (ribonuclease1), which belongs to the human secreted ribonuclease family. For more 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” refers to a patient who 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, an RNase1 expression level in plasma of ≥418 ng/ml or a positive immunohistochemical stain for RNase1, and a positive ALK Immunohistochemical staining results of phosphorylation.

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 ALK inhibitors can achieve effective therapeutic effects in patients with RDAA-positive diseases. Therefore, ALK inhibitors have good application prospects in the preparation of drugs for the treatment of RDAA-positive diseases.

In a first aspect of the present disclosure, a method for diagnosing and/or treating diseases can be provided, which at least includes the following steps: 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 both 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 a level of ALK phosphorylation that is negative for immunohistochemical staining for ALK phosphorylation. In some embodiments, the ALK phosphorylation level of the reference population is 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 <418 ng/ml or an immunohistochemical staining score for Rnasel <4 points.

In some embodiments, the threshold for ALK phosphorylation levels 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 protein 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.

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.

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 the subject's ALK gene rearrangement expression level. 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, when 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, it is determined that the subject is The subject has an RDAA-positive disease; and 5) Optionally, administer an ALK inhibitor to a subject with RDAA-positive disease.

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 a level of ALK phosphorylation that is negative for immunohistochemical staining for ALK phosphorylation. In some embodiments, the ALK phosphorylation level of the reference population is 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 <418 ng/ml or an immunohistochemical staining score for Rnasel <4 points.

In some embodiments, the threshold for ALK phosphorylation levels 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 protein 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.

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.

In the third party of the present disclosure, the use of ALK inhibitors in the preparation of drugs for the treatment of non-small cell lung cancer and/or pancreatic ductal adenocarcinoma can be provided.

In a fourth aspect of the present disclosure, an anti-RNase1 antibody or an antigen-binding portion thereof may be provided, comprising: A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and an amine group as shown in SEQ ID NO: 3 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:4, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:5, and a heavy chain CDR2 with an amino acid sequence as SEQ ID NO:5 Heavy chain CDR3 of the amino acid sequence shown in NO:6; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:9, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:10, and an amine group as shown in SEQ ID NO:11 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:12, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:13, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:13 Heavy chain CDR3 of the amino acid sequence shown in NO:14; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 18, and an amine group as shown in SEQ ID NO: 19 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:20, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:21, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:21 Heavy chain CDR3 of the amino acid sequence shown in NO:22; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:25, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:26, and an amine group as shown in SEQ ID NO:27 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:28, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:29, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:28 Heavy chain CDR3 of the amino acid sequence shown in NO:30; or A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:33, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:34, and an amine group as shown in SEQ ID NO:35 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:36, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:37, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:37 Heavy chain CDR3 of the amino acid sequence shown in NO:38.

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 shown in SEQ ID NO: 15, and a heavy chain variable region having an amino acid sequence 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 as set forth in SEQ ID NO: 31, and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 32; or A light chain variable region having an amino acid sequence as shown in SEQ ID NO:39, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:40.

In a fifth aspect of the present disclosure, an anti-ALK phosphorylated antibody or an antigen-binding portion thereof can be provided, which includes: A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:41, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:42, and an amine group as shown in SEQ ID NO:43 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:44, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:45, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:44 Heavy chain CDR3 of the amino acid sequence shown in NO:46; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 49, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 50, having an amine group as shown in SEQ ID NO: 51 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 52, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 53, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 52 Heavy chain CDR3 of the amino acid sequence shown in NO:54; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:57, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:58, and an amine group as shown in SEQ ID NO:59 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:60, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:61, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:61 Heavy chain CDR3 of the amino acid sequence shown in NO: 62; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:65, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:66, and an amine group as shown in SEQ ID NO:67 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:68, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:69, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:69 Heavy chain CDR3 of the amino acid sequence shown in NO:70; A light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:73, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:74, and an amine group as shown in SEQ ID NO:75 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:76, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:77, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:77 Heavy chain CDR3 of the amino acid sequence shown in NO:78; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 81, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 82, having an amine group as shown in SEQ ID NO: 83 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 84, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 85, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 85 NO: Heavy chain CDR3 of the amino acid sequence shown in 86; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:89, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:90, having an amine group as shown in SEQ ID NO:91 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:92, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:93, and a heavy chain CDR2 with an amino acid sequence as SEQ ID NO:93 Heavy chain CDR3 of the amino acid sequence shown in NO: 94; Light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:97, light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:98, having an amine group as shown in SEQ ID NO:99 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:100, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:101, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:101 Heavy chain CDR3 of the amino acid sequence shown in NO:102; or A light chain CDR1 having an amino acid sequence shown in SEQ ID NO: 105, a light chain CDR2 having an amino acid sequence shown in SEQ ID NO: 106, having an amine group shown in SEQ ID NO: 107 A light chain CDR3 with an acid sequence, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:108, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:109, and a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:109 Heavy chain CDR3 of the amino acid sequence shown in NO:110.

In some embodiments, the anti-ALK phosphorylated antibody or antigen-binding portion thereof comprises: A light chain variable region having an amino acid sequence shown in SEQ ID NO: 47, and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 48; A light chain variable region having an amino acid sequence shown in SEQ ID NO: 55, and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 56; A light chain variable region having an amino acid sequence shown in SEQ ID NO: 63, and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 64; A light chain variable region having an amino acid sequence shown in SEQ ID NO:71, and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:72; A light chain variable region having an amino acid sequence shown in SEQ ID NO:79, and a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:80; A light chain variable region having an amino acid sequence shown in SEQ ID NO: 87, and a heavy chain variable region having an amino acid sequence 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 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 shown in SEQ ID NO: 111, and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 112.

In a sixth aspect of the present disclosure, a kit may be provided, which may include: Reagents for detecting ALK phosphorylation levels; and A reagent for detecting RNase1 expression levels.

In some embodiments, the reagent for detecting the level of ALK phosphorylation, 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, a reagent for detecting ALK phosphorylation levels includes an anti-ALK phosphorylation antibody or an antigen-binding portion thereof as described in the present disclosure. In some embodiments, a reagent for detecting RNase1 expression levels includes 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 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, wherein patients with RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression level in plasma ≥418 ng/ml or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation.

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.

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 RNase1 expression level 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, plasma Rnase1 expression levels ≥418 ng/ml or positive immunohistochemical staining results for Rnase1, and positive ALK phosphorylation. Immunohistochemical staining results.

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.

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, ≥418 ng/ml. Rnase1 expression levels in plasma or positive immunohistochemical staining results for RNase1 and positive immunohistochemical staining results for ALK phosphorylation.

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.

In a tenth aspect of the present disclosure, a device for diagnosing RDAA-positive diseases can be provided, which includes: 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; Data acquisition department: collects the results output by the testing department; Data Analysis Department: analyzes the data collected by the Data Acquisition Department; and Result output section.

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 detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level are independently selected from one or more of protein 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.

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

In some embodiments, the threshold for ALK phosphorylation levels 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 ≥418 ng/ml or for Rnase1 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 ≥418 ng/ml or for When the immunohistochemical staining score of RNase1 is ≥4 points, it is determined to be RDAA-positive disease.

In some embodiments, the detection part, the data acquisition part, the data analysis part and the result output part can be separated into separate equipment units or form 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 phospho- EL antibodies or antigen-binding portions thereof, kits, uses and devices, and vice versa, and various embodiments resulting from such combinations are considered part of the disclosure of this application.

In particular, the present disclosure provides the following implementations:

1. 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 said patients with RDAA-positive disease 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 results for ALK phosphorylation, and wherein the patient with RDAA-positive disease is not a non-small cell lung cancer patient.

2. The method of embodiment 1, 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, lung or pancreatic cancer tissue sections and/or blood from the subject; Preferably, pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient.

3. The method of any one of the preceding embodiments, wherein the patient is a patient with pancreatic ductal adenocarcinoma.

4. The method of any one of the preceding embodiments, the ALK inhibitor comprising an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.

5. The method according to any one of the preceding embodiments, wherein the ALK inhibitor includes crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, or ensartinib. any one or combination thereof.

6. 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, ≥418 ng/ml of plasma or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation, and wherein the patient with RDAA-positive disease is not a non-small cell lung cancer patient.

7. The use of embodiment 6, 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, lung or pancreatic cancer tissue sections and/or blood from the subject; Preferably, pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient.

8. The use according to embodiment 6 or 7, wherein the patient is a patient with pancreatic ductal adenocarcinoma.

9. The use of any one of embodiments 6-8, wherein the ALK inhibitor includes crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensa any one or combination thereof.

10. The use of any one of embodiments 6-9, wherein the ALK inhibitor comprises an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.

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: 3 weeks later, 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 harvested 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 will make the surviving cells uniformly sensitive to HAT.) (2) Feeder cells: This study selected mouse peritoneal macrophages, with an amount of 10 ⁵ cells/well.

2. Steps of cell fusion (1) Prepare feeder cell layer: Mice of the same strain as the immunized mice, commonly used BALB/C mice, 8 weeks ↓ Sacrifice by neck pull, soak in 75% alcohol for 5 minutes ↓ Cut with sterile scissors Skin, expose peritoneum ↓ Use a sterile syringe to inject 6ml of pre-cooled culture medium (do not puncture the intestinal tube) ↓ Wash repeatedly and suck out the washing fluid ↓ Put the washing fluid into a 10ml centrifuge tube, 1200rpm/separate for 5min ↓ Use 20% fetal calf serum (FCS) ), adjust the number of cells to 1×10 ⁵ /ml ↓ Add 100 μl/well to a 96-well plate ↓ Place in a 37°C CO2 incubator for culture (2) Prepare immune splenocytes 3 days after the last booster immunization The mice were sacrificed by neck pulling↓ Take the spleen aseptically and wash it once with the culture medium↓ Grind the spleen and pass it through a stainless steel mesh↓ Centrifuge and wash the cells twice with the culture medium↓ Count and take 10 ⁸ spleen lymphocyte suspension for later use (3) Preparation of myeloma Centrifuge logarithmic growth of myeloma cells ↓ Wash twice with serum-free culture medium ↓ Count and obtain 1×10 ⁷ cells for later use (4) Fusion ① Mix myeloma cells and splenocytes in a ratio of 1:10 or 1:5 Together, wash once with serum-free incomplete culture medium in a 50 ml centrifuge tube, centrifuge at 1200 rpm for 8 minutes; discard the supernatant and use a pipette to absorb 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 days. After ~10 days, switch to HT culture medium, maintain it for another 2 weeks, and switch to general 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) 1 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 0.5ml Pristane (phytane) or liquid paraffin into BALB/C mice 2 weeks later, and then intraperitoneally inject 1×10 ⁶ hybridoma cells. 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, 10ml 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. SEQ ID NO amino acid sequence instruction 1 QSLLYSNGNTY light chain CDR1 Anti-RNase1 antibody CDC02 2 LVS light chain CDR2 3 FQGTHFPRT light chain CDR3 4 GYTFTNYG Heavy chain CDR1 5 LNTYTGES Heavy chain CDR2 6 CARGDYGYDDPLDY Heavy chain CDR3 7 DIVMTQTPLTLSVTIGQPASISCKSGQSLLYSNGNTYLSWFLQRPGQSPNRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDVGVYYCFQGTHFPRTFGGGTKLEIK light chain variable region 8 QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSETSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDYWGQGTTVTVSS heavy chain variable region 9 QSLLHSGGKTY light chain CDR1 Anti-RNase1 antibody CDC03 10 LVS light chain CDR2 11 CQGSHFPWT light chain CDR3 12 GFTFSNYW Heavy chain CDR1 13 IRLRSDNYGT Heavy chain CDR2 14 TRGFAY Heavy chain CDR3 15 DIQMTQTPLTLSVTIGQPASMSCKPSQSLLHSGGKTYLNWFLLRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGLYYCCQGSHFPWTFGGGTKLEIK light chain variable region 16 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLRSDNYGTHYAESVKGRFTISRDDSRSSVYLQVNNLRAEDTGIYYCTRGFAYWGQGTLVTVSST heavy chain variable region 17 QSLLHSGGKTY light chain CDR1 Anti-RNase1 antibody CDC04 18 LVSKLDSGVPD light chain CDR2 19 CQGSHFPWT light chain CDR3 20 GFTFSNYY Heavy chain CDR1 twenty one IKLKSNNYATHFAESVQG Heavy chain CDR2 twenty two EDTGIFYCTRG Heavy chain CDR3 twenty three DVVMTQTPLTLSVTIGQPASISCKSSQSLLHSGGKTYLNWFLQRPGQSPKRLMYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCCQGSHFPWTFGGGTKLEMKR light chain variable region twenty four EVQLQESGGGLVQPGGSMKLSCVASGFTFSNYYMNWVRQSPEKGLEWVAEIKLKSNNYATHFAESVQGRFTISRDDSKSSVYLQMNNLRAEDTGIFYCTRGFCLLGPRDSGHCLC heavy chain variable region 25 QSLLNSDGKTY light chain CDR1 Anti-RNase1 antibody CDC05 26 LVS light chain CDR2 27 CQGTHFPWT light chain CDR3 28 GFTFSNYW Heavy chain CDR1 29 IRLKSNYAT Heavy chain CDR2 30 TRGFAY Heavy chain CDR3 31 FPLTLSVTIGQPASISCKSSQSLLNSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCCQGTHFPWTTFGGGTKLEIKRRTELEIKQ light chain variable region 32 EVKIEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSCVYLQMNNLRTEDTGIYYCTRGFAYWGQGTLVTVSA heavy chain variable region 33 QSLLYGNGKTY light chain CDR1 Anti-RNase1 antibody CDC06 34 LVS light chain CDR2 35 MQGTHFPRT light chain CDR3 36 GYVFSNYW Heavy chain CDR1 37 IYPGDGNS Heavy chain CDR2 38 VRWDGDYAV Heavy chain CDR3 39 DVVMTQTPLTLSVTIGQPASISCKSSQSLLYGNGKTYLNWFLQRPGQSPKRLIYLVSKLDSGAPVRFTGSGSGTDFTLKISRVEAEDLGIYYCMQGTHFPRTFGGGTKLEMKR light chain variable region 40 QAYLQQSGAELVRPGSSVKISCKASGYVFSNYWMNWVKQRPGQGLEWLGQIYPGDGNSNYNGKFKGKATLTAVKSSSTAYMQLSSLTSEDSAVYFCVRWDGDYAVWGAGTTVTVSS heavy chain variable region

Use these 5 antibodies to perform protein immunoblot hybridization experiments (western blot). The experimental steps are as follows: 1. Configuration of protein lysis solution 1. Protein lysate 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 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 is 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 mix the added solution with the cells attached to the surface of the culture dish. Lyse and collect into EP tube; 4) Heating in a constant-temperature metal bath at 100°C for 10 minutes to denature the protein, instantaneous separation at 12,000rpm, and short-term storage at 4°C for later 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) Configure the 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 leave at room temperature for a short period of time Save for later use; 3) Starting from the first well, add the standard in decreasing volumes from 20 μl to 0 μl into the protein standard wells, 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 after detecting 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 protein detected in the experiment. This experiment detects proteins RNase1, p-ALK (Y1604), p-ALK (Y1282/1283) ) The required separation gel concentrations are 10%, 8%, and 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 Separation gel configuration system (5ml) Element Volume(ml)(8%/10%) Deionized water 30% acrylamide 1.5M Tris-HCl (pH=6.8) 10% SDS 10% ammonium persulfate TEMED 2.3/1.9 1.3/1.7 1.3/1.3 0.05/0.05 0.05/0.05 0.003/0.002

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 stand on a horizontal table at room temperature for 30-60 minutes; Table 2 Stacking gel configuration system (2ml) Element Volume (ml) Deionized water 30% acrylamide 1.5M Tris-HCl (pH=8.8) 10% SDS 10% ammonium persulfate TEMED 1.1 2.5 1.3 0.05 0.05 0.002

3) After the upper layer of concentrated gel 3) is solid, 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 it in the electrophoresis tank. Pour the appropriate amount of electrophoresis solution according to the number of gel 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 clear 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 startup, 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 "black glue white film" principle, use the transparent surface of the film transfer clip as the bottom layer, cover it with a sponge pad, and place the filter paper and PVDF film sandwiched in the previous step horizontally, then cover it with a layer of sponge pad, and put the film transfer clip Close the black surface and fasten the film clip buckle. 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-configure 5% skim milk with PBST and an appropriate amount of PBST for cleaning. After the transfer is completed, open the transfer clamp and carefully take out 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 the primary antibody: Use primary antibody dilution, 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 in 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 chemiluminescence matrix, Sizhengbai, Cat. No.: 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. SEQ ID NO amino acid sequence instruction 41 QDINSY light chain CDR1 Anti-ALK phosphorylated antibody CDA04 42 RANRLVDGVPS light chain CDR2 43 LQYEEFPLT light chain CDR3 44 GFTFSSFG Heavy chain CDR1 45 INSGSSSIFYADTVKG Heavy chain CDR2 46 EDTAMYYC Heavy chain CDR3 47 DTTVTQSPSSMYASLGERVTITCKASQDINSYLTWLQQKPGKTPTTLIHRANRLVDGVPSRFSGSGSGQDYSLTISSLEFEDMGIYYCLQYEEFPLTLGAGTKLEIKR light chain variable region 48 EVKLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYINSGSSSIFYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCTRRGAAYGSSPFSYWGQGTLVTVSA heavy chain variable region 49 QDINSY light chain CDR1 Anti-ALK phosphorylated antibody CDA01 50 RANRLVDGVPS light chain CDR2 51 LQYDEFPLT light chain CDR3 52 GFTFSSFG Heavy chain CDR1 53 INSASSTVYYADTLKG Heavy chain CDR2 54 TRRGHLGLPFAY Heavy chain CDR3 55 DTTVTQSPSSIYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAKTKLEMKR light chain variable region 56 EVKLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYINSASSTVYYADTLKGRFTISRDNPNNALFLQMTSLRSEDTAIYYCTRRGHLGLPFAYWGQGTLVTVSA heavy chain variable region 57 QSLVHSNGNTY light chain CDR1 Anti-ALK phosphorylated antibody CDB04 58 KVSN light chain CDR2 59 SQSTHVPRT light chain CDR3 60 GYSFPVYF Heavy chain CDR1 61 INPYNGDTFYNQKFQV Heavy chain CDR2 62 EDSAVYYCGGSGDGGAWFAY Heavy chain CDR3 63 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPRTFGGGTKLEI light chain variable region 64 EVQLQESGPELVKPGTSVKISCKASGYSFPVYFVNWVKQSHGKSLEWIGRINPYNGDTFYNQKFQVKTTLTVDKSSSTAHMELLSLTSEDSAVYYCGGSGDGGAWFAYWGQGTLVTVSA heavy chain variable region 65 QDINNY light chain CDR1 Anti-ALK phosphorylated antibody CDA05 66 RANRLVDGVPS light chain CDR2 67 LQYDEFPLT light chain CDR3 68 GFTFSSFG Heavy chain CDR1 69 ISRGSNSI Heavy chain CDR2 70 ARRGGDYGSSPFAY Heavy chain CDR3 71 DTTVTQSPSSMYASLGERVTITCKASQDINNYLTWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLEMKR light chain variable region 72 EVKLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISRGSNSIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARRGGDYGSSPFAYWGQGTLVTVSA heavy chain variable region 73 QDINGY light chain CDR1 Anti-ALK phosphorylated antibody CDA02 74 RANRLVDGVPS light chain CDR2 75 EDMEIYYCLQYDEFPLT light chain CDR3 76 GFTFSSFG Heavy chain CDR1 77 INSDSTNIYYADRVKG Heavy chain CDR2 78 EDTAMYFCARRGSLGMIYGY Heavy chain CDR3 79 DTTVTQSPSSMYASLGERVTITCKASQDINGYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMEIYYCLQYDEFPLTFGAGTKLEMKR light chain variable region 80 DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWIAYINSDSTNIYYADRVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYFCARRGSLGMIYGYWGQGTLVTVSA heavy chain variable region 81 QDINSY light chain CDR1 Anti-ALK phosphorylated antibody CDB03 82 RANRLVDGVPS light chain CDR2 83 LQYEEFPLT light chain CDR3 84 GFTFSSFG Heavy chain CDR1 85 ISRGSNSI Heavy chain CDR2 86 ARRGLLGMVYAY Heavy chain CDR3 87 DTTVTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISRLEYEDMGSYYCLQYEEFPLTFGAGTKLEIKR light chain variable region 88 EVKLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWIAYINSDSTNIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYFCARRGLLGMVYAYWGQGTLVTVSA heavy chain variable region 89 QSLVHSNGNTY light chain CDR1 Anti-ALK phosphorylated antibody CDB01 90 KVS light chain CDR2 91 EDRGVYFCSQSTHVPRT light chain CDR3 92 GYSLTAYF Heavy chain CDR1 93 INLNNNGDIVYNQKFKD Heavy chain CDR2 94 GKSGDGGAWFAY Heavy chain CDR3 95 DIVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDRGVYFCSQSTHVPRTFGGGTKLEIKRRTELEIKQ light chain variable region 96 DVQLQESGPEMVKPGASVKISCKASGYSLTAYFINWVKQSHGKSLEWIGRINLNNGDIVYNQKFKDKATLTVDKSSRSAHMEFRSLTSEDSAVYFCGKSGDGGAWFAYWGQGTLVTVSA heavy chain variable region 97 QSLVHSNGNTY light chain CDR1 Anti-ALK phosphorylated antibody CDB02 98 KVS light chain CDR2 99 QSTHVPRT light chain CDR3 100 GYPLPAYF Heavy chain CDR1 101 INNLNNNGDI Heavy chain CDR2 102 GKSGDGGAWFAY Heavy chain CDR3 103 DVVVTQTPLSLPVSLGDHASISCRSSQSLVHSNGNTYLHWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPRTFGGGTKLEIKR light chain variable region 104 EVQLQESGPEMVKPGASVKISCKASGYPLPAYFINWVKQSHGKRLEWIGRINLNNGDIFYNQNFKDKATLAVDRSSTSAHMELLSLTSEDSAVYYCGKSGDGGAWFAYWGQGTLVTVSA heavy chain variable region 105 QDINSY light chain CDR1 Anti-ALK phosphorylated antibody CDA03 106 RAN light chain CDR2 107 LQYEEFPLT light chain CDR3 108 GFTFSSFG Heavy chain CDR1 109 INSDSTNI Heavy chain CDR2 110 ARRGLLGMVYAY Heavy chain CDR3 111 DIQMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISRLEYEDMGSYYCLQYEEFPLTFGAGTKLEIK light chain variable region 112 EVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWIAYINSDSTNIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYFCARRGLLGMVYAYWGQGTLVTVSA heavy chain variable region

Use these 9 antibodies to perform protein immunoblot 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 thus 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.

The 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 Rnase1 Elisa kit (product name and product number: ELISA Kit for Ribonuclease1, human; Product Number: SEA297Hu)

1. Preparation of reagents and items (items in the kit) Reagents Number (dose) amount Reagents Number (dose) amount Preconditioning and preparing 96-well plates for use 1 96 well plate sealing 4 Standard product 2 Standard diluent 1×20mL Detection reagent A 1×120µL Assay Diluent A 1×12mL Detection reagent B 1×120µL Assay Diluent B 1×12mL TMB reaction matrix solution 1×9mL stop solution 1×6mL Wash buffer (30 × concentration) 1×20mL Instructions 1 serving

2. Experimental steps 1. Prepare all reagents, samples and standards; 2. Add 100 μL of standard or sample to each well and incubate at 37°C for 1 hour; 3. Extract the prepared detection reagent A and add 100 μL/well and incubate at 37°C for 1 hour. hours; 4. Aspirate and wash 3 times; 5. Add 100 μL/well of prepared detection reagent B and incubate at 37°C for 30 minutes; 6. Aspirate and wash 5 times; 7. Add 90 μL/well TMB Reaction matrix 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 are dewaxed with xylene and hydrated with ethanol of 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, microwave on high heat for 5 minutes, medium heat for 5 minutes (or high-pressure heating 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) Develop DAB chromogenic solution for 3 minutes and observe under the microscope; (11) Run water to stop color development and counterstain with hematoxylin for 1 minute; (12) Rinse with tap water and differentiate with differentiation solution for 5 seconds; (13) Rinse the sections, dehydrate with ethanol, clear with xylene, and seal with neutral gum.

Method for determining immunohistochemistry results: All hematoxylin-stained sections were reviewed independently by two pathologists. The tumor content of the cancer specimen is greater than 70%. 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: 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.

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 radishine 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 medium) , 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. Masses under the abdominal skin of the mice could be observed 10 days after inoculation. growth, 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) is 25 mg per kilogram of body weight per day, and the dose of placebo is 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). 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 tumor tissue sections showed that RNase1 expression was >4 points and ALK phosphorylation was >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, Aletinib ( 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 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 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).

without

Figure 1 shows the results of protein immunoblotting hybridization experiments of five RNase1 monoclonal antibodies in Example 1. Figure 2 shows the results of protein immunoblotting 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.

TW202404596A_112127312_SEQL.xml

Claims (10)

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 said patients with RDAA-positive disease 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 results for ALK phosphorylation, and wherein the patient with RDAA-positive disease is not a non-small cell lung cancer patient. The method of claim 1, 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, lung or pancreatic cancer tissue sections and/or blood from the subject; Preferably, pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient. The method according to claim 1 or 2, wherein the patient is a patient with pancreatic ductal adenocarcinoma. The method according to any one of claims 1-3, wherein the ALK inhibitor includes an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation. The method according to any one of claims 1-4, the ALK inhibitor includes crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensartinib any one or combination thereof. Use of an ALK inhibitor in the preparation of a medicament for the treatment of RDAA-positive disease, wherein a sample from a patient suffering from said RDAA-positive disease has a negative ALK gene rearrangement detection result, ≥418 ng/ml in plasma Rnase1 expression level or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation, and wherein the patient with RDAA-positive disease is not a non-small cell lung cancer patient. The use as described in claim 6, wherein the sample is from the patient's tissue, cells and/or body fluids; Preferably, tissue sections and/or blood from the patient; Preferably, tumor tissue sections and/or blood from the patient; Preferably, lung or pancreatic cancer tissue sections and/or blood from the subject; Preferably, pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient. For the use described in claim 6 or 7, the patient is a patient with pancreatic ductal adenocarcinoma. The use as described in any one of claims 6-8, wherein the ALK inhibitor includes crizotinib, ceritinib, lorlatinib, aletinib, brigatinib, ensarti any one or combination thereof. The use according to any one of claims 6-9, wherein the ALK inhibitor includes an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.