CN115993453A

CN115993453A - Methods and kits for diagnosis and treatment of RDAA positive diseases

Info

Publication number: CN115993453A
Application number: CN202210867236.XA
Authority: CN
Inventors: 刘伦旭; 查正宇
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-07-22
Filing date: 2022-07-22
Publication date: 2023-04-21
Also published as: WO2024017338A1

Abstract

The present disclosure provides a method for diagnosing and/or treating a disease comprising at least the steps of: detecting Anaplastic Lymphoma Kinase (ALK) gene rearrangement, anaplastic lymphoma kinase phosphorylation level, and RNase1 expression level in a subject, defining the disease as an RNase 1-driven disease (RDAA positive disease) if the detection of ALK gene rearrangement is negative and ALK phosphorylation level and RNase1 expression level are higher than a reference population level, and optionally administering an ALK inhibitor to the subject, wherein the RDAA positive disease is RDAA positive non-small cell lung cancer and the ALK inhibitor does not include ensatinib.

Description

Methods and kits for diagnosis and treatment of RDAA positive diseases

Technical Field

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

Background

ALK (anaplastic lymphoma kinase ) is a class of receptor tyrosine kinases, the variation of which is associated with the occurrence of a variety of cancers. Variant forms of ALK include ALK gene rearrangements; activating mutation of Alk gene; gene copy number variation, and the like. ALK gene rearrangement results in the development of a variety of cancers such as lymphoma, lung cancer, neuroblastoma, myofibroblastic tumor, the most common of which are Anaplastic Large Cell Lymphoma (ALCL) and non-small cell lung cancer (NSCLC), but ALK gene rearrangement is positive for only 6.7% of NSCLC patients and 50% of ALCL adult patients.

The treatment regimen and prognosis evaluation are different for different types of cancer. For example, ALK inhibitors may be used to treat patients with tumors that are positive for the ALK gene rearrangement. However, for tumor patients whose ALK is wild-type (i.e., ALK gene rearrangement negative), no relevant cancer-promoting mechanism has been found, nor has an effective targeted therapy been developed.

Disclosure of Invention

Aiming at the urgent problem that only effective treatment methods (namely, application of ALK inhibitor) for ALK gene rearrangement positive patients exist clinically at present, but no effective treatment means are found for ALK gene rearrangement negative patients, the inventor of the present disclosure identifies RDAA positive diseases in ALK gene rearrangement negative patient groups through ingenious selection of biomarkers, and further discovers that the application of ALK inhibitor effectively kills RDAA positive cancer cells, remarkably inhibits tumor growth of RDAA positive patients, thereby effectively treating RDAA positive diseases and bringing good news to patients. In particular, the present disclosure provides methods for diagnosing and/or treating RDAA positive diseases; wherein an anti-RNase 1 antibody or antigen-binding portion thereof, an anti-ALK phosphorylated antibody or antigen-binding portion thereof is used; kits and devices for diagnosing RDAA positive disease; use of a combination of a reagent for detecting ALK gene rearrangement, a reagent for detecting ALK phosphorylation level and a reagent for detecting RNase1 expression level in the preparation of a kit for detecting RDAA positive disease.

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

detecting Anaplastic Lymphoma Kinase (ALK) gene rearrangement, ALK phosphorylation levels, and RNase1 expression levels in the subject, defining the disease as a disease resulting from RNase 1-driven ALK activation (RDAA positive disease) if the detection of ALK gene rearrangement is negative and ALK phosphorylation levels and RNase1 expression levels are higher than the reference population levels, and optionally administering an ALK inhibitor to the subject.

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

1) Detecting ALK gene rearrangement in the subject, and if the detection result of ALK gene rearrangement is negative, performing the following steps:

2) Detecting ALK phosphorylation levels and RNase1 expression levels in the subject; and

3) Comparing the ALK phosphorylation level and the RNase1 expression level obtained by detection with the ALK phosphorylation level and the RNase1 expression level of a reference population respectively; and

4) Optionally, determining that the subject has an RDAA positive disease if the detected ALK phosphorylation level and RNase1 expression level are higher and reach or are above a set threshold, respectively, compared to the ALK phosphorylation level and RNase1 expression level of a reference population; and

5) Optionally, an ALK inhibitor is administered to a subject with an RDAA positive disease.

In a third aspect of the present disclosure, there may be provided the use of an ALK inhibitor in the manufacture 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, there may be provided an anti-RNase 1 antibody or antigen binding portion thereof 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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID 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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID 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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID 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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID NO. 30. Or alternatively

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, heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO. 36, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO. 37, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO. 38.

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

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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID NO. 46;

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, heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO. 52, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO. 53, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID 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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID 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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID 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, a light chain CDR3 having an amino acid sequence as 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 CDR3 having an amino acid sequence as shown in SEQ ID NO. 78;

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, heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO. 84, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO. 85, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO. 86;

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, heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO. 92, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO. 93, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO. 94.

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, heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO. 100, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO. 101, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO. 102; or alternatively

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, heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO. 108, heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO. 109, and heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO. 110.

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

1) A reagent for detecting ALK phosphorylation levels; and

2) And (3) an agent for detecting the expression level of RNase 1.

In a seventh aspect of the present disclosure, there may be provided the use of a combination of reagents for detecting ALK phosphorylation levels and reagents for detecting RNase1 expression levels in the preparation of a kit for detecting an RDAA positive disease in which patients have negative ALK gene rearrangement detection results, RNase1 expression levels in plasma of ∈418ng/ml or positive immunohistochemical staining results for RNase1, and positive immunohistochemical staining results for ALK phosphorylation.

In an eighth aspect of the present disclosure, there may be provided 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 the preparation of a kit for detecting an RDAA positive disease, a patient with a negative ALK gene rearrangement detection result, an RNase1 expression level in plasma of ∈418ng/ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation.

In a ninth aspect of the present disclosure, there may be provided the use of an ALK inhibitor in the manufacture of a medicament for the treatment of an RDAA positive disease in a patient with a negative ALK gene rearrangement detection result, an Rnase1 expression level in plasma of ≡418ng/ml or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation.

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

1) Detection unit: a reagent section and a measurement section for performing detection, wherein a reagent capable of detecting an ALK gene rearrangement expression level, a reagent for detecting an ALK phosphorylation level, and a reagent for detecting an RNase1 expression level are provided, respectively;

2) A data acquisition unit: collecting the result output by the detection part;

3) Data analysis unit: analyzing the data collected by the data collection part; and

4) And a result output unit.

Drawings

FIG. 1 shows the results of Western blot hybridization experiments of 5 RNase1 monoclonal antibodies of example 1.

FIG. 2 shows the results of Western blot hybridization experiments of 9 ALK protein phosphorylated monoclonal antibodies of example 2.

FIG. 3 shows the results of an experiment of the killing effect of ALK inhibitors on RDAA positive lung cancer cells.

Figure 4 shows the tumor growth inhibition effect of ALK inhibitors on RDAA positive tumor-bearing mice.

Figure 5 shows 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.

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

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein 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 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, the relevant information of which is detailed in https:// www.ncbi.nlm.nih.gov/Gene/238 (ALK ALK receptor tyrosine kinase [ Homo sapiens (human) ] Gene ID: 238), and homologs thereof.

As used herein, the term "ALK gene rearrangement" includes, but is not limited to, those described in U.S. patent No. 9,651,555 and Du et al (Thoracic cancer.9:423-430, 2018), which are incorporated herein by reference in their entirety. ALK gene rearrangements may be rearrangements 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, resulted in the formation of fusion oncogenes.

As used herein, the term "ALK gene rearrangement negative" refers to the gene rearrangement negative by FISH (Fluorescence in situ hybridization), NGS (Next-generation sequencing), IHC

The ALK gene rearrangement is negative when detected by the methods such as (immunochistochemical) method. For example, patient surgery or puncture of pathological tissue samples was diagnosed as ALK Molecular rearrangement (ALK rearrangement) negative using the Vysis ALK Break Apart FISH probe kit (Abbott Molecular, inc.); the patient operation or puncture pathological tissue sample is diagnosed to have no ALK gene related mutation by using a second generation high throughput gene detection technology (WUOGYO EW, yi Sharpness Biotechnology Co., shenzhen Dacron Gene Co., ltd, and the like) and is diagnosed to be negative for ALK molecular rearrangement; patient surgery or puncture pathology samples were diagnosed as negative for ALK molecular rearrangement using anti-ALK (D5F 3) rabbit monoclonal antibody reagent (immunohistochemistry) (VENTANA anti-ALK (D5F 3) Rabbit Monoclonal Primary Antibody).

As used herein, the term "RNase1" refers to ribonuclease1 (ribonuclease 1) belonging to the human secreted ribonuclease family, and the information on this is described in https:// www.ncbi.nlm.nih.gov/gene/6035

(RNASE 1 ribonuclease A family member 1,pancreatic[Homo sapiens (human)), and homologs thereof.

As used herein, "RDAA positive disease" refers to a disease associated with RNAse 1-driven ALK activation.

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

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

The inventors of the present disclosure have for the first time found that administration of ALK inhibitors can achieve an effective therapeutic effect in patients with RDAA positive diseases. Therefore, the ALK inhibitor has good application prospect in preparing medicaments for treating RDAA positive diseases.

Detecting A Lymphoma Kinase (ALK) gene rearrangement expression level, an ALK phosphorylation level, and an RNase1 expression level in the subject, defining the disease as a disease caused by RNase 1-driven ALK activation (RDAA positive disease) if the detection of ALK gene rearrangement is negative and the ALK phosphorylation level and the RNase1 expression level are higher than the reference population level, and optionally administering an ALK inhibitor to the subject.

In some embodiments, the subject may be a healthy person, or may be a suspected patient of having a disease (e.g., cancer) or a patient having a disease (e.g., cancer). In some embodiments, the subject may be a suspected cancer patient 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 test sample from a subject. In some embodiments, the test sample may be from a tissue, cell, and/or body fluid of a subject. In some embodiments, the sample to be tested may be from a tissue slice and/or blood of a subject. In some embodiments, the test sample may be from a tumor tissue section and/or blood of a subject. In some embodiments, the test sample may be from a lung cancer or pancreatic cancer tissue section and/or blood of a subject. In some embodiments, the test sample may be from a non-small cell lung cancer or pancreatic ductal adenocarcinoma tissue section and/or blood of a subject.

In some embodiments, the ALK phosphorylation level of the reference population is negative for immunohistochemical staining for ALK phosphorylation. In some embodiments, the ALK phosphorylation level of the reference population is an immunohistochemical staining score < 4 points for ALK phosphorylation.

In some embodiments, the reference population has an RNase1 expression level of < 418ng/ml RNase1 expression level in plasma or negative immunohistochemical staining for RNase 1. In some embodiments, the level of RNase1 expression in the reference population is < 418ng/ml RNase1 expression in plasma or an immunohistochemical staining score for RNase1 < 4 points.

In some embodiments, the set threshold value for ALK phosphorylation level is positive for immunohistochemical staining results for ALK phosphorylation. In some embodiments, the set threshold value for ALK phosphorylation level is an immunohistochemical staining score ≡4 score for ALK phosphorylation.

In some embodiments, the set threshold for RNase1 expression level is that the RNase1 expression level in plasma is greater than or equal to 418ng/ml or that the immunohistochemical staining for RNase1 is positive. In some embodiments, the set threshold for RNase1 expression level is at least 418ng/ml for RNase1 expression level in plasma or at least 4 points for immunohistochemical staining of RNase 1.

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

Detection of ALK gene rearrangement expression levels, ALK phosphorylation levels, and RNase1 expression levels may be accomplished by any method known in the art, e.g., using commercial kits, sequencing, etc. In some embodiments, the detection of ALK gene rearrangement expression level, ALK phosphorylation level, and RNase1 expression level is selected independently from one or more of western blotting, immunohistochemistry, 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 comprises an antibody or antigen-binding portion thereof capable of inhibiting ALK phosphorylation. In some embodiments, the ALK inhibitor comprises any one of crizotinib, ceritinib, loratidine, aletinib, bugantinib, ensartinib, or a combination thereof.

1) Detecting the ALK gene rearrangement expression level of the subject, and if the detection result of ALK gene rearrangement is negative, performing the following steps:

In some embodiments, the anti-RNase 1 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 set forth in SEQ ID NO. 15 and a heavy chain variable region having an amino acid sequence as set forth 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 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 alternatively

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

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 set forth in SEQ ID NO. 47 and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 48;

a light chain variable region having the amino acid sequence shown in SEQ ID NO. 55 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 56;

A light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 63 and a heavy chain variable region having an amino acid sequence as set forth 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 set forth in SEQ ID NO. 79 and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 80;

a light chain variable region having the amino acid sequence shown in SEQ ID NO. 87 and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 88;

a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 95 and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 96;

a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 103 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 104; or alternatively

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

1) A reagent for detecting ALK phosphorylation levels; and

2) And (3) an agent for detecting the expression level of RNase 1.

In some embodiments, the reagent that detects ALK phosphorylation levels, the reagent that detects RNase1 expression levels, the reagent that detects ALK gene rearrangement expression levels may be any reagent known in the art that is capable of performing this function. In some embodiments, the reagent that detects the level of ALK phosphorylation comprises an anti-ALK phosphorylated antibody, or antigen-binding portion thereof, described in the present disclosure. In some embodiments, the agent that detects the level of RNase1 expression comprises an anti-RNase 1 antibody or antigen binding portion thereof described in the present disclosure. In some embodiments, the kit further comprises a reagent that detects the level of ALK gene rearrangement expression.

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

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

The ALK inhibitor may be any substance known in the art that is effective in inhibiting ALK activity and/or expression, etc. In some embodiments, ALK inhibitors include, but are not limited to, any one of crizotinib, ceritinib, loratidine, aletinib, buganidine, ensartinib, or a combination thereof. In some embodiments, the ALK inhibitor comprises an antibody or antigen-binding portion thereof capable of inhibiting ALK phosphorylation. In some embodiments, an ALK inhibitor comprises an anti-ALK phosphorylated antibody described in the present disclosure, or an antigen-binding portion thereof.

1) Detection unit: a reagent section and a measurement section for performing detection, wherein a reagent capable of detecting ALK gene rearrangement, a reagent for detecting ALK phosphorylation level, and a reagent for detecting RNase1 expression level are provided, respectively;

2) A data acquisition unit: collecting the result output by the detection part;

4) And a result output unit.

In some embodiments, the detection of ALK gene rearrangement, ALK phosphorylation levels, and RNase1 expression levels are independently selected from one or more of western blotting, immunohistochemistry, 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 portion compares the collected data of ALK gene rearrangement expression level, ALK phosphorylation level, and RNase1 expression level with a set threshold.

In some embodiments, the set threshold value for ALK phosphorylation level is positive for immunohistochemical staining results for ALK phosphorylation.

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

In some embodiments, the set threshold for RNase1 expression level is that the RNase1 expression level in plasma is greater than or equal to 418ng/ml or that the immunohistochemical staining for RNase1 is positive.

In some embodiments, the set threshold for RNase1 expression level is at least 418ng/ml for RNase1 expression level in plasma or at least 4 points for immunohistochemical staining of RNase 1.

In some embodiments, an RDAA positive disease is determined when the analysis of the data analysis portion indicates that ALK gene rearrangement expression levels are negative, that immunohistochemical staining for ALK phosphorylation is positive, and that Rnase1 expression levels in plasma are greater than or equal to 418ng/ml or that immunohistochemical staining for Rnase1 is positive.

In some embodiments, when analysis by the data analysis portion indicates that ALK gene rearrangement expression levels are negative, immunohistochemical staining for ALK phosphorylation is greater than or equal to 4 points, and Rnase1 expression levels in plasma are greater than or equal to 418ng/ml or immunohistochemical staining for Rnase1 is greater than or equal to 4 points, an RDAA positive disease is determined.

In some embodiments, the detection portion, the data acquisition portion, the data analysis portion, and the result output portion may be separated into separate device units or form an integrated device.

The various embodiments and preferences described above for the methods of the present disclosure may be combined with one another (so long as they are not inherently contradictory to one another) and are equally applicable to the anti-RNase 1 antibodies or antigen-binding portions thereof, anti-ALK phosphorylating antibodies or antigen-binding portions thereof, kits, uses and devices of the present disclosure, and vice versa, and the various embodiments resulting from such combination are all considered a part of the disclosure of the present application.

In particular, the present disclosure provides the following embodiments:

1. a method for diagnosing a disease comprising at least the steps of:

1) Detecting Anaplastic Lymphoma Kinase (ALK) gene rearrangement, anaplastic lymphoma kinase phosphorylation level, and RNase1 expression level in a subject; and

2) If the detection result of anaplastic lymphoma kinase gene rearrangement is negative and the ALK phosphorylation level and the RNase1 expression level are higher than the reference population level, the disease is defined as a disease caused by RNase 1-driven ALK activation (RDAA positive disease).

2. A method for diagnosing a disease comprising at least the steps of:

1) Detecting Anaplastic Lymphoma Kinase (ALK) gene rearrangement in the subject, and if the detection result of ALK gene rearrangement is negative, performing the following steps:

4) Optionally, the subject is determined to have an RDAA positive disease in the event that the detected ALK phosphorylation level and RNase1 expression level are higher and reach or are above a set threshold, respectively, compared to the ALK phosphorylation level and RNase1 expression level of the reference population.

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

4. The method according to any one of the preceding embodiments, wherein the set threshold for the expression level of RNase1 is a plasma RNase1 expression level of ≡418ng/ml or a positive immunohistochemical staining result for RNase 1.

5. The method of any one of the preceding embodiments, wherein the detecting is performed on a test sample from a subject, the test sample being from a tissue, cell and/or body fluid of the subject;

preferably, tissue sections and/or blood from a subject;

preferably, tumor tissue sections and/or blood from a subject;

preferably, lung cancer or pancreatic cancer tissue sections and/or blood from a subject;

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

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

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

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

9. A method for treating a disease comprising at least the steps of:

administering an Anaplastic Lymphoma Kinase (ALK) inhibitor to a patient suffering from an RDAA positive disease, defined as a disease resulting from RNase 1-driven ALK activation,

wherein a sample from said patient with RDAA positive disease has a negative ALK gene rearrangement detection result, an RNase1 expression level in plasma of ≡418ng/ml or a positive immunohistochemical staining result for RNase1 and a positive immunohistochemical staining result for ALK phosphorylation, and

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

the ALK inhibitor does not include ensatinib.

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

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

preferably, tumor tissue sections and/or blood from a patient;

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

11. The method of

embodiments

9 or 10, wherein the ALK inhibitor comprises an antibody or antigen-binding portion thereof capable of inhibiting ALK phosphorylation.

12. The method of any one of embodiments 9-11, wherein the ALK inhibitor comprises any one of crizotinib, ceritinib, loratidine, aletinib, bujitinib, or a combination thereof.

13. An anti-RNase 1 antibody or antigen binding portion thereof comprising:

14. The anti-RNase 1 antibody or antigen binding portion thereof of embodiment 13 comprising:

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

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

17. A kit, comprising:

3) A reagent for detecting ALK phosphorylation levels; and

4) And (3) an agent for detecting the expression level of RNase 1.

18. The kit of embodiment 17, wherein

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

19. The kit of embodiment 17, wherein

The reagent for detecting the expression level of RNase1 comprising the anti-RNase 1 antibody or antigen-binding portion thereof according to any one of embodiments 13 to 14.

20. The kit of any one of embodiments 17-19, further comprising a reagent that detects 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 the preparation of a kit for detecting an RDAA positive disease, a patient with a negative ALK gene rearrangement detection result, an RNase1 expression level in plasma of ∈418ng/ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result 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 the preparation of a kit for detecting an RDAA positive disease, a patient with a negative ALK gene rearrangement detection result, an RNase1 expression level in plasma of ∈418ng/ml or a positive immunohistochemical staining result for RNase1, and a positive immunohistochemical staining result for ALK phosphorylation.

Use of an ALK inhibitor in the manufacture 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, an Rnase1 expression level in plasma of ≡418ng/ml or a positive immunohistochemical staining result for Rnase1, and a positive immunohistochemical staining result for ALK phosphorylation, and wherein said RDAA positive disease is RDAA positive non-small cell lung cancer and said ALK inhibitor does not include ensatinib.

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

preferably, tissue sections and/or blood from a patient;

preferably, tumor tissue sections and/or blood from a patient;

25. The use of embodiment 23 or 24, wherein the ALK inhibitor comprises any one of crizotinib, ceritinib, loratidine, aletinib, bujitinib, or a combination thereof.

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

28. An apparatus for diagnosing an RDAA positive disease, comprising:

2) A data acquisition unit: collecting the result output by the detection part;

4) And a result output unit.

29. The device of embodiment 28, wherein the detection portion is performed on a test sample from the subject, the test sample being from a tissue, cell, and/or body fluid of the subject;

preferably, tissue sections and/or blood from a subject;

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

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

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

30. The apparatus 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 apparatus of embodiment 28, wherein when the analysis by the data analysis portion indicates

4) The ALK gene rearrangement is negative and the ALK gene rearrangement is negative,

5) Immunohistochemical staining for ALK phosphorylation was positive and

6) When the expression level of the Rnase1 in the blood plasma is more than or equal to 418ng/ml or the immunohistochemical staining result aiming at the Rnase1 is positive,

then it is judged as an RDAA positive disease.

32. The device of embodiment 28, wherein the detection portion, the data acquisition portion, the data analysis portion, and the result output portion may be separated into individual device units or form an integrated device.

The technical aspects of the present disclosure will be more clearly and clearly illustrated below by way of example in conjunction with examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. The scope of the present disclosure is limited only by the claims.

Examples

Example 1: preparation and binding Properties of anti-RNase 1 antibodies

Preparation of RNase1 antibody

1. Selection of animals: pure BALB/C mice

2. Selection of RNase1 antibody immunization protocol

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

a: primary immunization antigen 50. Mu.g was injected subcutaneously or intraparenally (total 1ml,0.2 ml/spot) with Freund's complete adjuvant;

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

c: after 3 weeks, the third immunization dose is the same as above, no adjuvant is added, and the injection is carried out intraperitoneally;

d: after 3 weeks, the immunity is enhanced, the dosage is 0.5mg, and the medicine is injected intraperitoneally;

e: after 3 days, spleen was taken for fusion.

3. Cell fusion

1. Preparation before cell fusion

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

Culture of myeloma cells DMEM medium. Calf serum concentration was 10% and cell concentration was 1×10 ⁵ Preferably/ml. When the cells were in mid-log growth, the ratio was 1:3 proportion passage. Passaging was performed every 3 days. (periodic treatment with 8-azaguanine, leading to uniform sensitivity of surviving cells to HAT.)

(2) Feeder cells:

the amount of macrophages in the abdominal cavity of the mice is 10 in the study ⁵ Cells/wells.

2. Step of cell fusion

(1) Preparing a feeder cell layer:

(2) Preparation of immune spleen cells

(3) Preparation of myeloma cells

(4) Fusion of

(1) Myeloma cells and spleen cells were mixed at a ratio of 1:10 or 1:5, washing with serum-free incomplete culture solution 1 time in a 50ml centrifuge tube, centrifuging at 1200rpm for 8min; the supernatant was discarded and the residual liquid was blotted with a pipette to avoid affecting polyethylene glycol (PEG) concentration. The bottom of the centrifugal tube is lightly flicked to loosen the cell sediment slightly.

(2) 1ml of 45% PEG solution pre-warmed at 37℃was added over 90s with gentle shaking. The mixture was subjected to a water bath at 37℃for 90s.

(3) The incomplete culture solution was added at 37℃to terminate the PEG effect, and 1ml, 2ml, 3ml, 4ml, 5ml and 6ml were added every 2min, respectively.

(4) Centrifuge at 800rpm for 6min.

(5) The supernatant was filled and resuspended in HAT selection medium containing 20% calf serum.

(6) The cells were added to the 96-well plate of the existing feeder cell layer, and 100. Mu.l each well was added. One immunized spleen can be seeded with 4 96-well plates.

(7) The plates were incubated at 37℃in a 5% CO2 incubator.

3. Selection of hybridoma cells and antibody detection

1) The HAT selection hybridoma cells are cultured in the HAT selection culture solution, a large number of tumor cells die within 1-2 days of culture, the tumor cells disappear after 3-4 days, the hybridization cells form small colonies, the HT culture solution is replaced after the HAT selection culture solution is maintained for 7-10 days, and the HT culture solution is maintained for 2 weeks, and then the normal culture solution is used. During the selective culture, when the hybridoma cells are distributed to be 1/10 area of the hole bottom, specific antibodies can be detected, and the needed hybridoma cell lines can be screened out. During the selection culture, half of the culture medium is generally changed every 2 to 3 days.

4. Cloning of hybridoma producing RNase1 antibody

1) Feeder cell layers (confluent with cells) were prepared 1 day prior to cloning.

2) Hybridoma cells to be cloned were gently blow-dried from within the culture well and counted.

3) The cells were adjusted to 10 cells/ml.

4) The feeder cell layer cell culture plate prepared on the first day was taken, and 100. Mu.l of diluted cells were added to each well. Incubate in a 5% CO2 incubator at 37 ℃.

5) The liquid was changed on day 7, and then 1 time every 2 days.

6) Cell clone formation was seen for 8 days and antibody activity was detected in time.

7) Cells from the positive wells were transferred to 24-well plates for expansion.

8) Cell clones were frozen.

5. Mass production of RNase1 monoclonal antibodies

Inoculating hybridoma cells in vivo to prepare ascites.

Preparation of ascites: conventional intraperitoneal injection of 0.5ml Pristane or liquid Paraffin into BALB/C mice was performed 1X 10 by 2 weeks later ⁶ Ascites can be produced 10 days after inoculation of the hybridoma cells. Mice were sacrificed and ascites was aspirated into tubes with a dropper, typically 10ml of ascites was obtained in one mouse.

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

5 anti-RNase 1 antibodies were obtained, and specific amino acid sequences thereof are shown in the following table.

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Western blot hybridization experiments (western blot) were performed using these 5 antibodies, the experimental procedure was as follows:

1. Preparation of protein lysate

1. Protein lysate 2 Xsample buffer 40ml system configuration: the components are added as follows: 80% glycerol 10ml, sterilized water 1ml,10%SDS 24ml,1M Tris-HCL (pH 6.8) 5ml, and stored at room temperature after thoroughly mixing.

2. Preparation of protein loading buffer: fully mixing beta-mercaptoethanol and a proper amount of bromophenol blue powder in a 1.5ml EP tube, and conventionally preserving in a refrigerator at-20 ℃ for standby

2. Total protein extraction

1) Taking out the cell culture dish from the constant temperature incubator at 37 ℃ to evaluate the cell density under a microscope;

2) Placing the culture dish on ice (the whole operation steps are carried out on the ice), sucking the upper culture solution, and washing the culture dish with PBS (phosphate buffered saline) which is cooled for 2 times;

3) According to cell density, the ratio of 1:1, respectively adding 20-50 mu l of PBS and protein lysate into each hole, immediately scraping the added solution and cells attached to the surface of the culture dish by using a cell scraper after the liquid covers the surface of the culture dish, fully stirring, mixing and cracking, and collecting the mixture into an EP tube;

4) Heating in a metal bath at 100deg.C for 10 min to denature protein, instantaneous separating at 12000rpm, and storing at 4deg.C for short term.

3. Protein concentration quantification (BCA method)

1) Before the experiment, PBS and BSA stock solution are quantitatively mixed to prepare a BSA standard with the concentration of 0.5mg/ml, and the BSA standard is stored on ice or at the temperature of 4 ℃ for standby;

2) Preparing BCA mixed solution: preparing light green BCA mixed working solution according to the number of the detection samples and the corresponding gradient standard substances which are set in the experiment according to the ratio of BCA reagent to Cu reagent=50:1, and fully and uniformly mixing, and preserving at room temperature for a short time for later use;

3) Starting to add the standard substance into the protein standard substance wells from 20-0 μl decreasing volume in the first well, and then adding PBS to make up the liquid in each well to 20 μl, so as to form a series of concentration gradients of the standard substance in each well;

4) The enzyme label instrument uses the absorbance detected by 562nm wavelength, records the OD value and draws the protein standard curve;

5) Sample protein treatment: taking 96-well plates and prepared protein samples, adding 18 μl of PBS and 2 μl of the samples into each well, adding 200 μl of BCA mixed solution, and reacting in a 37 ℃ metal bath or incubator for 30 minutes;

6) Taking a sample after reaction, detecting to obtain an OD value, calculating the protein concentration of the sample according to a drawn protein standard curve, and calculating the sample volume during electrophoresis according to the protein loading sample volume, wherein the specific calculation formula is as follows: protein loading volume = protein loading amount/sample protein concentration;

7) Sample proteins were mixed with formulated protein loading buffer at 20:1, and the mixture is instantly separated from a refrigerator at the temperature of minus 20 ℃ at 12000rpm for standby.

4. SDS-PAGE gel electrophoresis

1) Preparation of the separation gel: the concentrations of the separation gel needed by the experimental detection proteins RNase1, p-ALK (Y1604) and p-ALK (Y1282/1283) are 10%, 8% and 8%. The separation gel system is shown in table 1. A glue making plate frame is arranged on the horizontal tabletop, mixed liquid (5 ml) is prepared according to a separation colloid system, and a reserved gap between glue making plates is quickly, carefully and stably driven in. Slowly pumping 100-200 mu l of isopropyl alcohol or absolute ethyl alcohol horizontally from left to right along the edge of the wall of the glue making plate to flatten the glue surface, slightly lifting and shaking a glue making frame at one side to discharge potential bubbles, and finally standing on a horizontal tabletop at normal temperature for 30-60 minutes to solidify the glue to be separated;

TABLE 1 Release gel configuration System (5 ml)

2) Preparation of concentrated glue: after the separation gel is solidified, carefully sucking off isopropanol or absolute ethyl alcohol, and airing under the condition of natural ventilation. Next, the upper layer of gum (2 ml) was prepared as a concentrated gum system (as shown in Table 2) and rapidly and smoothly poured into the gap of the gum plate until the liquid overflowed. Slowly and steadily insert 15-hole comb. Standing the horizontal table top for 30-60 minutes at room temperature;

TABLE 2 concentrated gel configuration system (2 ml)

3) After the upper concentrated glue layer is solidified, carefully taking down the glue plate from the glue making frame, tightly fixing the glue plate in the electrophoresis tank, carefully slowly and vertically pulling out the comb to leave a sample feeding hole, and pouring a proper amount of electrophoresis liquid into the electrophoresis tank according to the quantity of the glue plate;

4) Protein loading: taking out the protein sample stored in the refrigerator at-20 ℃ and returning to room temperature after thawing. Sequentially loading the protein sample according to the calculated protein sample loading volume in an equivalent manner, and reserving 1-2 holes and adding 5 μl of protein sample loading indicator (marker) according to experimental requirements;

5) Electrophoresis: after sample loading, starting electrophoresis with a constant voltage of 80V, marking the beginning of electrophoresis when beads-like bubbles are generated at the bottom of the glue making plate, then compressing bromophenol blue in a visible protein sample to form a straight line, adjusting the voltage to a constant voltage of 120V after the protein reaches a transparent separating glue interface with obvious distinction degree according to a marker, and stopping electrophoresis when a bromophenol blue indicator descends to the bottommost edge of the glue making plate to start film transferring;

6) Transferring: filter paper required for membrane transfer is prepared in advance, and a methanol box, a membrane transfer clamp, a PVDF membrane and the like for activation are arranged, and the membrane transfer liquid is precooled at 4 ℃ before membrane transfer. A piece of filter paper was placed in a horizontal container, and an appropriate amount of transfer solution was poured into the container to slightly ultrafiltration the paper thickness. The PVDF membrane was wetted and activated with methanol and then placed on top of the filter paper. And taking out the electrophoresed rubber plate, peeling the albumin glue from the rubber plate, properly trimming the boundary, placing the rubber plate on a PVDF film, covering a layer of filter paper on the PVDF film, and continuously removing generated bubbles during operation. Finally, according to the principle of 'black glue and white film', the transparent surface of the film transfer clamp is taken as the lowest layer, a sponge cushion is covered on the transparent surface of the film transfer clamp, the filter paper and the PVDF film clamped in the previous step are horizontally placed, a layer of sponge cushion is covered on the transparent surface of the film transfer clamp, and the black surface of the film transfer clamp is aligned and fastened with the film transfer clamp buckle. And (3) clamping the fastened film transfer in a film transfer groove, adding film transfer liquid until the film transfer liquid submerges a film transfer indication line, placing the film transfer groove in a proper container, starting film transfer, and adding ice water into the container for preliminary cooling. Using a 300mA constant flow membrane, selecting proper membrane transfer time according to the size of target protein, wherein the membrane transfer time of R1 and p-ALK proteins is respectively 1 and 2 hours;

7) Closing: 5% skim milk prepared with PBST and a proper amount of PBST for cleaning were prepared in advance. After the transfer is completed, the transfer clamp is opened, and the PVDF film after the transfer is carefully taken out. The membranes were washed 2-3 times in PBST for 2 minutes each. Then transferring into 5% skimmed milk prepared from PBST, placing on a shaking table, and sealing for 1 hr;

8) Incubating primary antibodies: primary antibodies were prepared using primary antibody dilutions, 5% milk or 5% bsa in proportion, at a concentration of 1:1000 for each antibody (the source of the antibodies in this experiment was self-made monoclonal murine antibodies), and 4ml for each antibody. The antibody is prepared before incubation, poured into the lattice of the corresponding strip of the incubation box and placed on ice to maintain the activity of the antibody at low temperature. The PVDF film in 5% skim milk was removed and rinsed slightly with PBST to remove residual milk. Detecting the size of protein strips according to experiments, taking a color development marker as a scale, forming the film into strips, and then placing the strips into a grid corresponding to the antibody. Sealing, and slowly shaking for 12 hours by a shaking table at 4 ℃;

9) Recovering primary antibody: recovering the primary antibody after 12 hours;

10 Incubation of secondary antibody: goat anti-mouse IgG (H+L) secondary antibody (Zhongsequoia gold bridge, cat# ZB-2305) was labeled with horseradish enzyme according to 1:5000 ratio corresponding secondary antibody dilutions were prepared in advance using 5% skim milk formulated with PBST. The PVDF membrane was rinsed with PBST at least 3 times, each for 5-10 minutes, and the liquid was poured off after rinsing. Then pouring the secondary anti-dilution liquid into PVDF membrane lattices of corresponding species, and slowly shaking the shaking table for 1 hour;

11 Recovering the secondary antibody: recovering the secondary antibody and rinsing the PVDF membrane again by PBST for 3 times, each time for 5-10 minutes;

12 ECL development: pre-preparing a super-sensitive ECL developing solution (UltraSignal super-sensitive ECL chemiluminescent substrate, sizhengbai, cat# 4AW 011-100), mixing ECL A solution and ECL B solution in an EP pipe in equal volume, and keeping away from light and low temperature for later use. Before development, the PVDF film was placed on a foam plate, the residual PBST was slightly wiped off, and then a prepared super-sensitive ECL developer was dropped thereon according to the size of the film, after the two sides were completely immersed. Exposure was performed in a Bio-rad imaging instrument.

The experimental results are shown in FIG. 1. The 5 kinds of RNase1 monoclonal antibodies can specifically recognize endogenous RNase1 protein, and the target band is clear and is consistent with the protein size of the RNase1, and no nonspecific band exists. The results show that: all of these 5 anti-RNase 1 antibodies specifically bind to RNase1 and can be effectively used for detecting RNase1 in a sample.

Example 2: preparation of anti-ALK phosphorylated antibodies

The preparation process of the antibody was the same as that of the RNase1 antibody described above, and was different only in terms of antigen use. The antigen used to prepare the ALK phosphorylated antibodies was a synthetic ALK protein phosphorylated peptide of 2 total ALK protein phosphorylated peptides, 5mg each.

9 anti-ALK phosphorylated antibodies were obtained, and specific amino acid sequences thereof are shown in the following table.

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Western blot hybridization experiments were performed using these 9 antibodies, and the experimental method was the same as that of the western blot procedure of RNase1 in example 1.

The experimental results are shown in FIG. 2. The 9 ALK phosphorylation monoclonal antibodies can specifically identify endogenous ALK phosphorylated proteins, and the target bands are clear and coincide with the ALK phosphorylated proteins in size without nonspecific bands. The results show that: each of these 9 anti-ALK phosphorylating antibodies specifically binds to phosphorylated ALK, and thus can be effectively used to detect ALK phosphorylation levels in a sample.

Example 3: killing of RDAA positive disease cells by ALK inhibitors

ALK gene rearrangement expression levels in tumor tissue section samples from 3 subjects were detected using a second generation sequencing method: patient surgery or puncture pathological tissue samples are diagnosed to be negative for ALK gene rearrangement by using a second-generation high-throughput gene detection technology (Shenzhen Hua big gene Co., ltd.) without ALK gene rearrangement and related mutation.

Subjects negative for ALK gene rearrangement continue with the following test.

The expression level of RNase1 in plasma samples from 3 ALK gene rearrangement negative subjects was detected using an enzyme-linked immunosorbent assay (ELISA). The detection method comprises the following steps:

Elisa detection reagent and flow were carried out using the Rnase1 Elisa kit (Product name and Product Number: ELISA Kit for Ribonuclease, human; product Number: SEA297 Hu)

1. Reagent and article preparation (article in kit)

Reagent(s)	Number (dose) amount	Reagent(s)	Number (dose) amount
				Pretreatment, ready-to-use 96-well plate	1 number of	96-well plate seal	4 pieces of
Standard substance	2 pieces of	Standard substance diluent	1×20mL
				Detection reagent A	1×120μL	Detection of Diluent A	1×12mL
Detection reagent B	1×120μL	Detection of dilution B	1×12mL
				TMB reaction substrate solution	1×9mL	Stop solution		1×6mL
Washing buffer (30X concentration)	1×20mL	Description							1 part of

2. Experimental procedure

1. Preparing all reagents, samples and standards;

2. 100 μl of standard or sample was added to each well and incubated for 1 hour at 37deg.C;

3. extracting 100 mu L/Kong Jiaru of the prepared detection reagent A, and incubating for 1 hour at 37 ℃;

4. sucking and washing 3 times;

5. adding 100 mu L/hole of the prepared detection reagent B, and incubating for 30 minutes at 37 ℃;

6. sucking and washing 5 times;

7. 90. Mu.L/well TMB reaction substrate solution was added. Incubation at 37 ℃ for 120 min;

8. stop solution was added at 50. Mu.L/well. The microplate reader reads at 450 nm.

And calculating the concentration of RNase1 in the sample according to the absorbance value and the dilution factor by taking the standard substance as a reference. A418 ng/ml concentration was used as a demarcation point to distinguish between high and low RNase1 expression. (average concentration of RNase1 in plasma of non-small cell lung cancer patients was 418 ng/ml).

The expression level of RNase1 in tumor tissue samples from 3 ALK gene rearrangement negative subjects was detected using Immunohistochemistry (IHC). Immunohistochemical experiment procedure:

(1) Baking the tissue slices for 3 hours by using a 65 ℃ oven;

(2) Dewaxing paraffin sections by using dimethylbenzene, and hydrating the paraffin sections by using ethanol with different concentrations;

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

(4) Washing with PBS for 3 times, and 5 minutes each time;

(5) Immersing the slices into a citric acid buffer solution for restoration by microwaves, wherein the microwaves are high-fire for 5 minutes and medium-fire for 5 minutes (or high-pressure heating is carried out at 120 ℃ for 2 minutes);

(6) After natural cooling to room temperature (about 60 minutes), the PBS was rinsed 3 times for 5 minutes each;

(7) Removing redundant liquid on the glass slide, dripping RNase1 monoclonal antibody, wherein the dilution concentration of the antibody is 1:100 to 1:1000, placing in a wet box at 4deg.C overnight;

(8) Taking out the solution on the second day, standing at room temperature for 1h, and washing with PBS for 3 times, each time for 5 minutes;

(9) Dropwise adding the polymerase-labeled secondary antibody, standing for 30 minutes at room temperature, and flushing with PBS for 3 times, wherein each time lasts for 5 minutes;

(10) Developing the DAB color development liquid for 3 minutes, and observing under a mirror;

(11) Stopping the color development by running water, and counterstaining with hematoxylin for 1 minute;

(12) Washing with tap water, and differentiating the differentiation liquid for 5s;

(13) Washing, slicing, dehydrating with ethanol, and sealing with xylene transparent and neutral resin.

Immunohistochemical outcome determination method: all hematoxylin stained sections were read by two pathologists alone. The tumor content of the cancer specimens is greater than 70%. The double-blind method was used for evaluation of RNase1 staining, and cell localization positive for RNase1 expression was observed and confirmed, which was expressed as brown or tan particles. Immunohistochemical staining scores were obtained by multiplying the number of positively stained cells by staining intensity. The tissue specimens were observed under an optical microscope and scored as percentage of positively stained cells: 0: negative expression; 1, the method comprises the following steps: positive cell number < 15%;2, the method comprises the following steps: 15-50% of positive cells; 3, the method comprises the following steps: positive cells are more than or equal to 50%; the staining intensity was scored according to the color shade: 0 point: no positive; 1, the method comprises the following steps: light yellow; 2, the method comprises the following steps: brown yellow; 3, the method comprises the following steps: tan brown. Immunohistochemical staining score = 4 was used as a demarcation point to distinguish between high and low levels of RNase1 expression.

ALK phosphorylation levels in tumor tissue samples from 3 subjects were detected using the HIC method described above using an ALK phosphorylation monoclonal antibody (CDA 04 out of the 9 ALK phosphorylation monoclonal antibodies displayed in the antibody preparation section). Immunohistochemical staining score = 4 was used as a demarcation point to distinguish between high and low ALK phosphorylation levels.

The following criteria were followed for classification into RDAA positive subjects and non-RDAA positive (RDAA negative) subjects:

RDAA positive subjects: the expression level of the Rnase1 in the blood plasma is more than or equal to 418ng/ml or the immunohistochemical staining score of the Rnase1 is more than or equal to 4 minutes; and the immunohistochemical staining score aiming at ALK phosphorylation is more than or equal to 4 minutes;

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

Cancer cells from 1 RDAA positive and 1 RDAA negative non-small cell lung cancer subjects were treated with six ALK inhibitors (final concentrations of 1 μg per ml of medium, cell densities of 5 ten thousand cells per ml of medium) with placebo (PBS buffer), crizotinib (Crizotinib), ceritinib (ceritinib), loratidinib (Loratinib), aletinib (aletinib), bugatinib (brinatinib), and Ensartinib, respectively, cell survival was observed, the number of surviving cells was calculated daily, and growth curves were plotted.

The results of the experiment are shown in FIG. 3, with the abscissa axis being the time of administration in days and the ordinate axis being the relative cell number. The results show that: placebo failed to 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 1 RDAA positive non-small cell lung cancer patient are inoculated into the abdominal subcutaneous tissue of a nude mouse, the number of inoculated cells is half ten thousand cells per mouse, and subcutaneous tumor growth of the abdomen of the mouse can be observed after 10 days of inoculation, so that the RDAA positive tumor-bearing mouse is constructed.

Tumor-bearing mice bearing RDAA positive tumors were randomly divided into seven groups (5 each) and each group was orally administered placebo (PBS buffer), crizotinib, ceritinib, loratidine, aletinib, bujitinib, or ensatinib (25 mg daily per kg body weight of ALK inhibitor was used, 2ul daily per mouse was used for placebo, dosing was started 2 weeks after tumor inoculation, and dosing was stopped the fourth week).

The experimental results are shown in fig. 4 and 5. The results show that: ALK inhibitors significantly inhibited tumor growth in RDAA positive tumor-bearing mice relative to placebo group mice (P < 0.01); meanwhile, the survival of the tumor-bearing mice is also obviously improved (P < 0.01).

Example 5: therapeutic effects of ALK inhibitors on RDAA positive patients

40 patients from the university of Sichuan Huaxi Hospital have been determined to be negative for ALK gene rearrangement according to the assay method in example 3. Then, the expression level of Rnase1 in the plasma sample of the patient and the expression level of Rnase1 protein and the ALK phosphorylation level in the tumor tissue sample were measured by using the kit, and classified into RDAA positive patients and non-RDAA positive patients according to the following criteria:

RDAA positive patients: the expression level of the Rnase1 in the blood plasma is more than or equal to 418ng/ml or the immunohistochemical staining score of the Rnase1 is more than or equal to 4 minutes; and the immunohistochemical staining score aiming at ALK phosphorylation is more than or equal to 4 minutes;

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

A total of 3 RDAA positive patients were identified, all non-small cell lung cancer patients. The 3 patients were administered crizotinib at a dose of 250 mg each time twice daily, and the CT results before and after 4 weeks of continuous use are shown in fig. 6. It can be clearly seen that: crizotinib significantly inhibited tumor growth in RDAA positive patients.

Example 6: therapeutic effects of ALK inhibitors on RDAA positive PDAC patients

10 PDAC patients whose ALK gene rearrangement was negative were determined by the second generation high throughput gene sequencing (NGS) method. Tumor tissue sections of these patients were subjected to the RNase1 and ALK phosphorylation immunohistochemical assays mentioned in the kit. Among them, 1 tumor tissue section immunohistochemical result is RNase1 expression >4 minutes and ALK phosphorylation >4 minutes, and is identified as RDAA positive PDAC patient. Tumor cells of this patient were cultured, and cells were treated with six ALK inhibitors (final concentrations of 1 μg per ml of medium and 5 ten thousand cells per ml of medium for each of the above drugs) respectively with placebo (control, PBS buffer), crizotinib, ceritinib (ceritinib), loratidinib (Loratinib), aletinib (aletinib), bujitinib (brinatinib), and Ensartinib (Ensartinib), and cell survival was observed, the number of surviving cells was calculated daily, and growth curves were plotted.

The results of the experiment are shown in FIG. 7, in which the axis of abscissas represents the time of administration in days and the axis of ordinates represents the relative cell number. The results show that: placebo failed to inhibit the growth of RDAA-positive PDAC cells, but various ALK inhibitors effectively killed RDAA-positive PDAC cells (P < 0.01).

Claims

1. A method for diagnosing a disease comprising at least the steps of:

2. A method for diagnosing a disease comprising at least the steps of:

3. The method of any one of the preceding claims, wherein the set threshold for ALK phosphorylation level is positive for immunohistochemical staining results for ALK phosphorylation.

4. The method of any one of the preceding claims, wherein the set threshold for RNase1 expression level is that the RNase1 expression level in plasma is ≡418ng/ml or that the immunohistochemical staining for RNase1 is positive.

5. The method of any one of the preceding claims, wherein the detecting is performed on a test sample from a subject, the test sample being from a tissue, cell and/or body fluid of the subject;

preferably, tissue sections and/or blood from a subject;

preferably, tumor tissue sections and/or blood from a subject;

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

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

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

9. A method for treating a disease comprising at least the steps of:

the ALK inhibitor does not include ensatinib.

10. The method of claim 9, wherein the sample is from a tissue, cell, and/or body fluid of a patient;

preferably, tissue sections and/or blood from a patient;

preferably, tumor tissue sections and/or blood from a patient;

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

12. The method of any one of claims 9-11, wherein the ALK inhibitor comprises any one of crizotinib, ceritinib, loratidine, aletinib, bujitinib, or a combination thereof.

13. An anti-RNase 1 antibody or antigen binding portion thereof comprising:

14. The anti-RNase 1 antibody or antigen binding portion thereof of claim 13 comprising:

16. The anti-ALK phosphorylated antibody or antigen-binding portion thereof of claim 15, which comprises:

17. A kit, comprising:

1) A reagent for detecting ALK phosphorylation levels; and

2) And (3) an agent for detecting the expression level of RNase 1.

18. The kit of claim 17, wherein

The reagent for detecting ALK phosphorylation levels comprises the anti-ALK phosphorylated antibody or antigen-binding portion thereof of any one of claims 15-16.

19. The kit of claim 17, wherein

The reagent for detecting the expression level of RNase1 comprising the anti-RNase 1 antibody or antigen-binding portion thereof according to any one of claims 13 to 14.

20. The kit of any one of claims 17-19, further comprising a reagent to detect ALK gene rearrangement.

24. The use of claim 23, wherein the sample is from a tissue, cell and/or body fluid of a patient;

preferably, tissue sections and/or blood from a patient;

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

25. The use of claim 23 or 24, wherein the ALK inhibitor comprises any one of crizotinib, ceritinib, loratidine, aletinib, bujitinib, or a combination thereof.

26. 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.

27. 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.

28. An apparatus for diagnosing an RDAA positive disease, comprising:

2) A data acquisition unit: collecting the result output by the detection part;

4) And a result output unit.

29. The device of claim 28, wherein the detection portion is for a test sample from a subject, the test sample being from a tissue, cell and/or body fluid of the subject;

preferably, tissue sections and/or blood from a subject;

30. The apparatus according to 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.

31. The apparatus of claim 28, wherein when the analysis by the data analysis portion indicates that

1) The ALK gene rearrangement is negative and the ALK gene rearrangement is negative,

2) Immunohistochemical staining for ALK phosphorylation was positive and

3) When the expression level of the Rnase1 in the blood plasma is more than or equal to 418ng/ml or the immunohistochemical staining result aiming at the Rnase1 is positive,

Then it is judged as an RDAA positive disease.

32. The device of claim 28, wherein the detection portion, the data acquisition portion, the data analysis portion, and the result output portion may be separated into separate device units or form an integrated device.