WO2024017352A1 - 用于rdaa阳性疾病的诊断和治疗的方法和试剂盒 - Google Patents
用于rdaa阳性疾病的诊断和治疗的方法和试剂盒 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present disclosure relates to the field of biomedical technology, specifically to the diagnosis and treatment of diseases, especially the specific application of ALK inhibitors in the treatment of RDAA-positive diseases.
- ALK anaplastic lymphoma kinase
- ALK is a type of receptor tyrosine kinase, and its mutations are associated with the occurrence of various cancers. Mutated forms of ALK include Alk gene rearrangements; activating mutations of the Alk gene; gene copy number variations, etc.
- ALK gene rearrangement can lead to the occurrence of lymphoma, lung cancer, neuroblastoma, myofibroblastoma and other cancers, the most common of which are anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC).
- ALK gene rearrangement positivity accounts for only 6.7% of NSCLC patients and 50% of adult patients with ALCL.
- Treatment options and prognosis assessments vary for different types of cancer. For example, patients with tumors that are positive for ALK gene rearrangement can be treated with ALK inhibitors. However, for tumor patients with wild-type ALK (i.e., ALK gene rearrangement negative), no relevant cancer-promoting mechanism has been discovered, and there is no effective targeted therapy.
- 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.
- a method for diagnosing and/or treating a disease may be provided, which at least includes the following steps:
- ALK anaplastic lymphoma kinase
- a method for diagnosing and/or treating diseases can be provided, which at least includes the following steps:
- an ALK inhibitor in the preparation of a medicament for the treatment of non-small cell lung cancer and/or pancreatic ductal adenocarcinoma.
- an anti-RNase1 antibody or an antigen-binding portion thereof comprising:
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 6 chain CDR3;
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10
- light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:20, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:21, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:22 chain CDR3;
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 25 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 26, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 27 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 28, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 29, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 30 chain CDR3; or
- an anti-ALK phosphorylated antibody or an antigen-binding portion thereof comprising:
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 105 Light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 106, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 107 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 108, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 109, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 110 Chain CDR3.
- a kit comprising:
- a combination of a reagent for detecting ALK phosphorylation level and a reagent for detecting RNase1 expression level in preparing a kit for detecting RDAA-positive disease in patients with negative RDAA-positive disease can be provided.
- the use of a combination of a reagent for detecting the expression level of ALK gene rearrangement, a reagent for detecting ALK phosphorylation level, and a reagent for detecting the expression level of RNase1 in preparing a kit for detecting RDAA-positive diseases can be provided.
- patients with the RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression levels in plasma of ⁇ 418ng/ml or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining for ALK phosphorylation. Histochemical staining results.
- the use of an ALK inhibitor in preparing a medicament for treating RDAA-positive disease can be provided, and the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result and a plasma concentration of ⁇ 418ng/ml.
- Rnase1 expression levels in or positive immunohistochemical staining results for Rnase1 and positive immunohistochemical staining results for ALK phosphorylation.
- a device for diagnosing RDAA-positive diseases 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 collection department collects the results output by the detection department
- Figure 1 shows the results of Western immunoblot hybridization experiments of five RNase1 monoclonal antibodies in Example 1.
- Figure 2 shows the results of Western immunoblot hybridization experiments of nine ALK protein phosphorylated monoclonal antibodies in Example 2.
- Figure 3 shows the experimental results of the killing effect of ALK inhibitors on RDAA-positive lung cancer cells.
- Figure 4 shows the inhibitory effect of ALK inhibitor on tumor growth in RDAA-positive tumor-bearing mice.
- Figure 5 shows the results of the survival effect of ALK inhibitors on RDAA-positive tumor-bearing mice.
- Figure 6 shows the tumor growth inhibitory effect of ALK inhibitors on RDAA-positive patients.
- Figure 7 shows the sensitivity of cells from RDAA-positive PDAC patients to ALK inhibitors.
- 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.
- 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.
- 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.
- the Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Inc.) was used to diagnose ALK rearrangement negative for the patient's surgical or puncture pathological tissue samples; the patient's surgical or puncture pathological tissue samples were diagnosed as negative for ALK rearrangement using the second-generation Qualcomm Quantitative genetic testing technology (Wuhan Angkeyi Technology Consulting Co., Ltd., Yishan Biotechnology Co., Ltd., Shenzhen BGI Co., Ltd., etc.) diagnosed no ALK gene-related mutations and was diagnosed as negative for ALK molecular rearrangement; the patient underwent surgery or puncture Pathological tissue samples were diagnosed as negative for ALK molecular rearrangement using anti-ALK(D5F3) Rabbit Monoclonal Antibody reagent (immunohistochemistry) (VENTANA anti-ALK(D5F3) Rabbit Monoclonal Primary Antibody).
- Rnase1 refers to ribonuclease 1 (ribonuclease1), which belongs to the human secreted ribonuclease family.
- ribonuclease1 ribonuclease 1
- RNASE1 ribonuclease A family member 1, pancreatic [Homo sapiens (human)]
- RDAA-positive disease refers to a disease associated with RNAse1-driven ALK activation.
- a “patient with RDAA-positive disease” means that the patient has a negative ALK gene rearrangement test result, an ALK phosphorylation level that is higher than that of the reference population, and an RNase1 expression that is higher than that of the reference population. level of RNase1 expression; specifically, the patient had a negative ALK gene rearrangement test result, a plasma RNase1 expression level ⁇ 418 ng/ml or a positive immunohistochemical stain for RNase1, and a positive ALK phospho immunohistochemical staining results.
- reference population refers to healthy individuals or patients with RDAA-negative disease.
- ALK inhibitors have good application prospects in the preparation of drugs for the treatment of RDAA-positive diseases.
- a method for diagnosing and/or treating a disease may be provided, which at least includes the following steps:
- ALK lymphoma kinase
- the subject may be a healthy person, a patient suspected of a disease (eg, cancer), or a patient suffering from a disease (eg, cancer).
- the subject may be a cancer suspect or a cancer patient.
- the cancer may be lung cancer or pancreatic cancer.
- the cancer may be non-small cell lung cancer or pancreatic ductal adenocarcinoma.
- the detection is performed on a sample from the subject to be tested.
- the sample to be tested may be from tissues, cells and/or body fluids of a subject.
- the sample to be tested may be from tissue sections and/or blood of the subject.
- the sample to be tested may be from a subject's tumor tissue section and/or blood.
- the sample to be tested may be from lung or pancreatic cancer tissue sections and/or blood of the subject.
- 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.
- the reference population has ALK phosphorylation levels that are negative for immunohistochemical staining for ALK phosphorylation. In some embodiments, the reference population has an ALK phosphorylation level with an immunohistochemical staining score for ALK phosphorylation of ⁇ 4 points.
- the RNase1 expression level in the reference population is an Rnasel expression level in plasma ⁇ 418 ng/ml or a negative immunohistochemical staining result for Rnasel. In some embodiments, the RNase1 expression level in the reference population is an Rnasel expression level in plasma of ⁇ 418 ng/ml or an immunohistochemical staining score for Rnasel of ⁇ 4 points.
- the set threshold for ALK phosphorylation level is a positive immunohistochemical stain for ALK phosphorylation. In some embodiments, the set threshold for ALK phosphorylation level is an immunohistochemical staining score for ALK phosphorylation ⁇ 4 points.
- 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.
- 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.
- 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.
- the detection of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level are independently selected from one or more of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay.
- detection of ALK gene rearrangement expression levels, ALK phosphorylation levels, and/or RNase1 expression levels is performed using antibodies.
- the ALK inhibitor includes an antibody or antigen-binding portion thereof capable of inhibiting ALK phosphorylation.
- the ALK inhibitor includes any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensartinib, or a combination thereof.
- a method for diagnosing and/or treating diseases can be provided, which at least includes the following steps:
- the subject may be a healthy person, a patient suspected of a disease (eg, cancer), or a patient suffering from a disease (eg, cancer).
- the subject may be a cancer suspect or a cancer patient.
- the cancer may be lung cancer or pancreatic cancer.
- the cancer may be non-small cell lung cancer or pancreatic ductal adenocarcinoma.
- the detection is performed on a sample from the subject to be tested.
- the sample to be tested may be from tissues, cells and/or body fluids of a subject.
- the sample to be tested may be from tissue sections and/or blood of the subject.
- the sample to be tested may be from a subject Tumor tissue sections and/or blood of the subject.
- the sample to be tested may be from lung or pancreatic cancer tissue sections and/or blood of the subject.
- 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.
- the reference population has ALK phosphorylation levels that are negative for immunohistochemical staining for ALK phosphorylation. In some embodiments, the reference population has an ALK phosphorylation level with an immunohistochemical staining score for ALK phosphorylation of ⁇ 4 points.
- the RNase1 expression level in the reference population is an Rnasel expression level in plasma ⁇ 418 ng/ml or a negative immunohistochemical staining result for Rnasel. In some embodiments, the RNase1 expression level in the reference population is an Rnasel expression level in plasma of ⁇ 418 ng/ml or an immunohistochemical staining score for Rnasel of ⁇ 4 points.
- the set threshold for ALK phosphorylation level is a positive immunohistochemical stain for ALK phosphorylation. In some embodiments, the set threshold for ALK phosphorylation level is an immunohistochemical staining score for ALK phosphorylation ⁇ 4 points.
- 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.
- 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.
- 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.
- the detection of ALK gene rearrangement expression level, ALK phosphorylation level and RNase1 expression level are independently selected from one or more of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay.
- detection of ALK gene rearrangement expression levels, ALK phosphorylation levels, and/or RNase1 expression levels is performed using antibodies.
- the ALK inhibitor includes an antibody or antigen-binding portion thereof capable of inhibiting ALK phosphorylation.
- the ALK inhibitor includes any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensartinib, or a combination thereof.
- an ALK inhibitor in the preparation of a medicament for the treatment of non-small cell lung cancer and/or pancreatic ductal adenocarcinoma.
- an anti-RNase1 antibody or an antigen-binding portion thereof comprising:
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 2, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 3 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 6 chain CDR3;
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 9 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 10
- light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 20, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 21, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 22 chain CDR3;
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 25 light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 26, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 27 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 28, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 29, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 30 chain CDR3; or
- the anti-RNase1 antibody or antigen-binding portion thereof comprises:
- an anti-ALK phosphorylated antibody or an antigen-binding portion thereof comprising:
- Light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 105 Light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 106, light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 107 , a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 108, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 109, and a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 110 Chain CDR3.
- the anti-ALK phosphorylated antibody or antigen-binding portion thereof comprises:
- a kit comprising:
- the reagent for detecting ALK phosphorylation level, the reagent for detecting RNase1 expression level, and the reagent for detecting ALK gene rearrangement expression level can be any reagent known in the art that can achieve this function.
- a reagent for detecting ALK phosphorylation levels includes an anti-ALK phosphorylation antibody or an antigen-binding portion thereof as described in this disclosure.
- reagents for detecting RNase1 expression levels include an anti-RNase1 antibody or an antigen-binding portion thereof as described in the present disclosure.
- the kit further includes a reagent for detecting the expression level of ALK gene rearrangement.
- a combination of a reagent for detecting ALK phosphorylation level and a reagent for detecting RNase1 expression level in preparing a kit for detecting RDAA-positive disease in patients with negative RDAA-positive disease can be provided.
- a positive immunohistochemical staining result for Rnase1 refers to an immunohistochemical staining score for Rnase1 of ⁇ 4 points.
- a positive immunohistochemical staining result for ALK phosphorylation refers to an immunohistochemical staining score for ALK phosphorylation ⁇ 4 points.
- the use of a combination of a reagent for detecting the expression level of ALK gene rearrangement, a reagent for detecting ALK phosphorylation level, and a reagent for detecting the expression level of RNase1 in preparing a kit for detecting RDAA-positive diseases can be provided.
- patients with the RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression levels in plasma of ⁇ 418ng/ml or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining for ALK phosphorylation. Histochemical staining results.
- a positive immunohistochemical staining result for Rnase1 refers to an immunohistochemical staining score for Rnase1 of ⁇ 4 points.
- a positive immunohistochemical staining result for ALK phosphorylation refers to an immunohistochemical staining score for ALK phosphorylation ⁇ 4 points.
- the use of an ALK inhibitor in preparing a medicament for treating RDAA-positive disease can be provided, and the patient with the RDAA-positive disease has a negative ALK gene rearrangement test result and a plasma concentration of ⁇ 418ng/ml.
- Rnase1 expression levels in or positive immunohistochemical staining results for Rnase1 and positive immunohistochemical staining results for ALK phosphorylation.
- a positive immunohistochemical staining result for Rnase1 refers to an immunohistochemical staining score for Rnase1 of ⁇ 4 points.
- 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.
- ALK inhibitors include, but are not limited to, any one of crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensartinib, or combinations thereof .
- ALK inhibitors include antibodies or antigen-binding portions thereof capable of inhibiting ALK phosphorylation.
- an ALK inhibitor includes an anti-ALK phosphorylated antibody of the present disclosure, or an antigen-binding portion thereof.
- a device for diagnosing RDAA-positive diseases which includes:
- Detection section including a reagent section and a measurement section for detecting reagents capable of detecting ALK gene rearrangement, reagents for detecting ALK phosphorylation levels and reagents for detecting RNase1 expression levels, respectively;
- Data collection department collects the results output by the detection department
- the subject may be a healthy person, a patient suspected of a disease (eg, cancer), or a patient suffering from a disease (eg, cancer).
- the subject may be a cancer suspect or a cancer patient.
- the cancer may be lung cancer or pancreatic cancer.
- the cancer may be non-small cell lung cancer or pancreatic ductal adenocarcinoma.
- the detection is performed on a sample from the subject to be tested.
- the sample to be tested may be from tissues, cells and/or body fluids of a subject.
- the sample to be tested may be from tissue sections and/or blood of the subject.
- the sample to be tested may be from a subject's tumor tissue section and/or blood.
- the sample to be tested may be from lung or pancreatic cancer tissue sections and/or blood of the subject.
- 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.
- the detection of ALK gene rearrangement, ALK phosphorylation level and RNase1 expression level are independently selected from one or more of Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. In some embodiments, detection of ALK gene rearrangement expression levels, ALK phosphorylation levels and/or RNase1 expression levels is performed using antibodies.
- the data analysis section compares the collected data on ALK gene rearrangement expression levels, ALK phosphorylation levels and RNase1 expression levels with set thresholds.
- the set threshold for ALK phosphorylation level is a positive immunohistochemical stain for ALK phosphorylation.
- the set threshold for ALK phosphorylation level is an immunohistochemical staining score for ALK phosphorylation ⁇ 4 points.
- 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.
- 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.
- the immunohistochemical staining result for ALK phosphorylation is positive, and the Rnase1 expression level in the plasma is ⁇ 418ng/ml or the Rnase1 expression level is negative.
- the immunohistochemical staining result is positive, it is determined to be RDAA-positive disease.
- the immunohistochemical staining score for ALK phosphorylation is ⁇ 4 points
- the Rnase1 expression level in the plasma is ⁇ 418ng/ml or for Rnase1
- the immunohistochemical staining score is ⁇ 4 points, it is determined to be RDAA-positive disease.
- the detection part, data acquisition part, data analysis part and result output part may be separated into separate equipment units or formed into an integrated equipment.
- a method for treating diseases which at least includes the following steps:
- anaplastic lymphoma kinase (ALK) inhibitors to patients with RDAA-positive disease, defined as disease resulting from RNase1-driven ALK activation,
- samples from patients with RDAA-positive disease have negative ALK gene rearrangement test results, Rnase1 expression levels in plasma of ⁇ 418ng/ml or positive immunohistochemical staining results for Rnase1, and positive for Immunohistochemical staining results of ALK phosphorylation, and
- the patient with RDAA-positive disease is not a non-small cell lung cancer patient.
- tissue sections and/or blood from the patient Preferably, tissue sections and/or blood from the patient;
- tumor tissue sections and/or blood from the patient Preferably, tumor tissue sections and/or blood from the patient;
- lung or pancreatic cancer tissue sections and/or blood from the subject Preferably, lung or pancreatic cancer tissue sections and/or blood from the subject;
- pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient Preferably, pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient.
- the ALK inhibitor comprising an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.
- ALK inhibitor includes crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, or ensartinib. any one or combination thereof.
- an ALK inhibitor in the preparation of a medicament for the treatment of RDAA-positive disease, wherein samples from patients suffering from said RDAA-positive disease have negative ALK gene rearrangement detection results, ⁇ 418ng/ml in plasma Rnase1 expression level or positive immunohistochemical staining results for Rnase1, and positive immunohistochemical staining results for ALK phosphorylation, and wherein the patient with RDAA-positive disease is not a non-small cell lung cancer patient.
- tissue sections and/or blood from the patient Preferably, tissue sections and/or blood from the patient;
- tumor tissue sections and/or blood from the patient Preferably, tumor tissue sections and/or blood from the patient;
- lung or pancreatic cancer tissue sections and/or blood from the subject Preferably, lung or pancreatic cancer tissue sections and/or blood from the subject;
- pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient Preferably, pancreatic ductal adenocarcinoma tissue sections and/or blood from the patient.
- ALK inhibitor includes crizotinib, ceritinib, lorlatinib, alectinib, brigatinib, ensa any one or combination thereof.
- ALK inhibitor comprises an antibody or an antigen-binding portion thereof capable of inhibiting ALK phosphorylation.
- Prokaryotic cell purified RNase1 protein was used as antigen (5 mg in total) in combination with adjuvant.
- adjuvants Commonly used adjuvants: Freund's complete adjuvant, Freund's incomplete adjuvant.
- the second immunization dose is the same as above, plus Freund's incomplete adjuvant for intraperitoneal injection, the dose is 0.3ml;
- the third immunization dose is the same as above, without adjuvant, and injected intraperitoneally;
- Myeloma cells were cultured in DMEM medium.
- the concentration of calf serum is 10% and the cell concentration is 1 ⁇ 10 5 /ml.
- the cells are in the mid-phase of logarithmic growth, passage them at a ratio of 1:3. Passage every 3 days. (Regular treatment with 8-azaguanine makes surviving cells uniformly sensitive to HAT.)
- Mouse peritoneal macrophages were used in this study, and the amount was 10 5 cells/well.
- HAT selects hybridoma cells and culture fusion cells in HAT selection culture medium.
- a large number of tumor cells will die within 1 to 2 days of culture. After 3 to 4 days, the tumor cells will disappear and the hybrid cells will form small colonies.
- the HAT selection culture medium will be maintained for 7 to 10 days. After 3 days, switch to HT culture medium, maintain it for another 2 weeks, and then switch to normal culture medium.
- 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.
- half of the culture medium is generally changed every 2 to 3 days.
- Hybridoma cells were inoculated in vivo to prepare ascites.
- Preparation of ascites Routinely intraperitoneally inject 0.5 ml of Pristane (phytane) or liquid paraffin into BALB/C mice. Two weeks later, 1 ⁇ 10 6 hybridoma cells are intraperitoneally injected. Ascites can be produced 10 days after the cells are inoculated. The mice were killed, and the ascites was sucked into the test tube with a dropper. Generally, 10 ml of ascites could be obtained from one mouse.
- Pristane phytane
- the monoclonal antibody content in ascites fluid can reach 5 mg/ml.
- Protein lysis solution 2 ⁇ sample buffer 40ml system configuration add the following ingredients: 10ml 80% glycerol, 1ml sterilized water, 24ml 10% SDS, 5ml 1M Tris-HCL (pH6.8), mix thoroughly and store at room temperature for later use. .
- Preparation of protein loading buffer Mix ⁇ -mercaptoethanol and appropriate amount of bromophenol blue powder in a 1.5ml EP tube, and store it in a -20°C refrigerator for routine use.
- the microplate reader uses a wavelength of 562nm to detect the absorbance, record the OD value, and draw a protein standard curve;
- Sample protein treatment Take the 96-well plate and the prepared protein sample, add 18 ⁇ l PBS and 2 ⁇ l sample to each well, and add 200 ⁇ l BCA mixture, and react in a 37°C metal bath or incubator for 30 minutes;
- 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 ⁇ l of protein loading indicator (marker) according to experimental needs;
- 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 reaches 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 transferring;
- Membrane transfer Prepare the filter paper required for membrane transfer in advance, install the methanol box for activation, transfer clamp, PVDF membrane, etc., and pre-cool the transfer liquid at 4°C before transfer. Place a piece of filter paper in a horizontal container and pour an appropriate amount of transfer solution so that the height slightly exceeds the thickness of the filter paper. Methanol wets and activates the PVDF membrane, which is then placed on filter paper. Take out the gel plate after electrophoresis, peel off the protein gel from the gel plate, trim the borders appropriately and place it on the PVDF membrane, cover it with a layer of filter paper, and continuously remove the bubbles generated during the operation.
- Incubate primary antibody Use primary antibody diluent, 5% milk or 5% BSA according to the proportion to configure the primary antibody.
- the concentration of the primary antibody configuration in this experiment is 1:1000 (the sources of the antibodies in this experiment are self-made monoclonal mouse antibodies). Each antibody is configured with 4ml.
- Prepare the antibody before incubation pour it into the corresponding compartment of the incubation box and place it on ice at low temperature to maintain antibody activity.
- 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;
- Recover the secondary antibody Recover the secondary antibody and rinse the PVDF membrane again with PBST 3 times, 5-10 minutes each time;
- ECL development Pre-configure the ultra-sensitive ECL developer (UltraSignal ultra-sensitive ECL chemiluminescent substrate, Sizhengbai, product number: 4AW011-100). Mix equal volumes of ECL A solution and B solution in an EP tube and protect from light. Reserve at low temperature. Before development, place the PVDF film on the foam board, dry the remaining PBST slightly, and then drop the prepared ultra-sensitive ECL developer on it according to the size of the film until both sides are completely infiltrated. Exposed in Bio-rad imaging instrument.
- the 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.
- 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.
- 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 item number: ELISA Kit for Ribonuclease1, human; Product Number: SEA297Hu)
- Immunohistochemistry was used to detect the expression level of RNase1 in tumor tissue samples from three ALK gene rearrangement-negative subjects. Immunohistochemistry experimental steps:
- 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.
- 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.
- RDAA-positive subjects 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;
- Rnase1 expression level in plasma is ⁇ 418ng/ml or immunohistochemical staining score for Rnase1 is ⁇ 4 points; and immunohistochemical staining score for ALK phosphorylation is ⁇ 4 points.
- Cancer cells from 1 RDAA-positive and 1 RDAA-negative non-small cell lung cancer subject were treated with placebo (PBS buffer), crizotinib, ceritininb, and loratinib, respectively.
- Cells were treated with six ALK inhibitors: Loratinib, Alectinib, Brigatinib and Ensartinib (the final concentration of the above drugs was 1 ⁇ g per ml of culture) Base, cell density is 50,000 cells per ml of culture medium), observe cell survival, count the number of surviving cells every day, and draw a growth curve.
- the experimental results are shown in Figure 3.
- the abscissa axis is the medication time in (days), and the ordinate axis is the relative cell number.
- the results showed that placebo could not inhibit the growth of RDAA-positive lung cancer cells, but various ALK inhibitors effectively killed RDAA-positive lung cancer cells (P ⁇ 0.01), but had no significant effect on RDAA-negative lung cancer cells.
- Example 4 Therapeutic effect of ALK inhibitor on RDAA-positive lung cancer mice
- RDAA-positive tumor-bearing mice were constructed.
- Tumor-bearing mice bearing RDAA-positive tumors were randomly divided into seven groups (5 mice in each group), and placebo (PBS buffer), crizotinib, ceritinib, lorlatinib, and aletinib were orally administered.
- the dose of ALK inhibitor, brigatinib, or ensartinib (ALK inhibitor) was 25 mg per kilogram of body weight per day, and the dose of placebo was 2 ⁇ l per mouse per day, starting 2 weeks after tumor inoculation. , stop administration from the fourth week).
- Example 5 Therapeutic effect of ALK inhibitor on RDAA positive patients
- 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.
- Example 6 Therapeutic effect of ALK inhibitor on patients with RDAA-positive PDAC
- NGS second-generation high-throughput gene sequencing
- the patient's tumor cells were cultured and treated with placebo control (PBS buffer), Crizotinib, Ceritininb, Loratinib, Alectinib ( Cells were treated with six ALK inhibitors (Alectinib), Brigatinib and Ensartinib (the final concentration of the above drugs was 1 ⁇ g per ml of culture medium, and the cell density was 50,000 cells per ml). base), observe cell survival, count the number of surviving cells every day, and draw a growth curve.
- PBS buffer placebo control
- Crizotinib Crizotinib
- Ceritininb Ceritininb
- Loratinib Loratinib
- Alectinib Cells were treated with six ALK inhibitors (Alectinib), Brigatinib and Ensartinib (the final concentration of the above drugs was 1 ⁇ g per ml of culture medium, and the cell density was 50,000 cells per ml). base), observe cell survival, count the number
- 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).
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Abstract
本公开提供了一种用于治疗疾病的方法,其至少包括以下步骤:对患有RDAA阳性疾病的患者施用间变性淋巴瘤激酶(ALK)抑制剂,所述RDAA阳性疾病定义为RNase1驱动的ALK活化所导致的疾病,其中来自所述患有RDAA阳性疾病的患者的样本具有阴性的ALK基因重排检测结果,≥418 ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果,且其中所述患有RDAA阳性疾病的患者不是非小细胞肺癌患者。
Description
本公开涉及生物医药技术领域,具体涉及疾病的诊断和治疗,特别是ALK抑制剂在治疗RDAA阳性疾病中的具体应用。
ALK(anaplastic lymphoma kinase,间变性淋巴瘤激酶)是一类受体酪氨酸激酶,其变异与多种癌症的发生相关。ALK的变异形式包括Alk基因重排;Alk基因的激活突变;基因拷贝数变异等。ALK基因重排会导致淋巴瘤、肺癌、神经母细胞瘤、肌纤维母细胞瘤等多种癌症的发生,其中最常见的是间变性大细胞淋巴瘤(ALCL)和非小细胞肺癌(NSCLC),但ALK基因重排阳性仅占NSCLC患者中的6.7%,占ALCL成年患者中的50%。
对于不同类型的癌症,治疗方案和预后评估都不尽相同。例如,对于ALK基因重排阳性的肿瘤患者,可以应用ALK抑制剂予以治疗。但对于ALK为野生型的肿瘤患者(即,ALK基因重排阴性),目前尚未发现相关的促癌机制,也没有有效的靶向治疗方法。
发明内容
针对目前临床上仅存在对ALK基因重排阳性患者有效治疗方法(即,施用ALK抑制剂),而对ALK基因重排阴性患者尚未发现有效治疗手段这一亟待解决的问题,本公开的发明人通过生物标记物的巧妙选择,在ALK基因重排阴性患者群体中鉴定出了RDAA阳性疾病,并进一步发现,施用ALK抑制剂有效杀伤了RDAA阳性癌细胞,显著抑制了RDAA阳性患者的肿瘤生长,从而能够有效治疗RDAA阳性疾病,为广大患者带来了福音。特别地,本公开提供了用于诊断和/或治疗RDAA阳性疾病的方法;其中使用的抗RNase1抗体或其抗原结合部分、抗ALK磷酸化抗体或其抗原结合部分;用于诊断RDAA阳性疾病的试剂盒、设备;检测ALK基因重排的试剂、检测ALK磷酸化水平的试剂与检测RNase1表达水平的试剂的组合在制备用于检测RDAA阳性疾病的试剂盒中的用途。
在本公开的第一方面,可以提供一种用于诊断和/或治疗疾病的方法,其至少包括以下步骤:
检测受试者的间变性淋巴瘤激酶(ALK)基因重排、ALK磷酸化水平和RNase1表达水平,若ALK基因重排的检测结果为阴性、且ALK磷酸化水平和RNase1表达水平均高于参考人群水平,则定义所述疾病为RNase1驱动的ALK活化所导致的疾病(RDAA阳性疾病),以及任选地对受试者施用ALK抑制剂。
在本公开的第二方面,可以提供一种用于诊断和/或治疗疾病的方法,其至少包括以下步骤:
1)检测受试者的ALK基因重排,若ALK基因重排的检测结果为阴性,则进行下述步骤:
2)检测受试者的ALK磷酸化水平和RNase1表达水平;以及
3)将检测得到的ALK磷酸化水平和RNase1表达水平分别与参考人群的ALK磷酸化水平和RNase1表达水平进行比较;以及
4)任选地,在检测得到的ALK磷酸化水平和RNase1表达水平分别与参考人群的ALK磷酸化水平和RNase1表达水平相比更高且达到或高于设定阈值的情况下,确定所述受试者患有RDAA阳性疾病;以及
5)任选地,向患有RDAA阳性疾病的受试者施用ALK抑制剂。
在本公开的第三方面,可以提供ALK抑制剂在制备用于治疗非小细胞肺癌和/或胰腺导管腺癌的药物中的用途。
在本公开的第四方面,可以提供一种抗RNase1抗体或其抗原结合部分,其包含:
具有如SEQ ID NO:1所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:2所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:3所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:4所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:5所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:6所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:9所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:10所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:11所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:12所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:13所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:14所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:17所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:18所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:19所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:20所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:21所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:22所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:25所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:26所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:27所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:28所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:29所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:30所示的氨基酸序列的重链CDR3;或者
具有如SEQ ID NO:33所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:34所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:35所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:36所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:37所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:38所示的氨基酸序列的重链CDR3。
在本公开的第五方面,可以提供一种抗ALK磷酸化抗体或其抗原结合部分,其包含:
具有如SEQ ID NO:41所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:42所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:43所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:44所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:45所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:46所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:49所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:50所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:51所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:52所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:53所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:54所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:57所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:58所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:59所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:60所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:61所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:62所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:65所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:66所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:67所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:68所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:69所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:70所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:73所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:74所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:75所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:76所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:77所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:78所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:81所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:82所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:83所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:84所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:85所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:86所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:89所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:90所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:91所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:92所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:93所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:94所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:97所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:98所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:99所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:100所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:101所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:102所示的氨基酸序列的重链CDR3;或者
具有如SEQ ID NO:105所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:106所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:107所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:108所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:109所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:110所示的氨基酸序列的重链CDR3。
在本公开的第六方面,可以提供一种试剂盒,其包含:
1)检测ALK磷酸化水平的试剂;和
2)检测RNase1表达水平的试剂。
在本公开的第七方面,可以提供检测ALK磷酸化水平的试剂与检测RNase1表达水平的试剂的组合在制备用于检测RDAA阳性疾病的试剂盒中的用途,所述RDAA阳性疾病的患者具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果。
在本公开的第八方面,可以提供检测ALK基因重排表达水平的试剂、检测ALK磷酸化水平的试剂与检测RNase1表达水平的试剂的组合在制备用于检测RDAA阳性疾病的试剂盒中的用途,所述RDAA阳性疾病的患者具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果。
在本公开的第九方面,可以提供ALK抑制剂在制备用于治疗RDAA阳性疾病的药物中的用途,所述RDAA阳性疾病的患者具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果。
在本公开的第十方面,可以提供一种用于诊断RDAA阳性疾病的设备,其包括:
1)检测部:包括其中分别设置有能够检测ALK基因重排表达水平的试剂、检测ALK磷酸化水平的试剂和检测RNase1表达水平的试剂的试剂部和进行检测的测定部;
2)数据采集部:对检测部输出的结果进行收集;
3)数据分析部:对数据采集部收集的数据进行分析;和
4)结果输出部。
图1示出了实施例1的5个RNase1单克隆抗体的蛋白质免疫印迹杂交实验结果。
图2示出了实施例2的9个ALK蛋白磷酸化单克隆抗体的蛋白质免疫印迹杂交实验结果。
图3示出了ALK抑制剂对RDAA阳性肺癌细胞的杀伤作用实验结果。
图4示出了ALK抑制剂对RDAA阳性肿瘤荷瘤小鼠肿瘤生长抑制效果。
图5示出了ALK抑制剂对RDAA阳性肿瘤荷瘤小鼠的生存影响结果。
图6示出了ALK抑制剂对RDAA阳性患者的肿瘤生长抑制效果。
图7示出了来自RDAA阳性PDAC患者的细胞对ALK抑制剂的敏感性。
除非另外定义,否则本公开使用的所有技术和科学术语具有与本公开所属领域中普通技术人员通常所理解的相同含义。本公开的描述中使用的术语仅用于描述具体实施方式的目的,而无意限制本公开。
在本文中使用时,术语“ALK”是指间变性淋巴瘤激酶,其相关信息详见https://www.ncbi.nlm.nih.gov/gene/238(ALK ALK receptor tyrosine kinase[Homo sapiens(human)]Gene ID:238),及其同源物。
在本文中使用时,术语“ALK基因重排”包括但不限于美国第9,651,555号专利和Du等人(Thoracic Cancer.9:423-430,2018)中所述的那些,这些文献以全文引用方式并入本文。ALK基因重排可以为ALK与选自由以下项组成的组的基因的重排:EML4、KIF5B、KLC1、TFG、TPR、HIP1、STRN、DCTN1、SQSTM1、NPM1、BCL11A、BIRC6、RANBP2、ATIC、CLTC、TMP4、和MSN,导致融合癌基因的形成。
在本文中使用时,术语“ALK基因重排阴性”是指通过FISH(Fluorescence in situ hybridization)法、NGS(Next-generation sequencing)法、IHC(Immunohistochemistry)法等方法检测时,结果为ALK基因重排阴性。例如,患者手术或穿刺病理组织样本使用Vysis ALK Break Apart FISH探针试剂盒(Abbott Molecular,Inc.)诊断为ALK分子重排(ALK rearrangement)阴性;患者手术或穿刺病理组织样本使用二代高通量基因检测技术(武汉昂科益科技咨询有限公司、益善生物技术股份有限公司、深圳华大基因股份有限公司等)诊断无ALK基因相关突变,诊断为ALK分子重排阴性;患者手术或穿刺病理组织样本使用抗ALK(D5F3)兔单克隆抗体试剂(免疫组织化学法)(VENTANA anti-ALK(D5F3)Rabbit Monoclonal Primary Antibody)诊断为ALK分子重排阴性。
在本文中使用时,术语“Rnase1”是指核糖核酸酶1(ribonuclease1),属于人体分泌型核糖核酸酶家族,其相关信息详见https://www.ncbi.nlm.nih.gov/gene/6035(RNASE1 ribonuclease A family member 1,pancreatic[Homo sapiens(human)]),及其同源物。
在本文中使用时,“RDAA阳性疾病”是指与RNAse1驱动的ALK活化相关的疾病。
在本文中使用时,“RDAA阳性疾病的患者”是指该患者具有阴性的ALK基因重排检测结果,高于参考人群的ALK磷酸化水平的ALK磷酸化水平,以及高于参考人群的RNase1表达水平的RNase1表达水平;特别地,该患者具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果。
在本文中使用时,“参考人群”是指健康人或RDAA阴性疾病患者。
本公开的发明人首次发现,对于RDAA阳性疾病的患者,施用ALK抑制剂能够达到有效的治疗效果。因此,ALK抑制剂在制备治疗RDAA阳性疾病的药物中具有良好的应用前景。
在本公开的第一方面,可以提供一种用于诊断和/或治疗疾病的方法,其至少包括以下步骤:
检测受试者的淋巴瘤激酶(ALK)基因重排表达水平、ALK磷酸化水平和RNase1表达水平,若ALK基因重排的检测结果为阴性、且ALK磷酸化水平和RNase1表达水平均高于参考人群水平,则定义所述疾病为RNase1驱动的ALK活化所导致的疾病(RDAA阳性疾病),以及任选地对受试者施用ALK抑制剂。
在一些实施方式中,所述受试者可以是健康人,也可以是疾病(例如,癌症)疑似患者或者患有疾病(例如,癌症)的患者。在一些实施方式中,所述受试者可以是癌症疑似患者或者癌症患者。在一些实施方式中,所述癌症可以是肺癌或胰腺癌。在一些实施方式中,所述癌症可以是非小细胞肺癌或胰腺导管腺癌。
在一些实施方式中,所述检测针对来自受试者的待测样本进行。在一些实施方式中,所述待测样本可以来自受试者的组织、细胞和/或体液。在一些实施方式中,所述待测样本可以来自受试者的组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的肿瘤组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的肺癌或胰腺癌组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的非小细胞肺癌或胰腺导管腺癌组织切片和/或血液。
在一些实施方式中,参考人群的ALK磷酸化水平为针对ALK磷酸化的免疫组织化学染色结果为阴性。在一些实施方式中,参考人群的ALK磷酸化水平为针对ALK磷酸化的免疫组织化学染色得分<4分。
在一些实施方式中,参考人群的RNase1表达水平为血浆中的Rnase1表达水平<418ng/ml或者针对Rnase1的免疫组织化学染色结果为阴性。在一些实施方式中,参考人群的RNase1表达水平为血浆中的Rnase1表达水平<418ng/ml或者针对Rnase1的免疫组织化学染色得分<4分。
在一些实施方式中,ALK磷酸化水平的设定阈值为针对ALK磷酸化的免疫组织化学染色结果为阳性。在一些实施方式中,ALK磷酸化水平的设定阈值为针对ALK磷酸化的免疫组织化学染色得分≥4分。
在一些实施方式中,RNase1表达水平的设定阈值为血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色结果为阳性。在一些实施方式中,RNase1表达水平的设定阈值为血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色得分≥4分。
在一些实施方式中,免疫组织化学染色是使用患病组织进行的。在一些实施方式中,免疫组织化学染色是使用肿瘤组织进行的。在一些实施方式中,免疫组织化学染色是使用肺癌或胰腺癌组织进行的。在一些实施方式中,免疫组织化学染色是使用非小细胞肺癌或胰腺导管腺癌组织进行的。
ALK基因重排表达水平、ALK磷酸化水平和RNase1表达水平的检测可以通过本领域已知的任何方法来完成,例如使用商业化的试剂盒、测序等。在一些实施方式中,ALK基因重排表达水平、ALK磷酸化水平和RNase1表达水平的检测彼此独立地选自蛋白质印迹法、免疫组织化学法、酶联免疫吸附法中的一种或多种。在一些实施方式中,ALK基因重排表达水平、ALK磷酸化水平和/或RNase1表达水平的检测是使用抗体进行的。
在一些实施方式中,所述ALK抑制剂包括能够抑制ALK磷酸化的抗体或其抗原结合部分。在一些实施方式中,所述ALK抑制剂包括克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、恩沙替尼中的任一种或其组合。
在本公开的第二方面,可以提供一种用于诊断和/或治疗疾病的方法,其至少包括以下步骤:
1)检测受试者的ALK基因重排表达水平,若ALK基因重排的检测结果为阴性,则进行下述步骤:
2)检测受试者的ALK磷酸化水平和RNase1表达水平;以及
3)将检测得到的ALK磷酸化水平和RNase1表达水平分别与参考人群的ALK磷酸化水平和RNase1表达水平进行比较;以及
4)任选地,在检测得到的ALK磷酸化水平和RNase1表达水平分别与参考人群的ALK磷酸化水平和RNase1表达水平相比更高且达到或高于设定阈值的情况下,确定所述受试者患有RDAA阳性疾病;以及
5)任选地,向患有RDAA阳性疾病的受试者施用ALK抑制剂。
在一些实施方式中,所述受试者可以是健康人,也可以是疾病(例如,癌症)疑似患者或者患有疾病(例如,癌症)的患者。在一些实施方式中,所述受试者可以是癌症疑似患者或者癌症患者。在一些实施方式中,所述癌症可以是肺癌或胰腺癌。在一些实施方式中,所述癌症可以是非小细胞肺癌或胰腺导管腺癌。
在一些实施方式中,所述检测针对来自受试者的待测样本进行。在一些实施方式中,所述待测样本可以来自受试者的组织、细胞和/或体液。在一些实施方式中,所述待测样本可以来自受试者的组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受
试者的肿瘤组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的肺癌或胰腺癌组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的非小细胞肺癌或胰腺导管腺癌组织切片和/或血液。
在一些实施方式中,参考人群的ALK磷酸化水平为针对ALK磷酸化的免疫组织化学染色结果为阴性。在一些实施方式中,参考人群的ALK磷酸化水平为针对ALK磷酸化的免疫组织化学染色得分<4分。
在一些实施方式中,参考人群的RNase1表达水平为血浆中的Rnase1表达水平<418ng/ml或者针对Rnase1的免疫组织化学染色结果为阴性。在一些实施方式中,参考人群的RNase1表达水平为血浆中的Rnase1表达水平<418ng/ml或者针对Rnase1的免疫组织化学染色得分<4分。
在一些实施方式中,ALK磷酸化水平的设定阈值为针对ALK磷酸化的免疫组织化学染色结果为阳性。在一些实施方式中,ALK磷酸化水平的设定阈值为针对ALK磷酸化的免疫组织化学染色得分≥4分。
在一些实施方式中,RNase1表达水平的设定阈值为血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色结果为阳性。在一些实施方式中,RNase1表达水平的设定阈值为血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色得分≥4分。
在一些实施方式中,免疫组织化学染色是使用患病组织进行的。在一些实施方式中,免疫组织化学染色是使用肿瘤组织进行的。在一些实施方式中,免疫组织化学染色是使用肺癌或胰腺癌组织进行的。在一些实施方式中,免疫组织化学染色是使用非小细胞肺癌或胰腺导管腺癌组织进行的。
ALK基因重排表达水平、ALK磷酸化水平和RNase1表达水平的检测可以通过本领域已知的任何方法来完成,例如使用商业化的试剂盒、测序等。在一些实施方式中,ALK基因重排表达水平、ALK磷酸化水平和RNase1表达水平的检测彼此独立地选自蛋白质印迹法、免疫组织化学法、酶联免疫吸附法中的一种或多种。在一些实施方式中,ALK基因重排表达水平、ALK磷酸化水平和/或RNase1表达水平的检测是使用抗体进行的。
在一些实施方式中,所述ALK抑制剂包括能够抑制ALK磷酸化的抗体或其抗原结合部分。在一些实施方式中,所述ALK抑制剂包括克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、恩沙替尼中的任一种或其组合。
在本公开的第三方面,可以提供ALK抑制剂在制备用于治疗非小细胞肺癌和/或胰腺导管腺癌的药物中的用途。
在本公开的第四方面,可以提供一种抗RNase1抗体或其抗原结合部分,其包含:
具有如SEQ ID NO:1所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:2所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:3所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:4所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:5所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:6所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:9所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:10所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:11所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:12所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:13所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:14所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:17所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:18所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:19所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:20所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:21所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:22所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:25所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:26所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:27所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:28所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:29所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:30所示的氨基酸序列的重链CDR3;或者
具有如SEQ ID NO:33所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:34所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:35所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:36所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:37所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:38所示的氨基酸序列的重链CDR3。
在一些实施方式中,所述抗RNase1抗体或其抗原结合部分包含:
具有如SEQ ID NO:7所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:8所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:15所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:16所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:23所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:24所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:31所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:32所示的氨基酸序列的重链可变区;或者
具有如SEQ ID NO:39所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:40所示的氨基酸序列的重链可变区。
在本公开的第五方面,可以提供一种抗ALK磷酸化抗体或其抗原结合部分,其包含:
具有如SEQ ID NO:41所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:42所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:43所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:44所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:45所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:46所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:49所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:50所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:51所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:52所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:53所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:54所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:57所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:58所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:59所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:60所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:61所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:62所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:65所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:66所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:67所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:68所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:69所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:70所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:73所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:74所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:75所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:76所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:77所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:78所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:81所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:82所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:83所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:84所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:85所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:86所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:89所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:90所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:91所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:92所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:93所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:94所示的氨基酸序列的重链CDR3;
具有如SEQ ID NO:97所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:98所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:99所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:100所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:101所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:102所示的氨基酸序列的重链CDR3;或者
具有如SEQ ID NO:105所示的氨基酸序列的轻链CDR1,具有如SEQ ID NO:106所示的氨基酸序列的轻链CDR2,具有如SEQ ID NO:107所示的氨基酸序列的轻链CDR3,具有如SEQ ID NO:108所示的氨基酸序列的重链CDR1,具有如SEQ ID NO:109所示的氨基酸序列的重链CDR2,以及具有如SEQ ID NO:110所示的氨基酸序列的重链CDR3。
在一些实施方式中,所述抗ALK磷酸化抗体或其抗原结合部分包含:
具有如SEQ ID NO:47所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:48所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:55所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:56所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:63所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:64所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:71所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:72所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:79所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:80所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:87所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:88所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:95所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:96所示的氨基酸序列的重链可变区;
具有如SEQ ID NO:103所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:104所示的氨基酸序列的重链可变区;或者
具有如SEQ ID NO:111所示的氨基酸序列的轻链可变区,以及具有如SEQ ID NO:112所示的氨基酸序列的重链可变区。
在本公开的第六方面,可以提供一种试剂盒,其包含:
1)检测ALK磷酸化水平的试剂;和
2)检测RNase1表达水平的试剂。
在一些实施方式中,检测ALK磷酸化水平的试剂、检测RNase1表达水平的试剂、检测ALK基因重排表达水平的试剂可以是本领域已知的任何能够实现该功能的试剂。在一些实施方式中,检测ALK磷酸化水平的试剂包括本公开所述的抗ALK磷酸化抗体或其抗原结合部分。在一些实施方式中,检测RNase1表达水平的试剂包括本公开所述的抗RNase1抗体或其抗原结合部分。在一些实施方式中,所述试剂盒还包含检测ALK基因重排表达水平的试剂。
在本公开的第七方面,可以提供检测ALK磷酸化水平的试剂与检测RNase1表达水平的试剂的组合在制备用于检测RDAA阳性疾病的试剂盒中的用途,所述RDAA阳性疾病的患者具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果。
在一些实施方式中,阳性的针对Rnase1的免疫组织化学染色结果是指≥4分的针对Rnase1的免疫组织化学染色得分。
在一些实施方式中,阳性的针对ALK磷酸化的免疫组织化学染色结果是指≥4分的针对ALK磷酸化的免疫组织化学染色得分。
在本公开的第八方面,可以提供检测ALK基因重排表达水平的试剂、检测ALK磷酸化水平的试剂与检测RNase1表达水平的试剂的组合在制备用于检测RDAA阳性疾病的试剂盒中的用途,所述RDAA阳性疾病的患者具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果。
在一些实施方式中,阳性的针对Rnase1的免疫组织化学染色结果是指≥4分的针对Rnase1的免疫组织化学染色得分。
在一些实施方式中,阳性的针对ALK磷酸化的免疫组织化学染色结果是指≥4分的针对ALK磷酸化的免疫组织化学染色得分。
在本公开的第九方面,可以提供ALK抑制剂在制备用于治疗RDAA阳性疾病的药物中的用途,所述RDAA阳性疾病的患者具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果。
在一些实施方式中,阳性的针对Rnase1的免疫组织化学染色结果是指≥4分的针对Rnase1的免疫组织化学染色得分。
在一些实施方式中,阳性的针对ALK磷酸化的免疫组织化学染色结果是指≥4分的针对ALK磷酸化的免疫组织化学染色得分。
ALK抑制剂可以是本领域已知的任何能够有效抑制ALK的活性和/或表达等的物质。在一些实施方式中,ALK抑制剂包括但不限于克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、恩沙替尼中的任一种或其组合。在一些实施方式中,ALK抑制剂包括能够抑制ALK磷酸化的抗体或其抗原结合部分。在一些实施方式中,ALK抑制剂包括本公开所述的抗ALK磷酸化抗体或其抗原结合部分。
在本公开的第十方面,可以提供一种用于诊断RDAA阳性疾病的设备,其包括:
1)检测部:包括其中分别设置有能够检测ALK基因重排的试剂、检测ALK磷酸化水平的试剂和检测RNase1表达水平的试剂的试剂部和进行检测的测定部;
2)数据采集部:对检测部输出的结果进行收集;
3)数据分析部:对数据采集部收集的数据进行分析;和
4)结果输出部。
在一些实施方式中,所述受试者可以是健康人,也可以是疾病(例如,癌症)疑似患者或者患有疾病(例如,癌症)的患者。在一些实施方式中,所述受试者可以是癌症疑似患者或者癌症患者。在一些实施方式中,所述癌症可以是肺癌或胰腺癌。在一些实施方式中,所述癌症可以是非小细胞肺癌或胰腺导管腺癌。
在一些实施方式中,所述检测针对来自受试者的待测样本进行。在一些实施方式中,所述待测样本可以来自受试者的组织、细胞和/或体液。在一些实施方式中,所述待测样本可以来自受试者的组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的肿瘤组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的肺癌或胰腺癌组织切片和/或血液。在一些实施方式中,所述待测样本可以来自受试者的非小细胞肺癌或胰腺导管腺癌组织切片和/或血液。
在一些实施方式中,ALK基因重排、ALK磷酸化水平和RNase1表达水平的检测彼此独立地选自蛋白质印迹法、免疫组织化学法、酶联免疫吸附法中的一种或多种。在一些实施方式中,ALK基因重排表达水平、ALK磷酸化水平和/或RNase1表达水平的检测是使用抗体进行的。
在一些实施方式中,数据分析部将收集的ALK基因重排表达水平、ALK磷酸化水平和RNase1表达水平的数据与设定阈值进行比较。
在一些实施方式中,ALK磷酸化水平的设定阈值为针对ALK磷酸化的免疫组织化学染色结果为阳性。
在一些实施方式中,ALK磷酸化水平的设定阈值为针对ALK磷酸化的免疫组织化学染色得分≥4分。
在一些实施方式中,RNase1表达水平的设定阈值为血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色结果为阳性。
在一些实施方式中,RNase1表达水平的设定阈值为血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色得分≥4分。
在一些实施方式中,当数据分析部的分析表明ALK基因重排表达水平为阴性,针对ALK磷酸化的免疫组织化学染色结果为阳性,并且血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色结果为阳性时,判定为RDAA阳性疾病。
在一些实施方式中,当数据分析部的分析表明ALK基因重排表达水平为阴性,针对ALK磷酸化的免疫组织化学染色得分≥4分,并且血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色得分≥4分时,判定为RDAA阳性疾病。
在一些实施方式中,检测部、数据采集部、数据分析部和结果输出部可以分离成单独的设备单元或形成集成设备。
上文针对本公开的方法所述的各种实施方式和优选项可以相互组合(只要它们彼此之间不是内在矛盾的),并且同样适用于本公开的抗RNase1抗体或其抗原结合部分、抗ALK磷酸化抗体或其抗原结合部分、试剂盒、用途和设备,反之亦然,由此组合而形成的各种实施方式都视为本申请公开的一部分。
特别地,本公开提供了以下实施方案:
1.一种用于治疗疾病的方法,其至少包括以下步骤:
对患有RDAA阳性疾病的患者施用间变性淋巴瘤激酶(ALK)抑制剂,所述RDAA阳性疾病定义为RNase1驱动的ALK活化所导致的疾病,
其中来自所述患有RDAA阳性疾病的患者的样本具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果,且
其中所述患有RDAA阳性疾病的患者不是非小细胞肺癌患者。
2.实施方案1所述的方法,其中所述样本来自患者的组织、细胞和/或体液;
优选地,来自患者的组织切片和/或血液;
优选地,来自患者的肿瘤组织切片和/或血液;
优选地,来自受试者的肺癌或胰腺癌组织切片和/或血液;
优选地,来自患者的胰腺导管腺癌组织切片和/或血液。
3.前述实施方案中任一项所述的方法,所述患者为胰腺导管腺癌患者。
4.前述实施方案中任一项所述的方法,所述ALK抑制剂包括能够抑制ALK磷酸化的抗体或其抗原结合部分。
5.前述实施方案中任一项所述的方法,所述ALK抑制剂包括克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、恩沙替尼中的任一种或其组合。
6.ALK抑制剂在制备用于治疗RDAA阳性疾病的药物中的用途,其中来自患有所述RDAA阳性疾病的患者的样本具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果,且其中所述患有RDAA阳性疾病的患者不是非小细胞肺癌患者。
7.实施方案6所述的用途,其中所述样本来自患者的组织、细胞和/或体液;
优选地,来自患者的组织切片和/或血液;
优选地,来自患者的肿瘤组织切片和/或血液;
优选地,来自受试者的肺癌或胰腺癌组织切片和/或血液;
优选地,来自患者的胰腺导管腺癌组织切片和/或血液。
8.实施方案6或7所述的用途,所述患者为胰腺导管腺癌患者。
9.实施方案6-8中任一项所述的用途,其中所述ALK抑制剂包括克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、恩沙替尼中的任一种或其组合。
10.实施方案6-9中任一项所述的用途,其中所述ALK抑制剂包括能够抑制ALK磷酸化的抗体或其抗原结合部分。
下面将结合实施例以例证的方式更清楚、明确地阐述本公开的技术方案。应该理解的是,这些实施例仅用于例证的目的,绝不旨在限制本公开的保护范围。本公开的保护范围仅通过权利要求来限定。
实施例
实施例1:抗RNase1抗体的制备和结合性能
RNase1抗体的制备
一、动物的选择:纯种BALB/C小鼠
二、RNase1抗体免疫方案的选择
原核细胞纯化的RNase1蛋白作为抗原(共5mg)与佐剂联用。常用佐剂:福氏完全佐剂、福氏不完全佐剂。
a:初次免疫抗原50μg加福氏完全佐剂皮下多点注射或脾内注射(共1ml,0.2ml/点);
b:3周后,第二次免疫剂量同上,加福氏不完全佐剂腹腔内注射,剂量为0.3ml;
c:3周后,第三次免疫剂量同上,不加佐剂,腹腔内注射;
d:3周后,加强免疫,剂量0.5mg,腹腔内注射;
e:3天后,取脾融合。
三、细胞融合
1.细胞融合前准备
(1)骨髓瘤细胞系的选择:P3/X63-Ag8(X63)(BALB/C骨髓瘤MOPC-21来源)。
骨髓瘤细胞的培养用DMEM培养基。小牛血清的浓度在10%,细胞浓度1×105/ml为宜。当细胞处于对数生长的中期时,按1:3比例传代。每3天传代一次。(定期用8-氮鸟嘌呤进行处理,使生存的细胞对HAT呈均一的敏感性。)
(2)饲养细胞:
本研究选用小鼠腹腔巨噬细胞,量为105细胞/孔。
2.细胞融合的步骤
(1)制备饲养细胞层:
(2)制备免疫脾细胞
(3)制备骨髓瘤细胞
(4)融合
①将骨髓瘤细胞与脾细胞按1:10或1:5的比例混合在一起,在50ml离心管中用无血清不完全培养液洗1次,离心,1200rpm,8min;弃上清,用吸管吸净残留液体,以免影响聚乙二醇(PEG)浓度。轻轻弹击离心管底,使细胞沉淀略松动。
②90s内加入37℃预温的1ml 45%PEG溶液,边加边轻微摇动。37℃水浴作用90s。
③加37℃预温的不完全培养液以终止PEG作用,每隔2min分别加入1ml、2ml、3ml、4ml、5ml和6ml。
④离心,800rpm,6min。
⑤充上清,用含20%小牛血清HAT选择培养液重悬。
⑥将上述细胞,加到已有饲养细胞层的96孔板内,每孔加100μl。一个免疫脾脏可接种4块96孔板。
⑦将培养板置37℃、5%CO2培养箱中培养。
3.选择杂交瘤细胞及抗体检测
1)HAT选择杂交瘤细胞于HAT选择培养液中培养融合细胞,培养1~2天内有大量瘤细胞死亡,3~4天后瘤细胞消失,杂交细胞形成小集落,HAT选择培养液维持7~10天后换用HT培养液,再维持2周,改用一般培养液。在选择培养期间,杂交瘤细胞布满孔底1/10面积时,即可开始检测特异性抗体,筛选出所需要的杂交瘤细胞系。在选择培养期间,一般每2~3天换一半培养液。
四、产生RNase1抗体杂交瘤的克隆化
1)克隆前1天制备饲养细胞层(同细胞融合)。
2)将要克隆的杂交瘤细胞从培养孔内轻轻吹干,计数。
3)调整细胞为10个细胞/ml。
4)取头天准备的饲养细胞层的细胞培养板,每孔加入稀释细胞100μl。孵育于37℃、5%CO2孵箱中。
5)在第7天换液,以后每2天换液1次。
6)8天可见细胞克隆形成,及时检测抗体活性。
7)将阳性孔的细胞移至24孔板中扩大培养。
8)冻存细胞克隆。
五、RNase1单克隆抗体的大量生产
体内接种杂交瘤细胞,制备腹水。
腹水的制备:常规先腹腔注射0.5ml Pristane(降植烷)或液体石蜡于BALB/C鼠,2周后腹腔注射1×106个杂交瘤细胞,接种细胞10天后可产生腹水。处死小鼠,用滴管将腹水吸入试管中,一般一只小鼠可获10ml腹水。
腹水中单克隆抗体含量可达到5mg/ml。
获得了5种抗RNase1抗体,其具体氨基酸序列如下表所示。
利用这5个抗体进行蛋白质免疫印迹杂交实验(western blot),实验步骤如下:
一、蛋白裂解液的配置
1.蛋白裂解液2×sample buffer 40ml体系配置:按以下成分添加:80%甘油10ml,灭菌水1ml,10%SDS 24ml,1M Tris-HCL(pH6.8)5ml,充分混合后常温储存备用。
2.蛋白上样缓冲液的配置:将β-巯基乙醇与适量溴酚蓝粉末于1.5mlEP管内充分混合,于-20℃冰箱常规保存备用
二、总蛋白提取
1)从37℃恒温孵箱中取出需要提取蛋白的细胞培养皿,于显微镜下评估细胞密度;
2)将培养皿置于冰上(整个操作步骤均在冰上进行),吸去上层培养液,用遇冷的PBS清洗2次;
3)根据细胞密度按1:1的比例分别每孔加入20-50μl的PBS和蛋白裂解液,在液体覆盖培养皿表面后立即用细胞刮将所加溶液与培养皿表面附着细胞充分搅拌混合裂解,并收集至EP管内;
4)100℃恒温金属浴加热10分钟使蛋白变性,12000rpm瞬离,于4℃条件下短期保存备用。
三、蛋白浓度定量(BCA法)
1)实验前将PBS与BSA原液定量混合配置成浓度为0.5mg/ml的BSA标准品,冰上或4℃保存备用;
2)配置BCA混合液:根据实验设置的检测样本和对应梯度标准品的数目,按BCA试剂:Cu试剂=50:1的比例配制成淡绿色BCA混合工作液,充分混匀后室温短期保存备用;
3)于第一个孔开始将标准品从20μl-0μl递减的体积加入蛋白标准品孔中,随后加PBS补足每孔液体至20μl,使各孔内标准品的浓度形成一系列浓度梯度;
4)酶标仪使用562nm的波长检测的吸光度,记录OD值,并绘制蛋白标准曲线;
5)样品蛋白处理:取96孔板及制备好的蛋白样品,每孔加入18μl PBS和2μl样本,并加入200μl的BCA混合液,于37℃金属浴或孵箱中反应30分钟;
6)取反应后样本,检测得到OD值后,根据绘制完成的蛋白标准曲线计算样本蛋白浓度,并根据蛋白上样量计算电泳时的样本体积,具体计算公式:蛋白上样体积=蛋白上样量/样本蛋白浓度;
7)将样本蛋白与配好的蛋白上样缓冲液按20:1的比例充分混合,12000rpm瞬离于-20℃冰箱保存备用。
四、SDS-PAGE凝胶电泳
1)分离胶的制备:根据实验检测蛋白分子量的大小来选择不同浓度的分离胶,本实验检测蛋白RNase1、p-ALK(Y1604)、p-ALK(Y1282/1283)所需分离胶浓度为10%、8%、8%。分离胶体系如表1所示。于水平桌面安装好制胶板架,按分离胶体系配置好混合液(5ml),迅速小心平稳地打入制胶板间预留缝隙。随后沿制胶板壁边缘自左向右水平缓慢打入100-200μl体积异丙醇或无水乙醇压平胶面,轻微抬起并晃动一侧制胶架使其排出潜在的气泡,最后静置于水平桌面常温30-60分钟待分离胶凝固;
表1分离胶配置体系(5ml)
2)浓缩胶的制备:待分离胶凝固后,小心地吸去异丙醇或无水乙醇,自然通风条件下晾干。随后按浓缩胶体系(如表2所示)配置上层胶(2ml),并迅速平稳地打入制胶板间隙至液体溢出。缓慢、平稳插入15孔梳。室温下水平桌面静置30-60分钟;
表2浓缩胶配置体系(2ml)
3)待上层浓缩胶凝固后,将胶板从制胶架上小心取下,严密地固定于电泳槽内,小心缓慢且垂直地拔出梳子留出上样孔,并在电泳槽内依据胶板的数量倒入适量的电泳液;
4)蛋白上样:取出-20℃冰箱内保存的蛋白样品,待其解冻恢复至室温。根据计算好的蛋白上样体积等量地依次上样,并根据实验需要预留1-2孔加5μl蛋白上样指示剂(marker);
5)电泳:上样后以80V电压恒压开始电泳,当见到制胶板底部开始产生串珠样气泡即标志着电泳开始,随后可见蛋白样品内的溴酚蓝被压缩形成一条直线,根据marker判断蛋白到达一条明显区分度的透明分离胶界面后,调整电压至120V恒压,当溴酚蓝指示剂下降制胶板最底缘时便停止电泳开始转膜;
6)转膜:预先准备好转膜需要的滤纸,装有激活用的甲醇盒,转膜夹,PVDF膜等,并于转膜前在4℃环境下预冷转膜液。将一片滤纸置于水平容器中,倒入适量转膜液,使其高度略微超过滤纸厚度。甲醇润湿并活化PVDF膜,接着将其置于滤纸之上。取出电泳完毕的胶板,从胶板上剥离蛋白胶,适当修剪边界后置于PVDF膜上,在其上再覆一层滤纸,操作期间不断去除产生的气泡。最后遵循“黑胶白膜”原则,将转膜夹透明面作为最下层,其上覆海绵垫并水平置入前一步夹好的滤纸和PVDF膜,再于其上盖一层海绵垫,转膜夹黑色面对齐合上扣紧转膜夹卡扣。将扣好的转膜夹固定于转膜槽内,加入转膜液直到其淹没转膜指示线,将转膜槽置于合适的容器之后便开始转膜,容器内加入冰水预备降温。使用300mA恒流转膜,根据目标蛋白的大小选择合适的转膜时间,R1和p-ALK蛋白的转膜时间分别为1和2小时;
7)封闭:预先配置用PBST配置的5%脱脂牛奶及适量清洗用的PBST。转膜完成后打开转膜夹,小心取出完成转膜的PVDF膜。将膜置于PBST中清洗2-3次,每次2分钟。随后移入由PBST配成的5%脱脂牛奶内,放置于摇床上,封闭1小时;
8)孵育一抗:按照比例使用一抗稀释液、5%牛奶或5%BSA配置一抗,本实验一抗配置浓度均为1:1000(本实验抗体来源均为自制单克隆鼠抗),每支抗体配置4ml。孵育前配好所用抗体,倒入孵育盒对应条带的格内并置于冰上低温维持抗体活性。将5%脱脂牛奶内的PVDF膜取出并使用PBST略加漂洗以去除残留牛奶。根据实验检测蛋白条带大小,以显色marker为刻度将膜才成条带状后,放入对应抗体的格内。密封后置于4℃摇床缓慢摇动12小时;
9)回收一抗:12小时后将一抗回收;
10)孵育二抗:使用辣根酶标记山羊抗鼠IgG(H+L)二抗(中杉金桥,货号:ZB-2305)按照1:5000的比例使用PBST配成的5%脱脂牛奶提前配置相应二抗稀释液。PVDF膜用PBST漂洗至少3次,每次5-10分钟,漂洗完倒去液体。随后将二抗稀释液倒入对应种属的PVDF膜格内,摇床缓慢摇动1小时;
11)回收二抗:将二抗回收并用PBST再次漂洗PVDF膜3次,每次5-10分钟;
12)ECL显影:预先配置超敏ECL显影液(UltraSignal超敏ECL化学发光底物,四正柏,货号:4AW011-100),将ECL A液和B液等体积于EP管内混合后,避光低温备用。显影前,将PVDF膜置于泡沫板上,使残留的PBST略微拭干,随后根据膜的大小于其上滴下配好的超敏ECL显影液,待其两面完全浸润后。于Bio-rad成像仪器中曝光。
实验结果见图1。这5种RNase1单克隆抗体均能特异性的识别内源性的RNase1蛋白,目的条带清晰且与RNase1的蛋白大小吻合、无非特异性条带。结果表明:这5种抗RNase1抗体均与RNase1发生特异性结合,从而能够有效用于检测样本中的RNase1。
实施例2:抗ALK磷酸化抗体的制备
该抗体的制备过程与上述RNase1抗体的制备过程相同,仅在抗原使用上不同。制备ALK磷酸化抗体使用的抗原是合成的ALK蛋白磷酸化肽段,共2种ALK蛋白磷酸化肽段,每种5mg。
获得了9种抗ALK磷酸化抗体,其具体氨基酸序列如下表所示。
利用这9个抗体进行蛋白质免疫印迹杂交实验,实验方法与实施例1中RNase1的western blot过程相同。
实验结果见图2。这9种ALK磷酸化单克隆抗体均能特异性的识别内源性的ALK磷酸化蛋白,目的条带清晰且与ALK磷酸化蛋白大小吻合、无非特异性条带。结果表明:这9种抗ALK磷酸化抗体均与磷酸化ALK发生特异性结合,从而能够有效用于检测样本中的ALK磷酸化水平。
实施例3:ALK抑制剂对RDAA阳性疾病细胞的杀伤作用
利用二代测序的方法检测来3名受试者的肿瘤组织切片样品中ALK基因重排表达水平:患者手术或穿刺病理组织样本使用二代高通量基因检测技术(深圳华大基因股份有限公司)诊断无ALK基因重排及相关突变,诊断为ALK基因重排阴性。
对ALK基因重排阴性的受试者继续进行以下检测。
利用酶联免疫吸附(ELISA)法检测来自3名ALK基因重排阴性受试者的血浆样品中RNase1的表达水平。检测方法如下:
Elisa检测试剂及流程采用Rnase1 Elisa试剂盒(产品名及货号:ELISA Kit for Ribonuclease1,human;Product Number:SEA297Hu)
一、试剂及物品准备(试剂盒内物品)
二、实验步骤
1.准备所有试剂、样品和标准品;
2.每孔加入100μL标准品或样品,37℃孵育1小时;
3.抽取制备好的检测试剂A 100μL/孔加入,37℃孵育1小时;
4.抽吸并清洗3次;
5.加入制备好的检测试剂B 100μL/孔,37℃孵育30分钟;
6.抽吸并清洗5次;
7.加入90μL/孔TMB反应底物溶液。37℃孵育120分钟;
8.添加终止液50μL/孔。酶标仪450nm读数。
根据吸光度数值,以标准品为参照,以及稀释倍数,计算样品中RNase1的浓度。将418ng/ml浓度作为区分RNase1表达高与低的分界点。(非小细胞型肺癌患者血浆中RNase1的浓度平均值为418ng/ml)。
利用免疫组织化学(IHC)法检测来自3名ALK基因重排阴性受试者的肿瘤组织样品中RNase1的表达水平。免疫组织化学实验步骤:
(1)组织切片用65℃的烤箱烘烤3小时;
(2)石蜡切片经二甲苯脱蜡,不同浓度乙醇水化;
(3)3%过氧化氢抑制内源性过氧化物酶15分钟;
(4)PBS洗3次,每次冲洗5分钟;
(5)将切片浸入柠檬酸缓冲液用微波进行修复,微波高火5分钟,中火5分钟(或进行高压加热120℃,2分钟);
(6)自然冷却至室温后(约60分钟),PBS冲洗3次,每次5分钟;
(7)除去载玻片上多余液体,滴加RNase1单克隆抗体,抗体稀释浓度为1:100至1:1000,置于湿盒中,4℃过夜;
(8)第二日取出室温放置1h后PBS洗3次,每次5分钟;
(9)滴加多聚酶标二抗室温放置30分钟,PBS冲洗3次,每次5分钟;
(10)DAB显色液显色3分钟,镜下观察;
(11)流水终止显色,苏木素复染1分钟;
(12)自来水冲洗,分化液分化5s;
(13)冲洗切片,乙醇脱水、二甲苯透明、中性树胶封片。
免疫组织化学结果判定方法:所有的苏木素染色的切片都被两个病理医师单独的阅片。癌症标本的肿瘤含量大于70%。双盲法进行RNase1染色的评估,观察确定RNase1表达阳性的细胞定位,其表现为有棕黄色或棕褐色颗粒。免疫组织化学染色得分通过阳性染色细胞数乘以染色强度得到。光学显微镜下观察组织标本,按阳性染色细胞所占的百分比计分:0:阴性表达;1分:阳性细胞数为<15%;2分:阳性细胞15-50%;3分:阳性细胞≥50%;染色强度按照显色深浅计分:0分:无阳性;1分:浅黄;2分:棕黄;3分:棕褐。将免疫组织化学染色得分=4分作为区分RNase1表达水平高与低的分界点。
采用上述HIC法,使用ALK磷酸化单克隆抗体(抗体制备部分展示的9种ALK磷酸化单克隆抗体中的CDA04)检测来自3名受试者的肿瘤组织样品中的ALK磷酸化水平。将免疫组织化学染色得分=4分作为区分ALK磷酸化水平高与低的分界点。
按照以下标准划分为RDAA阳性受试者和非RDAA阳性(RDAA阴性)受试者:
RDAA阳性受试者:血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色得分≥4分;并且针对ALK磷酸化的免疫组织化学染色得分≥4分;
RDAA阴性受试者:血浆中的Rnase1表达水平<418ng/ml或者针对Rnase1的免疫组织化学染色得分<4分;并且针对ALK磷酸化的免疫组织化学染色得分<4分。
将来自1名RDAA阳性和1名RDAA阴性的非小细胞型肺癌受试者的癌细胞分别用安慰剂(PBS缓冲液)、克唑替尼(Crizotinib)、色瑞替尼(Ceritininb)、劳拉替尼(Loratinib)、阿来替尼(Alectinib)、布加替尼(Brigatinib)和恩沙替尼(Ensartinib)六种ALK抑制剂处理细胞(上述药物使用终浓度均为1微克每毫升培养基、细胞密度为5万个细胞每毫升培养基),观察细胞存活、每天计算存活细胞数量、并绘制生长曲线。
实验结果如图3所示,横坐标轴为用药的时间,单位为(天),纵坐标轴为相对细胞数量。结果显示:安慰剂无法抑制RDAA阳性肺癌细胞的生长,但各种ALK抑制剂均有效杀伤了RDAA阳性肺癌细胞(P<0.01),但对RDAA阴性肺癌细胞没有显著作用。
实施例4:ALK抑制剂对RDAA阳性肺癌小鼠的治疗效果
将来自1名RDAA阳性的非小细胞型肺癌患者的癌细胞接种至裸鼠腹部皮下组织,接种细胞数量为五十万细胞每只小鼠,接种10天后可观察到小鼠腹部皮下有肿块生长,即构建出RDAA阳性的荷瘤小鼠。
将负荷RDAA阳性肿瘤的荷瘤小鼠随机分成七组(每组5只),分别口服施用安慰剂(PBS缓冲液)、克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、或恩沙替尼(ALK抑制剂的使用剂量为25毫克每日每公斤体重,安慰剂的使用剂量为2ul每日每只小鼠,肿瘤接种2周后开始给药,第四周起停止给药)。
实验结果如图4、图5所示。结果表明:相对于用安慰剂组小鼠,ALK抑制剂显著抑制了RDAA阳性肿瘤荷瘤小鼠的肿瘤生长(P<0.01);同时,该荷瘤小鼠的生存也显著得到改善(P<0.01)。
实施例5:ALK抑制剂对RDAA阳性患者的治疗效果
来自四川大学华西医院的40名患者已按照实施例3中的检测方法确定为ALK基因重排阴性。然后,利用本试剂盒对患者的血浆样本中的Rnase1表达水平和肿瘤组织样本中的RNase1蛋白表达水平及ALK磷酸化水平进行测定,按照以下标准划分为RDAA阳性患者和非RDAA阳性患者:
RDAA阳性患者:血浆中的Rnase1表达水平≥418ng/ml或者针对Rnase1的免疫组织化学染色得分≥4分;并且针对ALK磷酸化的免疫组织化学染色得分≥4分;
RDAA阴性患者:血浆中的Rnase1表达水平<418ng/ml或者针对Rnase1的免疫组织化学染色得分<4分;并且针对ALK磷酸化的免疫组织化学染色得分<4分。
共鉴定出3名RDAA阳性患者,均为非小细胞型肺癌患者。对这3名患者以每次250毫克,每天两次的剂量施用克唑替尼,连续使用4周后前后的CT结果如图6所示。能够明显看出:克唑替尼显著抑制了RDAA阳性患者的肿瘤生长。
实施例6:ALK抑制剂对RDAA阳性PDAC患者的治疗效果
通过第二代高通量基因测序(NGS)的方法,确定了10名ALK基因重排为阴性的PDAC患者。将这些患者的肿瘤组织切片进行本试剂盒中提到的RNase1和ALK磷酸化免疫组化检测。其中1名肿瘤组织切片免疫组化结果为RNase1表达>4分且ALK磷酸化>4分,鉴定为RDAA阳性的PDAC患者。将该患者的肿瘤细胞培养,分别以安慰剂对照(control,PBS缓冲液)、克唑替尼(Crizotinib)、色瑞替尼(Ceritininb)、劳拉替尼(Loratinib)、阿来替尼(Alectinib)、布加替尼(Brigatinib)和恩沙替尼(Ensartinib)六种ALK抑制剂处理细胞(上述药物使用终浓度均为1微克每毫升培养基、细胞密度为5万个细胞每毫升培养基),观察细胞存活、每天计算存活细胞数量、并绘制生长曲线。
实验结果如图7所示,横坐标轴为用药的时间,单位为(天),纵坐标轴为相对细胞数量。结果显示:安慰剂无法抑制RDAA阳性PDAC细胞的生长,但各种ALK抑制剂均有效杀伤了RDAA阳性PDAC细胞(P<0.01)。
Claims (10)
- 一种用于治疗疾病的方法,其至少包括以下步骤:对患有RDAA阳性疾病的患者施用间变性淋巴瘤激酶(ALK)抑制剂,所述RDAA阳性疾病定义为RNase1驱动的ALK活化所导致的疾病,其中来自所述患有RDAA阳性疾病的患者的样本具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果,且其中所述患有RDAA阳性疾病的患者不是非小细胞肺癌患者。
- 权利要求1所述的方法,其中所述样本来自患者的组织、细胞和/或体液;优选地,来自患者的组织切片和/或血液;优选地,来自患者的肿瘤组织切片和/或血液;优选地,来自受试者的肺癌或胰腺癌组织切片和/或血液;优选地,来自患者的胰腺导管腺癌组织切片和/或血液。
- 前述权利要求中任一项所述的方法,所述患者为胰腺导管腺癌患者。
- 前述权利要求中任一项所述的方法,所述ALK抑制剂包括能够抑制ALK磷酸化的抗体或其抗原结合部分。
- 前述权利要求中任一项所述的方法,所述ALK抑制剂包括克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、恩沙替尼中的任一种或其组合。
- ALK抑制剂在制备用于治疗RDAA阳性疾病的药物中的用途,其中来自患有所述RDAA阳性疾病的患者的样本具有阴性的ALK基因重排检测结果,≥418ng/ml的血浆中的Rnase1表达水平或者阳性的针对Rnase1的免疫组织化学染色结果,以及阳性的针对ALK磷酸化的免疫组织化学染色结果,且其中所述患有RDAA阳性疾病的患者不是非小细胞肺癌患者。
- 权利要求6所述的用途,其中所述样本来自患者的组织、细胞和/或体液;优选地,来自患者的组织切片和/或血液;优选地,来自患者的肿瘤组织切片和/或血液;优选地,来自受试者的肺癌或胰腺癌组织切片和/或血液;优选地,来自患者的胰腺导管腺癌组织切片和/或血液。
- 权利要求6或7所述的用途,所述患者为胰腺导管腺癌患者。
- 权利要求6-8中任一项所述的用途,其中所述ALK抑制剂包括克唑替尼、色瑞替尼、劳拉替尼、阿来替尼、布加替尼、恩沙替尼中的任一种或其组合。
- 权利要求6-9中任一项所述的用途,其中所述ALK抑制剂包括能够抑制ALK磷酸化的抗体或其抗原结合部分。
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CN103937877A (zh) * | 2010-02-04 | 2014-07-23 | 自治医科大学 | 对alk抑制剂具有先抗性或后抗性癌症的识别,判断和治疗 |
CN104603620A (zh) * | 2012-09-24 | 2015-05-06 | 文塔纳医疗系统公司 | 使用间变性淋巴瘤激酶(alk)作为标志物鉴定治疗响应性非小细胞肺癌的方法 |
CN109312406A (zh) * | 2016-06-01 | 2019-02-05 | 豪夫迈·罗氏有限公司 | 预测肺癌患者中对alk抑制剂疗法的响应的间变性淋巴瘤激酶中的新型突变 |
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CN112996534A (zh) * | 2018-08-07 | 2021-06-18 | 因斯瑞拜奥有限公司 | 结合间变性淋巴瘤激酶抑制剂进行egf/egfr通路抑制的方法和组合物 |
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