CN116377069A - Application of ADAR1 as biomarker and target for predicting lung adenocarcinoma immunotherapy effect - Google Patents
Application of ADAR1 as biomarker and target for predicting lung adenocarcinoma immunotherapy effect Download PDFInfo
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Abstract
The invention discloses application of ADAR1 as a biomarker and a target for predicting lung adenocarcinoma immunotherapy effect. In particular to application of a biomarker and/or a substance for detecting the biomarker in preparing a product for predicting or assisting in predicting curative effect of lung adenocarcinoma immune single drug therapy. The invention also discloses application of ADAR1 and an inhibitor thereof in lung adenocarcinoma immunotherapy. Experiments show that the ADAR1 inhibitor can inhibit the occurrence and development of tumors, is used for preventing or treating lung adenocarcinoma, and can enhance the anti-tumor effect of the immune checkpoint inhibitor by the synergistic effect of the ADAR1 inhibitor and a lung adenocarcinoma immune therapeutic drug (such as an immune checkpoint inhibitor). The predictive biomarker developed by the invention can maximize the curative effect of the immune checkpoint inhibitor, screen out the crowd which can benefit from ICIS treatment, expand the response crowd of ICIS treatment through multi-target combined treatment, and bring survival benefit to more patients.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of ADAR1 as a biomarker and a target for predicting lung adenocarcinoma immunotherapy effect.
Background
Lung cancer is the malignant tumor with highest incidence and mortality rate in China, and the survival rate of patients needs to be improved. Lung adenocarcinomas (lung adenocarcinoma, LUAD) account for approximately 50% -55% of the most common histological types, according to histopathological classification. In recent years, molecular targeting, immunotherapy and the like are continuously emerging, so that survival improvement of lung adenocarcinoma patients is obvious. Targeting therapies such as tyrosine kinase inhibitors (Tyrosine kinase inhibitor, TKI) achieve good efficacy but are directed to specific targets only and resistance occurs rapidly. The immune therapy mainly aims at the immune microenvironment around the tumor, blocks the combination of tumor cells and the surface of immune cells through an immune checkpoint inhibitor, and inhibits signal transduction, so that the activity of the immune cells is activated, and the aim of killing the tumor is fulfilled by means of autoimmunity. Immune checkpoint inhibitor therapy (Immune checkpoint inhibitors, ICIs) is a new breakthrough in tumor therapy, for example, monoclonal antibodies targeting PD-1 or PD-L1 can release T cell immunosuppressive signals and exert antitumor effects. Clinical trials and real world studies indicate that immunotherapy can benefit patients for long-term survival, but only a fraction of patients exhibit sensitive therapeutic responses, and there is still an urgent need for reliable markers to screen this fraction of the population. And immune checkpoint inhibitors are extremely expensive, it is therefore important to study and develop biomarkers that better predict the efficacy of immunotherapy, accurately identifying patients who can benefit from PD-1/PD-L1 inhibitors.
Tumor immunotherapy requires extensive biomarkers and detection methods to guide the clinic compared to targeted therapies. One is because of the different mechanisms of action of different immunotherapeutic approaches, such as targeted activation or inhibition of T cell receptors (CTLA-4 and PD-1). Secondly, there are a number of mechanisms of immunosuppression in the tumor microenvironment. Thus, biomarkers of immunotherapy are more complex than targeted drugs. There are a number of popular markers currently emerging to predict the efficacy of immunotherapy, such as PD-L1 at protein levels associated with tumor inflammatory microenvironment, representing tumor mutational burden at DNA levels of the newly added antigen level (Tumor mutation burden, TMB), but there are still shortcomings. PD-L1 expression levels are one predictive marker uniquely approved by the U.S. FDA. However, PD-L1 sensitivity and specificity are limited, and the measurement of PD-L1 expression level is affected by various factors, the variety of antibodies and the inconsistency of cut off values lead to some inaccuracy in the quantification of PD-L1, so that the single use of this index cannot fully reflect the immune microenvironment. At the gene level, TMB can reflect the neoantigen and tumor immunogenicity produced by somatic cells to a certain extent, so that the reactivity of patients to immunotherapy is predicted, but the processes of antigen production, presentation and immune response excitation cannot be completely characterized by only using TMB, and people sensitive to treatment and insensitive to treatment cannot be distinguished by using TMB alone. Establishing predictive biomarkers may maximize the efficacy of immune checkpoint inhibitors.
Therefore, searching for biomarkers which are more accurate and have clinical transformation potential can guide lung adenocarcinoma patients to receive ICIs treatment, screen out people who can benefit from the ICIs treatment and drug-resistant people, and expand the response people of the ICIs treatment through multi-target combined treatment, thereby bringing survival benefit to more patients.
Disclosure of Invention
The invention aims to provide a biomarker which can be used for predicting or assisting in predicting the curative effect of lung adenocarcinoma immune single drug treatment and/or application of the biomarker serving as a target in improving the curative effect of lung adenocarcinoma immune single drug treatment. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
To achieve the above object, the present invention provides, first of all, any one of the following uses of a biomarker and/or a substance detecting said biomarker:
a1 The application of the composition in preparing a product for predicting or assisting in predicting the curative effect of lung adenocarcinoma immune single drug treatment;
a2 The use of a pharmaceutical composition for the manufacture of a product for assessing or aiding in the assessment of the efficacy of a lung adenocarcinoma immune single drug therapy;
a3 Use of a composition for the preparation of a product for predicting or aiding in the prediction of the progression-free survival of a lung adenocarcinoma immune single drug therapy;
the biomarker may be ADAR1.
Further, the ADAR1 comprises an ADAR1 protein or an ADAR1 gene.
Further, the products include, but are not limited to, reagents, kits, chips, or dipsticks.
The preparation includes development and/or screening.
The nucleotide sequence of the ADAR1 gene mRNA can be SEQ ID No.1, the amino acid sequence of the ADAR1 protein can be SEQ ID No.2, and the 988 th-3780 th positions of the SEQ ID No.1 code for the ADAR1 protein shown in the SEQ ID No. 2.
Further, the detecting the substance of the biomarker may include detecting the substance of the biomarker by an Immunohistochemical (IHC) technique.
Further, the substance for detecting the biomarker can be a substance for detecting the ADAR1 expression amount in tumor tissues (such as baseline tumor tissues) of a lung adenocarcinoma patient to be detected.
In the above application, the substance for detecting the biomarker may include a reagent for detecting an expression amount of the ADAR1 protein or an ADAR1 protein content.
In the above applications, the agent may comprise an antibody, polypeptide, protein, or nucleic acid molecule that binds to an ADAR1 protein.
Antibodies that bind to an ADAR1 protein as described herein include antibodies against an ADAR1 protein or functional fragments thereof (e.g., antibody variable regions Fv, single chain antibodies ScFv, antigen binding fragments Fab or Fab ', F (ab ') 2, fab ' -SH, and like antibody fragments).
The invention also provides a kit, which may comprise any of the substances described herein for detecting the biomarker, which may have at least one of the following uses:
b1 Predicting or assisting in predicting the efficacy of lung adenocarcinoma immune single drug treatment;
b2 Assessing or aiding in assessing the efficacy of lung adenocarcinoma immune single drug therapy;
b3 Predicting or aiding in predicting the length of progression free survival of lung adenocarcinoma immune single drug therapy.
The kit may be a efficacy prediction kit, efficacy assessment kit, or companion diagnostic kit.
The test sample of the kit may be a tumor tissue sample.
Further, the kit may include an ADAR1 mab or ADAR1 polyclonal antibody, and the kit may further include an immunohistochemical reagent.
The invention also provides any one of the following applications of the biomarker ADAR1 as a target point:
c1 The application of the composition in preparing a product for improving the curative effect of lung adenocarcinoma immune single drug treatment;
c2 Use in the manufacture of a product for the treatment or co-treatment of lung adenocarcinoma;
c3 The use of a pharmaceutical composition for inhibiting the development and/or progression of lung adenocarcinoma tumors;
c4 For use in the manufacture of a product for use in combination with a lung adenocarcinoma immune single agent therapy.
The invention also provides any one of the following uses of the ADAR1 inhibitor:
d1 The application of the composition in preparing a product for improving the curative effect of lung adenocarcinoma immune single drug treatment;
d2 Use in the manufacture of a product for the treatment or co-treatment of lung adenocarcinoma;
d3 The use of a pharmaceutical composition for inhibiting the development and/or progression of lung adenocarcinoma tumors;
d4 For use in the manufacture of a product for use in combination with a lung adenocarcinoma immune single agent therapy.
Further, the product may be an agent or a drug.
The ADAR1 inhibitor may have at least any one of the following effects:
e1 Inhibiting or reducing the expression or activity of an ADAR1 gene;
e2 Inhibiting or reducing the transcription of ADAR1 gene into mRNA;
e3 Inhibiting or reducing translation of an ADAR1 gene into a protein;
e4 Inhibiting or reducing the activity or function of an ADAR1 protein.
In the above applications, the ADAR1 inhibitor may include a substance that reduces the expression amount of an ADAR1 protein or the content of an ADAR1 protein, or a substance that inhibits the expression of an ADAR1 gene, which may include one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule compound, an antibody, a polypeptide, a protein, a gene editing vector, a lentivirus, or an adeno-associated virus.
Further, the inhibition of ADAR1 gene expression may be achieved by gene mutation, gene silencing, gene knockout, gene editing, or gene knockdown techniques well known to those skilled in the art. Specific knockdown or shut down of expression of specific genes, for example, using RNA interference (RNAi) technology; the tool utilizing the gene editing technology may be, but is not limited to, CRISPR/Cas9 technology, zinc Finger Nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) technology, and the like. Techniques for inactivating or silencing ADAR1 gene expression from the post-transcriptional or translational level using gene knock-down techniques are well known to those skilled in the art. Such gene knockdown techniques include, but are not limited to, RNA interference, morpholino interference, antisense nucleic acids, ribozymes, or dominant negative mutations.
Silencing of genes using shRNA or siRNA expressed by viruses (e.g., lentiviruses, adeno-associated viruses) to inhibit gene expression is well known to those skilled in the art.
The nucleic acid molecule may comprise shRNA, microRNA, siRNA and/or antisense oligonucleotides.
Further, the shRNA (short hairpin RNA), microRNA (micro RNA), siRNA (small interfering RNA) and/or antisense oligonucleotide (e.g., antisense RNA) are used to inhibit expression of an ADAR1 gene.
In the above applications, the nucleic acid molecule may comprise an shRNA, which may comprise any of the following:
f1 The shRNA targeting interferes with expression of an ADAR1 gene;
f2 The coding DNA sequence of the shRNA is SEQ ID No.3.
The ADAR1 inhibitor may also be an antibody, which may be an antibody against an ADAR1 protein or a functional fragment thereof.
The ADAR1 inhibitor can also be a lentivirus or an adeno-associated virus, which can be a recombinant lentivirus or a recombinant adeno-associated virus expressing an shRNA (shRNA as shown in SEQ ID No. 3) for knocking down the ADAR1 gene.
Herein, the immune single agent treatment may be an immune checkpoint inhibitor treatment.
Herein, the immune monotherapy may include anti-PD-1 immunotherapy or anti-PD-L1 immunotherapy.
Herein, the drug employed in the immune monotherapy may be a PD-1 inhibitor and/or a PD-L1 inhibitor.
The invention also provides a combination for preventing or treating lung adenocarcinoma, which can include any of the ADAR1 inhibitors described herein and a lung adenocarcinoma immunotherapeutic agent.
Further, the lung adenocarcinoma immunotherapeutic agent may be an immune checkpoint inhibitor comprising a PD-1 inhibitor (e.g., an anti-PD-1 antibody) and/or a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody).
Further, the lung adenocarcinoma immunotherapeutic agent includes, but is not limited to, palbociclib, na Wu Liyou mab, singal Li Shan antibody, terlipressin Li Shan antibody, or Dewaruzumab.
The purpose of the above-mentioned application may be a disease diagnosis purpose, a disease prognosis purpose and/or a disease treatment purpose, and their purpose may also be a non-disease diagnosis purpose, a non-disease prognosis purpose and a non-disease treatment purpose; their direct purpose may be information of intermediate results of obtaining disease diagnosis results, disease prognosis results and/or disease treatment results, and their direct purpose may be non-disease diagnosis purpose, non-disease prognosis purpose and/or non-disease treatment purpose.
The invention detects ADAR1 expression level of tumor tissues from lung adenocarcinoma patients by utilizing an immunohistochemical method, finds that the ADAR1 expression level is obviously related to progression-free survival (PFS) of lung adenocarcinoma patients receiving ICIs, develops a biomarker for predicting or assisting in predicting lung adenocarcinoma immune single drug treatment efficacy 2 based on the ADAR1 expression level, and further researches the application of ADAR1 and an inhibitor thereof in improving lung adenocarcinoma immune single drug treatment efficacy based on ADAR1 as a target point. Experiments of the invention prove that: 1. ADAR1 can be used as a biomarker for predicting or assisting in predicting the curative effect of immune single drug treatment, and the curative effect of immune single drug treatment of a lung adenocarcinoma patient to be detected can be predicted or assisting in predicting by detecting the expression quantity of ADAR1 in tumor tissues of the lung adenocarcinoma patient to be detected, and the judgment standard is as follows: the immune single drug treatment effect of the patient to be detected in the ADAR1 low expression group is better than or the candidate is better than that of the patient to be detected in the ADAR1 high expression group; the curative effect of the immune single drug treatment is expressed in the progression-free survival rate or progression-free survival time; under the same follow-up time, the survival rate of the patients to be tested in the ADAR1 high expression group is less than or the candidates are less than the patients to be tested in the low expression group, or the survival time of the patients to be tested in the low expression group is less than or the candidates are less than the patients to be tested in the low expression group. 2. Reducing the expression of ADAR1 can inhibit the growth of tumors, so as to treat lung adenocarcinoma, namely the ADAR1 inhibitor can inhibit the growth of tumors, thereby inhibiting the occurrence and/or development of tumors, and is used for preventing or treating lung adenocarcinoma. 3. The synergistic immune single drug therapy (such as anti-PD-L1 immune therapy) with reduced ADAR1 expression can remarkably inhibit tumor growth, improve the curative effect of the immune single drug therapy, and enhance the anti-tumor effect of the immune therapy, namely, the combination of the ADAR1 inhibitor and lung adenocarcinoma immune therapy drug (such as immune checkpoint inhibitor) can remarkably improve the curative effect of the immune single drug therapy and enhance the anti-tumor effect of the immune therapy.
In conclusion, ADAR1 can be used as a curative effect marker for predicting or assisting in predicting the curative effect of lung adenocarcinoma immune single drug treatment. ADAR1 inhibitors can inhibit the occurrence and/or development of tumors, and are useful for preventing or treating lung adenocarcinoma, and the synergistic effect of ADAR1 inhibitors and immune checkpoint inhibitors can enhance the anti-tumor effect of immune checkpoint inhibitors.
Drawings
Fig. 1 is that high expression of ADAR1 correlates with poorer efficacy of lung adenocarcinoma immunotherapy. Wherein, A in FIG. 1 is the ADAR1 expression level of PR, SD, PD patients; FIG. 1B is a graph showing ADAR1 expression levels and progression-free survival analysis of lung adenocarcinoma patients receiving immunotherapy; FIG. 1C shows ADAR1 high and low expression sets and CD8 + Percent T cell infiltration; d in FIG. 1 is the PD-L1 level of ADAR1 high and low expression sets. (indicating that the difference reached significant levels (p < 0.05), indicating that the difference reached significant levels (p < 0.01)).
FIG. 2 is a graph showing synergistic effects of combined inhibition of ADAR1 expression on enhancing immunotherapy in an animal model of lung adenocarcinoma. Wherein a in fig. 2 is tumor volume; in FIG. 2, B is the tumor growth condition of mice in the isotype control group and the anti-PD-L1 mab group.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The nucleotide sequence of the ADAR1 gene mRNA in the following examples is SEQ ID No.1, and the amino acid sequence of the ADAR1 protein is SEQ ID No.2, wherein positions 988-3780 of SEQ ID No.1 encode the ADAR1 protein shown in SEQ ID No. 2.
Statistical analysis was performed using SPSS Statistics (version 25.0), graphPad Prism 8 (GraphPad Software, inc.) and R (version 3.6.2) software. Student's t-test was used to compare data between the two groups and chi-square test was used to analyze tumor microenvironment. Survival curves were plotted using the Kaplan-Meier method. * : p <0.05,: p <0.01,: p <0.001,: p <0.0001.
Example 1, application of ADAR1 in predicting efficacy of Lung adenocarcinoma immune Single drug therapy
Case selection in this example: this example is a retrospective, non-interventional clinical study. The study has been approved by the ethical committee of the tumor hospital at the national academy of medicine (approval number: 20/242-2438) following the principles of declaration of helsinki and informed consent has been obtained for all subjects.
This example is mainly incorporated in patients with lung adenocarcinoma treated with anti-PD-1/PD-L1 antibodies at the national academy of medical science oncology hospital from month 4 of 2016 to month 12 of 2019. The group entering standard is as follows: 1) Clinically diagnosing patients with localized advanced or advanced lung adenocarcinoma; 2) The first time an immunotherapy is received, but the number of treatment lines is not limited. The exclusion criteria were: 1) Other types of breast tumors such as thymus cancer and pleural mesothelioma; 2) No baseline treatment specimens. Patients enrolled in the study were treated once every two/three weeks with anti-PD-1 antibody (once per two weeks of treatment with nal Wu Liyou mab at 3 mg/kg/time or 240 mg/time; once every three weeks of treatment with palbociclib, melitt Li Shan antibody and terlipressin Li Shan antibody were all 200 mg/time). Usually, the treatment effect is evaluated by enhancing CT of neck, chest and abdomen every six weeks, and the improvement of the treatment effect by enhancing MRI of the skull if necessary is evaluated.
Typical conditions for incorporation of this example into a patient include initial age, sex, ECOG PS score, smoking status, pathology type, and mutation type. Clinical data included the number of immunotherapy lines and antibiotic treatment history. The antibiotic treatment history is limited to use during the period from 1 week prior to receiving immunotherapy to the progression of the immunotherapy disease.
Patient efficacy assessment during treatment was assessed according to solid tumor efficacy assessment standard version 1.1 (Response Evaluation Criteria in Solid Tumors, RECIST version 1.1). Efficacy evaluation criteria included complete remission (complete response, CR), partial Remission (PR), stable Disease (SD), and disease progression (progressive disease, PD).
Progression-free survival (PFS) is defined as the time from the beginning of a patient receiving immune monotherapy to disease progression or death.
The data are obtained by inquiring the medical history of hospitalization and carrying out telephone follow-up. The last follow-up time for this study was 2022, 9, 27 days. The operation tissue specimens before the immune single drug treatment of the patients are collected in the study, the specimens are immediately obtained according to the operation standard after being isolated, and wax block embedding is carried out. All tissue specimens were examined as lung adenocarcinoma by pathological tissue sections. The specimen obtaining and operating process is approved by the ethical committee of tumor hospitals of the national academy of medical science. The provider of the specimen had informed consent.
The study was incorporated into 20 lung adenocarcinoma immunotherapy patients, and the baseline data are shown in Table 1.
TABLE 1 baseline data for lung adenocarcinoma immunotherapy patients
1. Detection of ADAR1 protein expression levels in tissues of 20 lung adenocarcinoma patients
Expression of ADAR1 protein in the puncture tissues (baseline tumor tissues) of 20 lung adenocarcinoma patients was detected by the Human anti-ADAR1 monoclonal antibody (Abcam company product, cat#ab 88574) using an Immunohistochemical (IHC) technique, wherein the ADAR1 antibody was used at a concentration of 1:200, the expression level was expressed as an ADAR1 staining score, and the expression level was high as a score.
ADAR1 protein expression was scored according to the following principle: staining score (ADAR 1 staining score) =staining intensity x percent positive tumor cells x 100. Wherein, the staining intensity score is: no color development was 0 (negative), pale yellow was 1 (weak positive), yellow was 2 (medium positive), brown yellow was 3 (strong positive); the percentage of positive tumor cells was determined by examining ten randomly selected fields under a high power microscope (x 400), calculating the percentage of stained positive tumor cells (weak positive + medium positive + strong positive) in the field to all tumor cells in the field, and using the average of the percentages of ten field positive tumor cells as the percentage of positive tumor cells.
The results are shown in fig. 1 a and table 2, where ADAR1 expression was significantly higher in patients with disease progression group (PD).
TABLE 2 staining scores and progression-free survival of ADAR1 in tumor tissues of 20 lung adenocarcinoma patients
In the table above, 1 in the progression status in column 3 indicates recurrence during the 4 th column follow-up time, and 0 indicates no recurrence or no follow-up during the 4 th column follow-up time.
2. Survival curve
Based on the optimal cutoff (ADAR 1 staining score of 6), the group-in patients were divided into a high baseline ADAR1 expression level group (higher than 6) and a low baseline ADAR1 expression level group (lower than or equal to 6).
Survival curves were plotted according to status of progression and time to progression free survival, and the results of the analysis are shown in FIG. 1B, suggesting that the survival rate of progression free for the low group of ADAR1 expression levels was significantly increased (p < 0.001) at the same follow-up time.
3. Relationship between ADAR1 expression and tumor immune microenvironment
PD-L1 ratio and CD8 + T cell proportion was assessed based on the results of immunohistochemical staining. Tumor ratio score (TPS)>1% of the samples were defined as PD-L1 + A sample; CD8 + T cell infiltration>10% of the samples were defined as CD8TIL + And (3) a sample. The results indicate that CD8 in the low group of baseline ADAR1 expression levels + T cell infiltration ratio (C in FIG. 1) and PD-L1 expression level (D in FIG. 1) were higher.
The expression level of ADAR1 in the tumor tissue was expressed by a staining score.
From the results, the expression level of ADAR1 in the tumor tissue of the lung adenocarcinoma patient to be detected can be detected to predict or assist in predicting the immune single drug treatment effect of the lung adenocarcinoma patient to be detected, and the judgment standard is as follows:
the immune single drug treatment effect of the patient to be detected in the ADAR1 low expression group is better than or the candidate is better than that of the patient to be detected in the ADAR1 high expression group;
the curative effect of the immune single drug treatment is expressed in the progression-free survival rate or progression-free survival time; at the same follow-up time, the non-progression survival rate of the test patients in the ADAR1 high expression group is less than or the candidate is less than the test patients in the low expression group.
Thus, ADAR1 can be used as a biomarker to assess or aid in assessing the efficacy of immune monotherapy.
Example 2 synergistic enhancement of the anti-tumor Effect of immunotherapy in combination with ADAR1 inhibitor and immune monotherapy
The ADAR1 inhibitor may be a substance that inhibits the expression, silencing, or knocking out of an ADAR1 gene, or may be a substance that inhibits or reduces the content and/or activity of an ADAR1 protein.
1. Cell line and animal selection
The mouse LLC cell line used in this study was derived from the American ATCC cell resource center.
Male C57 mice of 5 to 6 weeks of age used in this study were purchased from Fukang, beijing. Mice used in the experiments were all kept in an SPF-rated environment. All procedures in the study strictly followed the regulations set by the animal ethics committee.
2. Construction of LLC cells knocked down with ADAR1 Gene
According to the mouse ADAR1 gene sequence and RNAi principle, the expression of ADAR1 gene is inhibited by knocking down ADAR1 gene in LLC cells by using short hairpin RNA (shRNA) sequence. The coding DNA sequence of the designed shRNA is as follows: 5'-CCGGCCTCAGTGCTGATTGACTTCTCAAGAGAAAGTCAATCAGCACTGAGGTTTTTTG-3' (SEQ ID No. 3).
The coding DNA (SEQ ID No. 3) of the shRNA designed as described above was constructed into a lentiviral cloning vector to give a recombinant lentiviral vector designated shADAR1, which was supplied by Summit Biotechnology (Shanghai) Inc.
The recombinant lentiviral vector shADAR1 and the packaging plasmid are transfected into HEK293T cells, and packaging is carried out in the HEK293T cells. And (3) packaging to obtain recombinant lentivirus expressing shRNA interfering with the human ADAR1 gene, namely RNAi virus, and transfecting LLC cells with the RNAi virus to obtain LLC cells knocked down with the ADAR1 gene.
3. Mouse lung adenocarcinoma subcutaneous transplantation tumor model
A tumor-bearing mouse model is established by subcutaneously transplanting wild LLC cells of a mouse lung adenocarcinoma cell line and LLC cell-derived tumor blocks knocked down by ADAR1 genes into male C57 mice of 5 to 6 weeks of age, and the total number of the mice is 20 in each group. Wherein, the tumor-bearing mice (10) established by the tumor blocks derived from wild LLC cells are simply called wild group tumor-bearing mice, and the tumor-bearing mice (10) established by the tumor blocks derived from LLC cells knocked down ADAR1 genes are simply called knockdown group tumor-bearing mice.
The size of the tumor is measured every three days by using a vernier caliper, and a specific method for constructing a mouse model can be referred to in the literature of "Chengming Liu, sufei Zheng, runsen Jin, et al, the superior efficacy of anti-PD-1/PD-L1immunotherapy in KRAS-mutant non-small cell lung cancer that correlates with an inflammatory phenotype and increased immunology. Cancer Lett.2020; 470:95-105.").
The volume calculation formula of the tumor is as follows: volume = long diameter x short diameter 2 /2
4. Antitumor treatment of tumor-bearing mice
After the maximum diameter of the tumor of the mice is about 0.5cm, the wild tumor-bearing mice and the knockdown tumor-bearing mice are randomly divided into two groups according to the tumor size of the mice, four groups are divided, and 5 mice are treated in each group according to different treatment modes:
(1) Control group (5 wild group tumor-bearing mice): each mouse was intraperitoneally injected with 100ul of phosphate buffered saline (1 XPBS (0.1M, pH 7.4) manufactured by Jiangsu Kaiki Biotechnology Co., ltd.) on days 1,4,7, 10, respectively, from which administration was started.
(2) ADAR1 knockout group (5 knockout group tumor-bearing mice): each mouse was intraperitoneally injected with 100ul of phosphate buffered saline (1 XPBS (0.1M, pH 7.4)) at days 1,4,7, and 10, respectively, at which administration was started.
(3) anti-PD-L1 mab group (5 wild group tumor-bearing mice): each mouse was intraperitoneally injected with 100ul of anti-PD-L1 mAb (InVitomab anti-mouse PD-L1, cat#BE0101, product of BioXell Co., U.S.A.) on days 1,4,7, and 10, respectively, of initial dosing.
(4) anti-PD-L1 mab combined with ADAR1 knockout group (5 knockout group tumor bearing mice): each mouse was intraperitoneally injected with 100ul of anti-PD-L1 mAb (InVitomab anti-mouse PD-L1, cat#BE0101, product of BioXell Co., U.S.A.) on days 1,4,7, and 10, respectively, of initial dosing.
Each of the 5 groups was administered by intraperitoneal injection, and the specific dose was 10 mg/kg/dose of anti-PD-L1 monoclonal antibody (InVivoMab anti-mouse PD-L1, cat#BE0101, product of BioXell Co., U.S.A.).
The major and minor diameters of the mouse subcutaneous tumor were measured every two days using vernier calipers, and volume = major diameter x minor diameter calculated 2 2 and the growth curves were plotted with tumor volumes and the survival of each group of mice was observed. Tumors were grown to a maximum diameter of 2cm, mice were more than 2g weight loss, or death was identified as endpoint events. Dissecting and sampling after the end of the experiment by carbon dioxide anesthesia, and relieving the pain of animals.
The results were as follows:
in the mouse lung adenocarcinoma subcutaneous engrafting tumor model, the ADAR1 knockdown group can reduce the tumor growth rate compared with the control group, and after the knockdown ADAR1 combined anti-PD-L1 mab (abbreviated as double-antibody treatment group) treatment, the tumor growth rate and the tumor size are significantly reduced compared with the single-drug treatment group (anti-PD-L1 mab group) and the single knockdown group (ADAR 1 knockdown group) (a and B in fig. 2).
The results show that the knockdown ADAR1 can inhibit tumor growth and treat lung adenocarcinoma, and the knockdown ADAR1 or the ADAR1 expression-inhibiting synergistic anti-PD-L1 monoclonal antibody can obviously inhibit tumor growth, improve the curative effect of immune single drug treatment and enhance the anti-tumor effect of immune treatment.
In conclusion, the ADAR1 inhibitor can inhibit tumor growth, further inhibit occurrence and/or development of tumors, is used for preventing or treating lung adenocarcinoma, and can remarkably improve the curative effect of immune single drug treatment and enhance the anti-tumor effect of immune treatment when being combined with lung adenocarcinoma immune treatment drugs.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. Use of a biomarker and/or a substance detecting said biomarker for any of the following:
a1 The application of the composition in preparing a product for predicting or assisting in predicting the curative effect of lung adenocarcinoma immune single drug treatment;
a2 The use of a pharmaceutical composition for the manufacture of a product for assessing or aiding in the assessment of the efficacy of a lung adenocarcinoma immune single drug therapy;
a3 Use of a composition for the preparation of a product for predicting or aiding in the prediction of the progression-free survival of a lung adenocarcinoma immune single drug therapy;
the biomarker is ADAR1.
2. The use according to claim 1, wherein the substance for detecting the biomarker comprises a reagent for detecting the expression level of ADAR1 protein or the content of ADAR1 protein.
3. The use according to claim 2, wherein the agent comprises an antibody, polypeptide, protein or nucleic acid molecule that binds to an ADAR1 protein.
4. A kit comprising a substance for detecting said biomarker according to any of claims 1 to 3, said kit having at least one of the following uses:
b1 Predicting or assisting in predicting the efficacy of lung adenocarcinoma immune single drug treatment;
b2 Assessing or aiding in assessing the efficacy of lung adenocarcinoma immune single drug therapy;
b3 Predicting or aiding in predicting the length of progression free survival of lung adenocarcinoma immune single drug therapy.
5. Use of a biomarker as claimed in claim 1 as a target for any of the following:
c1 The application of the composition in preparing a product for improving the curative effect of lung adenocarcinoma immune single drug treatment;
c2 Use in the manufacture of a product for the treatment or co-treatment of lung adenocarcinoma;
c3 The use of a pharmaceutical composition for inhibiting the development and/or progression of lung adenocarcinoma tumors;
c4 For use in the manufacture of a product for use in combination with a lung adenocarcinoma immune single agent therapy.
Use of any of the following adar1 inhibitors:
d1 The application of the composition in preparing a product for improving the curative effect of lung adenocarcinoma immune single drug treatment;
d2 Use in the manufacture of a product for the treatment or co-treatment of lung adenocarcinoma;
d3 The use of a pharmaceutical composition for inhibiting the development and/or progression of lung adenocarcinoma tumors;
d4 For use in the manufacture of a product for use in combination with a lung adenocarcinoma immune single agent therapy.
7. The use according to claim 6, wherein the ADAR1 inhibitor comprises a substance that reduces the amount of ADAR1 protein expression or the content of ADAR1 protein, or a substance that inhibits the expression of ADAR1 gene, said substance comprising one or more of a nucleic acid molecule, a carbohydrate, a lipid, a small molecule compound, an antibody, a polypeptide, a protein, a gene editing vector, a lentivirus, or an adeno-associated virus.
8. The use of claim 7, wherein the nucleic acid molecule comprises an shRNA comprising any one of:
f1 The shRNA targeting interferes with expression of an ADAR1 gene;
f2 The coding DNA sequence of the shRNA is SEQ ID No.3.
9. The use according to any one of claims 1-3 or 5-8, or the kit according to claim 4, wherein the immune monotherapy comprises an anti-PD-1 immunotherapy or an anti-PD-L1 immunotherapy.
10. A combination for use in the prevention or treatment of lung adenocarcinoma, characterized in that it comprises an ADAR1 inhibitor according to any one of claims 6 to 8 and a lung adenocarcinoma immunotherapeutic agent.
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