Marker for evaluating curative effect and drug resistance of tumor accurate treatment drug

Abstract

The invention discloses a marker for evaluating the curative effect and the drug resistance of a tumor precise treatment drug, which comprises four types of extracellular vesicle marker molecules, microenvironment cell marker molecules, tumor key target molecules and immunoregulation related molecules, and is used for detecting the marker in body fluid samples such as blood, hydrothorax and ascites, urine and the like. The expression condition of the molecules can be reflected from the perspective of liquid biopsy, and then the curative effect of multiple precise drug schemes on tumor treatment is judged, including but not limited to treatment means with molecules such as PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET and CLDN18.2 as targets, so that effective crowds are divided, the treatment effect is predicted, drug resistance is monitored, and the treatment effect of tumor treatment is improved. The biomarker is expected to be used as a universal liquid biopsy marker for various treatment schemes so as to assist in treatment decision and improve benefits of patients.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis technology, and specifically, relates to a marker for evaluating the efficacy and drug resistance of precision tumor treatment drugs. Background Art

[0002] Advanced malignant tumors have lost the chance of surgery, and drug therapy is the key means. As tumors enter the era of precision treatment, a large number of new targets have emerged, with representative molecular targets such as PD1, PD-L1, CLDN18.2, etc. Treatment plans based on multiple targets have become an indispensable means of precision tumor treatment. At present, there is no panel design in the field of liquid biopsy that can judge the expression of multiple targets from the RNA or protein expression level as a marker for the corresponding precision targeted treatment. Summary of the invention

[0003] The purpose of the present invention is to provide a marker for evaluating the efficacy and drug resistance of precision tumor treatment drugs.

[0004] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a marker for evaluating the efficacy and drug resistance of precision tumor treatment drugs, wherein the marker is at least one of PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, and CLDN18.2 molecules from tumor patient samples.

[0005] Preferably, the marker is PD-L1 and / or CLDN18.2 molecule.

[0006] Preferably, the markers are a panel of PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET and CLDN18.2 molecules.

[0007] The sample may be from body fluids of tumor patients, including whole blood, plasma, serum, ascites, pleural effusion, urine, feces, etc.

[0008] In a second aspect, the present invention provides a method for constructing a scoring model for evaluating the efficacy and drug resistance of precision tumor treatment drugs, which is a scoring model established based on the expression levels of the markers mentioned above in tumor patient samples.

[0009] In a third aspect, the present invention provides the use of biological products for detecting the markers in the preparation of therapeutic efficacy, sensitivity and / or prognosis evaluation reagents for precision tumor treatment drugs; Among them, the efficacy includes treatment efficacy, progression-free survival (PFS), and overall survival (OS).

[0010] Furthermore, the biological product is a substance that can detect the molecule at the RNA or protein level, including antibodies, protein expression chips, RNA expression chips, and reagents and / or chips used for RNA and protein detection by means of RNA sequencing, single cell sequencing, mass spectrometry protein sequencing, qPCR, western blot, and ELISA.

[0011] Furthermore, the tumor precision treatment drug is a therapeutic drug targeting at least one of PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, CLDN18.2 molecules, and any combination therapy related thereto, for example, targeted CLDN18.2 therapy, targeted HER2 therapy, etc.

[0012] In the present invention, the drugs include cell therapy drugs and / or immunotherapy drugs.

[0013] In the present invention, the tumor is any tumor suitable for receiving the precision treatment, without limitation to tumor type and specific patient characteristics.

[0014] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The markers and their combination solutions (panel) provided by the present invention can be used to simultaneously judge the efficacy of multiple precision drug solutions for treating tumors from the perspective of liquid biopsy, including the current most important precision treatment target-related drug solutions. Specifically, the detection of the expression levels of all or part of the molecules in the panel, as well as the scoring of their derivative combinations or the construction of mathematical statistical models, can be used as markers to judge the efficacy and drug resistance of multiple tumor precision treatment drug solutions, thereby dividing the effective population, predicting the treatment effect, and monitoring the occurrence of drug resistance. Therefore, the panel is expected to serve as a universal liquid biopsy marker for multiple treatment options, thereby assisting treatment decisions and increasing patient benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result of a comprehensive analysis conducted in combination with the patient's plasma molecular expression detection data and clinical efficacy data in a preferred embodiment of the present invention, and the PD-L1 molecular expression carried by the panel is identified as a marker for tumor immunotherapy.

[0016] Figure 2 This is a comprehensive analysis result carried out in combination with the patient plasma molecular expression detection data and clinical efficacy data in a preferred embodiment of the present invention, and the CLDN18.2 molecular expression carried by the panel is used as a marker for the tumor-targeted CLDN18.2 regimen. DETAILED DESCRIPTION

[0017] The present invention aims to provide a multi-purpose liquid biopsy molecular panel design, which includes four major categories: extracellular vesicle marker molecules, microenvironment cell marker molecules, tumor key target molecules and immune regulation-related molecules. This makes it easier to judge the therapeutic efficacy of tumor precision treatment drug regimens targeting molecules including PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, CLDN18.2, etc. from the perspective of liquid biopsy, thereby providing treatment markers.

[0018] Further, the marker is a scoring model established based on the expression level of any one or several molecules in the panel. The therapeutic efficacy judged by it includes treatment efficiency, progression-free survival PFS, and overall survival OS. The biological product described in the present invention is a substance that can detect the above-mentioned multiple molecules at the RNA (transcription) or protein (translation) level, including antibodies, protein expression chips, RNA expression chips, RNA sequencing, single cell sequencing, mass spectrometry protein sequencing, qPCR, western blot, ELISA and other means of detecting RNA and protein. Further, the substances used for expression detection include antibodies and primer sequences of any type and brand with the panel molecule as the detection target. The liquid biopsy molecular panel can be used as a marker for a variety of tumor precision treatment schemes, including treatments targeting molecules such as PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, CLDN18.2, and any combination schemes related thereto. The tumors involved are any tumors that are suitable for the above-mentioned precision treatment regimens, without restrictions on tumor types and specific patient characteristics.

[0019] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0020] Example 1 Markers for evaluating the efficacy and drug resistance of precision tumor treatment drugs (development of a panel)

[0021] The present invention provides a multi-purpose liquid biopsy molecular panel, which can be used as a marker to simultaneously guide the efficacy of multiple precision treatment plans. Specifically related to: 1. This panel can detect the expression of four categories of extracellular vesicle marker molecules, microenvironment cell marker molecules, tumor key target molecules and immune regulation-related molecules based on patient body fluid specimens; 2. Based on the detection results of this panel, any score or model can be established for the expression level of any one or more molecules in the panel, which can determine the therapeutic efficacy of precision tumor treatment drug regimens targeting molecules including PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, CLDN18.2, etc., thereby providing treatment markers.

[0022] The specific plan is as follows: The present invention provides a multi-purpose liquid biopsy molecular panel as a companion diagnostic indicator for determining the efficacy of tumor immunotherapy (including effective rate, progression-free survival (PFS), and overall survival (OS)) to guide clinical and scientific research decisions. As shown in Tables 1 and 2, the panel includes four categories: extracellular vesicle marker molecules, microenvironment cell marker molecules, tumor key target molecules, and immune regulation-related molecules.

[0023] Table 1 Panel design and example antibody selection and cutoff values

[0024] Table 2 Panel design and example antibody selection and cutoff values

[0025] The present invention provides a multi-purpose liquid biopsy molecular panel as a companion diagnostic indicator for determining the efficacy of tumor immunotherapy (including efficacy, progression-free survival (PFS), and overall survival (OS)) to guide clinical and scientific research decisions, such as Figure 1 As shown, high expression of EV-PD-L1 corresponds to low efficacy of tumor immunotherapy, short progression-free survival (PFS), and short overall survival (OS).

[0026] The present invention provides a multi-purpose liquid biopsy molecular panel as a companion diagnostic indicator for determining the efficacy of tumor-targeted CLDN18.2 treatment regimens (including efficacy, progression-free survival (PFS), and overall survival (OS)) to guide clinical and scientific research purposes, such as Figure 2 As shown, high expression of EV-CLDN18.2 corresponds to low efficacy of tumor-targeted CLDN18.2 treatment, short progression-free survival (PFS), and short overall survival (OS).

[0027] The present invention provides a multi-purpose liquid biopsy molecular panel as a companion diagnostic indicator for determining the efficacy of tumor-targeted HER2 treatment regimens (including efficacy, progression-free survival (PFS), and overall survival (OS)) so as to guide clinical and scientific research decision-making. As shown in the figure, high expression of EV-HER2 corresponds to low efficacy, short progression-free survival (PFS), and short overall survival (OS) of tumor-targeted HER2 treatment.

[0028] Furthermore, the definition of respective molecular expression refers to the protein and RNA expression levels of the above-mentioned various panel molecules detected by all possible means of detecting RNA and protein, such as antibodies, protein expression chips, RNA expression chips, RNA sequencing, single-cell sequencing, mass spectrometry protein sequencing, qPCR, western blot, ELISA, etc.

[0029] Furthermore, the antibodies or primers used for detecting protein or RNA expression of related molecules include antibodies of any type and brand or specific primers of any sequence that use the related molecules as detection targets.

[0030] Furthermore, tumor diseases are defined as any tumor type (including but not limited to gastrointestinal tumors, breast cancer, lung cancer, etc.), and its various tissue, pathological, and genetic molecular subtypes.

[0031] Furthermore, precision therapy is defined as: precision tumor treatment drug regimens targeting molecules including PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, CLDN18.2, etc., as well as various combination drug regimens involving the above-mentioned therapeutic drugs.

[0032] Example 2 Protein expression antibody chip to detect the expression of patient plasma protein panel molecules

[0033] (1) Preparation of protein expression antibody chip

[0034] The present invention uses multi-purpose panel-related antibodies to make chips, and the specific list and antibody item numbers are shown in Table 1. The specific antibodies are dissolved in PBS containing 5% glycerol, with a final concentration of 200-400 μg / ml to obtain different types of antibody solvents. The negative control is PBS solution. Use an automatic spotter to spot different types of the above-mentioned antibody solvents on a coated glass slide in a certain arrangement. The coated glass slide is a NEXTERION epoxy-coated glass slide from SCHOTT, Germany. Each antibody solvent spot is set with 3 replicates to obtain a protein expression antibody chip, and each chip can be spotted with 16 arrays. The antibody chip is stored in an environment of 2-8°C for standby use.

[0035] (2) Working principle and cutoff value of protein expression antibody chip to detect plasma protein expression

[0036] The protein expression antibody chip was used to detect the expression levels of the above-mentioned multiple proteins in plasma. 10 μL of plasma was taken from each patient and diluted to 100 μL using the sample diluent PBS to obtain a reaction solution. The antibody chip was taken out, and 100 μL of blocking solution was added to each well on the antibody chip. The blocking solution was 3% BSA. After incubation at room temperature for 30 min, the blocking solution in each well was removed to obtain a treatment chip. 100 μL of the prepared reaction solution was taken, and the reaction solution was added to each well on the treatment chip. After incubation at room temperature for 60 min, the reaction chip was obtained. The liquid in each well of the reaction chip was removed, and the detection antibody labeled with Biotin was added. After incubation at room temperature for 1 h, the chip to be tested was obtained. The liquid in each well of the chip to be tested was removed, and then 100 μL of Cy3-Streptavidin was added to each well. After incubation at room temperature for 30 min in the dark, the detection chip was obtained. The detection chip is dried, and a laser scanner, such as Axon GenePix, is used to scan the signal using a channel with an excitation frequency of 532nm. GenePix software is used to read the fluorescence value of the antibody chip to obtain a quantitative matrix of each protein expression. We used 71 peripheral blood specimens from untreated patients with gastrointestinal tumors and selected the median of each protein expression value as the cutoff value for determining high and low. Expressions above this cutoff are considered high expression, and those below this cutoff are considered low expression. The specific values ​​of the median are shown in Table 1.

[0037] Example 3 Prediction of immunotherapy efficacy by patient plasma PD-L1 molecule expression

[0038] Patients who received immunotherapy (including anti-PD1 / anti-PD-L1 / anti-CTLA-4 and various combination therapies) were selected from gastric cancer patients treated in the Department of Digestive Oncology, Peking University Cancer Hospital. Dynamic blood samples were collected from baseline to progression of the 62 patients who received immunotherapy, and the expression of PD-L1 protein in plasma was detected to determine the correlation between its expression level and progression-free survival (PFS) and overall survival (OS). It was found that patients with higher PD-L1 expression had poorer treatment-related PFS and OS, suggesting that higher expression of PD-L1 in plasma indicates poorer efficacy of immunotherapy, such as Figure 1 shown.

[0039] Figure 1 It is the result of a comprehensive analysis based on the patient's plasma molecular expression test data and clinical efficacy data. The results show that high expression of plasma PD-L1 corresponds to a short progression-free survival (PFS) and short overall survival (OS) in tumor immunotherapy, and plasma PD-L1 is an unfavorable factor in tumor immunotherapy.

[0040] Example 4 Prediction of therapeutic efficacy of targeted CLDN18.2 therapy by patient plasma CLDN18.2 molecule expression

[0041] Patients who received cell therapy (including CT041 CAR-T cell therapy) or antibody therapy (including monoclonal antibody, bile duct antibody, ADC) targeting CLDN18.2 were selected from patients with tumors (including gastric cancer, pancreatic cancer, biliary tumors, esophageal cancer, colorectal cancer, etc.) treated in the Department of Digestive Oncology, Peking University Cancer Hospital. Dynamic blood specimens from baseline to progression were collected for 31 patients who received CAR-T cell therapy targeting CLDN18.2 and 34 patients who received antibody therapy targeting CLDN18.2. CLDN18.2-targeted treatment methods include: CAR-T cell therapy targeting CLDN18.2, monoclonal antibody targeting CLDN18.2, bile duct targeting CLDN18.2, ADC targeting CLDN18.2, and the combination of the above-mentioned treatment drugs with any other treatment regimens or drugs. The expression of CLDN18.2 protein in plasma was detected to determine the correlation between its expression level and progression-free survival (PFS) and overall survival (OS). It was found that patients with higher CLDN18.2 expression had poorer treatment-related PFS and OS, suggesting that higher expression of CLDN18.2 in plasma indicates poorer efficacy of CLDN18.2-targeted treatment, such as Figure 2 shown.

[0042] Figure 2 It is the result of a comprehensive analysis based on the patient's plasma molecular expression test data and clinical efficacy data. The results show that high expression of plasma CLDN18.2 corresponds to low efficacy, short progression-free survival (PFS), and short overall survival (OS) of tumor-targeted CLDN18.2 therapy (including cell therapy and antibody therapy), and plasma CLDN18.2 is an unfavorable factor for tumor-targeted CLDN18.2 therapy.

[0043] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A marker for evaluating the efficacy and drug resistance of precision tumor treatment drugs, characterized in that: The marker is at least one of PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, and CLDN18.2 molecules from tumor patient samples.

2. The marker according to claim 1, characterized in that The markers are PD-L1 and / or CLDN18.2 molecules.

3. The marker according to claim 1, characterized in that The markers are a combination of PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET and CLDN18.2 molecules.

4. The marker according to any one of claims 1 to 3, characterized in that The samples are from body fluids of tumor patients, including whole blood, plasma, serum, ascites, pleural effusion, urine, and feces.

5. A method for constructing a scoring model for evaluating the efficacy and drug resistance of precision tumor treatment drugs, characterized in that: It is a scoring model established based on the expression level of the marker as described in any one of claims 1 to 4 in tumor patient samples.

6. Use of a biological product for detecting the marker according to any one of claims 1 to 4 in the preparation of a reagent for evaluating the efficacy, sensitivity and / or prognosis of a precise tumor treatment drug; in, The efficacy includes treatment efficacy, progression-free survival, and overall survival.

7. The use according to claim 6, characterized in that: The biological product is a substance that can detect the molecule at the RNA or protein level, including antibodies, protein expression chips, RNA expression chips, and reagents and / or chips used for RNA and protein detection by means of RNA sequencing, single cell sequencing, mass spectrometry protein sequencing, qPCR, western blot, and ELISA.

8. The use according to claim 6, characterized in that: The tumor precision treatment drug is a therapeutic drug targeting at least one of PD1, PD-L1, CTLA-4, TIM3, LAG3, TIGIT, HER2, HER3, EGFR, FGFRs, VEGFR, MET, and CLDN18.2 molecules, as well as any combination therapy regimens related thereto.

9. The use according to claim 6, characterized in that: The drugs include cell therapy drugs and / or immunotherapy drugs.

10. The use according to any one of claims 6 to 9, characterized in that: The tumor is any tumor suitable for receiving the precision treatment.

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