Biomarker for monitoring PD-1 antibody curative effect of PD-L1Positive gastric cancer patient

Abstract

The invention discloses a biomarker for monitoring the curative effect of a PD-1 antibody of a PD-L1Positive gastric cancer patient, and relates to the technical field of biomedical detection, and the biomarker is laminin gamma 2. According to the invention, the laminin gamma2 is used as a novel biomarker for monitoring the curative effect of the PD-1 treatment antibody of the PD-L1Positive gastric cancer patient, experiments prove that the serum level of the laminin gamma2 is higher than that of healthy people in the gastric cancer patient and is in positive correlation with a prognosis related factor sPD-L1, the tissue expression of the PD-L1Positive patient is higher, and the serum level of a person without PD-1 antibody treatment response is higher; it is proved that the marker has good progress monitoring value, treatment reaction differences can be effectively distinguished, and the blank of lack of PD-1 antibody curative effect monitoring markers for patients of the type is filled up.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, and particularly to a PD-L1 Positive A PD-1 antibody efficacy monitoring biomarker for gastric cancer patients. BACKGROUND

[0002] According to the data released by the National Cancer Center in 2024, the incidence and mortality of gastric cancer ranks fifth and third respectively among all types of cancers. The American Society of Clinical Oncology's Annual Report on Cancer Research Progress evaluates immunotherapy as the biggest breakthrough in cancer research in 2015. Cell magazine reports that the interaction between the immune system and cancer cells is continuous and dynamic, from the generation of cancer cells to the development of metastatic diseases, which depends on immune escape, and the continuous expression of programmed death receptor-ligand 1 (PD-L1) by cancer cells may be one of the important factors for tumor immune escape and low efficiency of immunotherapy. Currently, there are reports that anti-PD-L1 antibodies are effective for about 20% of lung cancer patients, with an effective rate of nearly 90% for Hodgkin's lymphoma treatment, and a treatment effective rate of 40%-50% for melanoma patients, and a treatment effective rate of 20% for liver cancer patients. For patients who respond to treatment, anti-PD-L1 treatment can significantly prolong their survival. Among them, about 40% of gastric cancer patients have positive PD-L1 expression, and plasma free PD-L1 (soluble PD-L1, sPD-L1) or exosome-carrying PD-L1 is associated with overall survival, and PD-L1 is considered an independent prognostic predictor. Current clinical trial results show that anti-PD-1 / PD-L1 monoclonal antibody treatment has an objective response rate (ORR) of about 20% in gastric cancer, and targeted treatment of tumor cells PD-1 / PD-L1 still has problems such as low effectiveness and easy drug resistance. SUMMARY

[0003] The present application aims to provide a PD-L1 Positive A PD-1 antibody efficacy monitoring biomarker for gastric cancer patients, which solves the problem of lack of PD-L1 Positive A PD-1 antibody efficacy monitoring biomarker for gastric cancer patients, which solves the problem of lack of PD-L1

[0004] To achieve the above object, the present application provides the following technical scheme: a PD-L1 Positive A PD-1 antibody efficacy monitoring biomarker for gastric cancer patients, wherein the biomarker is laminin gamma 2.

[0005] The above biomarker is used for preparing a PD-L1 PositiveThe application of the product for monitoring the curative effect of PD-1 treatment antibody on gastric cancer patients.

[0006] Preferably, the product comprises detection reagents for quantitatively detecting the biomarker.

[0007] Preferably, the detection reagents comprise reagents for detecting the biomarker at the gene level or the protein level.

[0008] Preferably, the detection reagents are reagents for one or more detection techniques or methods selected from the group consisting of enzyme-linked immunosorbent assay, immunofluorescence method, radioimmunoassay method, immunoprecipitation method, immunoblotting method, high-performance liquid chromatography method, capillary gel electrophoresis method, near-infrared spectroscopy method, mass spectrometry method, immunocherniluminescence method, colloidal gold immunotechnology, fluorescent immunochromatography technology, surface plasmon resonance technology, immun-PCR technology or biotin-avidin technology.

[0009] Preferably, the product comprises at least one of reagents, kits, test papers and chips.

[0010] A product for monitoring the curative effect of PD-L1 Positive The product for monitoring the curative effect of PD-1 treatment antibody on gastric cancer patients comprises reagents for detecting the biomarker laminin gamma 2.

[0011] Preferably, the product further comprises a sample collection device, a sample detection device and a monitoring device.

[0012] The sample collection device is a device for collecting a blood sample of a subject, and the subject is a gastric cancer patient.

[0013] The sample detection device is a device for detecting the protein or nucleic acid content / expression of the biomarker laminin gamma 2 in the blood sample.

[0014] The monitoring device comprises a data acquisition module and a monitoring module, and the data acquisition module is used for acquiring data detected by the sample detection device.

[0015] Compared with the prior art, the product has the following beneficial effects:

[0016] 1. In the application, laminin gamma 2 is used as a biomarker for monitoring the curative effect of PD-L1 Positive The new biomarker for monitoring the curative effect of PD-1 treatment antibody on gastric cancer patients has been verified by experiments, and its serum level is higher than that of healthy people, positively correlated with the prognostic factor sPD-L1, and PD-L1 PositiveThe patient tissue expresses higher, the serum level of the non-responder of PD-1 antibody treatment is higher, the marker has good progress monitoring value, can effectively distinguish the treatment response difference, and fills the blank of the lack of PD-1 antibody curative effect monitoring marker for the patient.

[0017] 2、In the application, laminin gamma 2 is applied to PD-L1 Positive The serum detection mode is provided for the PD-1 antibody curative effect monitoring of the gastric cancer patient, and the serum detection mode is simple, non-invasive and long-term available: tumor tissue is not needed to be obtained, and the detection can be completed only by collecting venous blood; sample processing is simple, operation is convenient, the long-term and multiple monitoring demand of the patient during treatment can be met, compared with the existing tissue detection mode, the patient burden is greatly reduced, and the monitoring feasibility is improved.

[0018] 3、In the application, the curative effect of the PD-1 treatment antibody can be fed back in real time through quantitative detection of laminin gamma 2, precise biomarker basis is provided for the clinician to adjust the treatment scheme, blind treatment is avoided, and ultimately the individualized treatment of the gastric cancer patient is achieved. Positive The clinical application prospect is clear. DETAILED DESCRIPTION

[0019] Figure 1 A graph for detecting the serum laminin gamma 2 level of a healthy person and a gastric cancer patient by ELISA in the embodiment is shown in the figure;

[0020] Figure 2 A graph for analyzing the correlation between the serum sPD-L1 and laminin gamma 2 levels of a gastric cancer patient in the embodiment is shown in the figure;

[0021] Figure 3 A graph for detecting PD-L1 in the gastric cancer tissue of a gastric cancer patient by immunohistochemical staining in the embodiment is shown in the figure; Negative The gastric cancer patient and PD-L1 Positive A graph for showing the laminin gamma 2 expression level in the gastric cancer tissue of a gastric cancer patient is shown in the figure;

[0022] Figure 4 A graph for detecting the serum laminin gamma 2 level of a gastric cancer patient with response and non-response after PD-1 antibody treatment by ELISA in the embodiment is shown in the figure. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0024] Unless otherwise indicated, the reagents, methods, and apparatuses employed in the present application are of a type that is conventional in the art. Unless otherwise indicated, the reagents and materials used in the following examples were obtained from vendors unless otherwise indicated. The methods of the experiments were carried out according to conventional procedures or as recommended by the manufacturer, unless otherwise indicated.

[0025] The present application provides a technical solution: a product for monitoring the efficacy of PD-L1 Positive The present application provides a technical solution: a product for monitoring the efficacy of PD-L1

[0026] The present application provides a technical solution: a product for monitoring the efficacy of PD-L1 Positive The present application provides a technical solution: a product for monitoring the efficacy of PD-L1

[0027] The product comprises a detection reagent for quantitatively detecting the biomarker, and the detection reagent comprises a reagent for detecting the biomarker at the genetic level or the protein level.

[0028] The detection reagent is a reagent used in one or more detection techniques or methods selected from the group consisting of enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunoprecipitation, immunoblotting, high-performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunotechnology, fluorescent immunochromatography, surface plasmon resonance technology, immuno-PCR technology, or biotin-avidin technology.

[0029] The product comprises at least one of a reagent, a kit, a test paper, and a chip.

[0030] The present application provides a technical solution: a product for monitoring the efficacy of PD-L1 Positive The present application provides a technical solution: a product for monitoring the efficacy of PD-L1

[0031] The product further comprises a sample collection device, a sample detection device, and a monitoring device.

[0032] The sample collection device is a device for collecting a blood sample of a subject, and the subject is a gastric cancer patient.

[0033] The sample detection device is a device for detecting the protein or nucleic acid content / expression of the biomarker laminin γ2 in the blood sample.

[0034] The monitoring device comprises a data acquisition module and a monitoring module, and the data acquisition module is used to acquire data detected by the sample detection device.

[0035] Embodiment:

[0036] This example takes the health check-up persons and gastric cancer patients in the First People's Hospital of Kunshan City as the research object, a total of 23 health check-up persons and 36 gastric cancer patients are included.

[0037] ELISA method was used to detect the content of sPD-L1 and laminin γ2 in serum:

[0038] 1. Sample collection: venous blood was drawn into blood collection tubes containing separation gel, centrifuged at 3500 rpm for 15 min, the upper serum was aspirated and centrifuged at 13000 rpm for 10 min, the supernatant was collected and detected on the same day and stored in the 4℃ refrigerator. Or stored in the -80℃ refrigerator for a long time, before use, it needs to be centrifuged at 13000 rpm for 10 min again, and the supernatant is collected.

[0039] 2. ELISA operation steps: PD-L1 and laminin γ2 antibodies were coated in the well plate respectively, patient serum was added, incubated at 37℃ for 1h, and the plate was washed with deionized water for 3-5 times, each time for 3-5 min. PD-L1 and laminin γ2 enzyme-labeled antibodies were added respectively, incubated at 37℃ for 30 min, and the plate was washed with deionized water for 3-5 times, each time for 3-5 min. Developing solution was added respectively, incubated at 37℃ for 15 min, and stop solution was added, and the absorbance was detected at 450 nm using an enzyme-labeled instrument. Each time of detection, standard curve was drawn using standard sample.

[0040] 3. Analysis of test results: the concentration of sPD-L1 and laminin γ2 in patient serum was calculated according to the standard curve.

[0041] Immunohistochemical staining to detect PD-L1 Negative Gastric cancer patients and PD-L1 Positive Expression level of laminin γ2 in gastric cancer tissues of gastric cancer patients:

[0042] Clinical gastric cancer tissue specimens soaked in 4% paraformaldehyde were embedded with paraffin, and after cooling, continuous sections were cut, and the sections were spread in a water bath and adhered to polylysine-coated glass slides.

[0043] (1) high temperature baking in oven for more than 2h;

[0044] (2) the sections were immersed in xylene for 10 min for deparaffinization, repeated 2 times;

[0045] (3) the sections were placed in gradient alcohol for hydration: 100% alcohol for 5 min, repeated 2 times, 95% alcohol for 5 min, repeated 2 times, 80% alcohol for 5 min, 75% alcohol for 5 min;

[0046] (4) After dewaxing and dehydration, the sections are gently rinsed with running water, then placed in a pressure cooker and steamed at high temperature for 15 minutes with freshly prepared antigen retrieval solution. After cooling at room temperature, the sections are washed with PBS on a shaker for 5 minutes. This process is repeated 3 times. The tissue is then dried with a paper towel and the tissue block is circled with a crayon.

[0047] (5) Cover the tissue with 5% BSA for 20 minutes to block the tissue;

[0048] (6) Incubate the primary antibody directly without washing. The anti-lamininγ2 antibody is diluted 1:200 and incubated overnight at 4°C or for 2 hours at 37°C.

[0049] (7) Wash with PBS for 5 min, repeat 3 times, incubate with secondary antibody at 37℃ for 30 min;

[0050] (8) Wash with PBS for 5 min, repeat 3 times, develop color with DAB chromogenic kit, observe under a microscope upright, and immediately place the slide in water to stop the color development after the color development is completed.

[0051] (9) After counterstaining with hematoxylin for 30-60 seconds, rinse gently with running water.

[0052] (10) Soak the slices in 0.3% hydrochloric acid alcohol for 30 seconds to differentiate the nucleus and cytoplasm, and then rinse gently with running water;

[0053] (11) Soak the slices in 75%, 80%, 95%, and 100% alcohol for 2 minutes each to dehydrate them, then soak them in xylene for 2 minutes. Repeat this process twice to make the slices transparent.

[0054] (12) Quickly drop a drop of neutral resin onto the tissue site and seal it with a siliconized anti-detachment coverslip. Observe and photograph under a microscope.

[0055] The test results showed that the serum concentration of lamininγ2 in gastric cancer patients was higher than that in healthy individuals. Figure 1 As shown; and through correlation analysis, it was found that the serum lamininγ2 concentration in gastric cancer patients was positively correlated with sPD-L1, R 2 =0.3586, such as Figure 2 As shown; immunohistochemical staining results showed that PD-L1 Positive Lamininγ2 in gastric cancer tissue compared to PD-L1 in gastric cancer patients Negative The incidence rate was high in gastric cancer patients (P<0.05). Figure 3 As shown; serum lamininγ2 levels were measured in gastric cancer patients who responded to and did not respond to PD-1 antibody therapy. It was found that patients with gastric cancer who did not respond to PD-1 antibody therapy had higher serum lamininγ2 levels. Figure 4 As shown.

[0056] The application discloses a PD-L1 Positive The application discloses a PD-1 therapeutic antibody efficacy monitoring biomarker for gastric cancer patients, and particularly provides a biomarker or a detection reagent thereof in the preparation of a product for PD-L1 Positive The application discloses a PD-1 therapeutic antibody efficacy monitoring biomarker for gastric cancer patients, and particularly provides a biomarker or a detection reagent thereof in the preparation of a product for PD-L1 Positive The application discloses a PD-1 therapeutic antibody efficacy monitoring biomarker for gastric cancer patients, and particularly provides a biomarker or a detection reagent thereof in the preparation of a product for PD-L1 Positive The application discloses a PD-1 therapeutic antibody efficacy monitoring biomarker for gastric cancer patients, and particularly provides a biomarker or a detection reagent thereof in the preparation of a product for PD-L1

[0057] Although the application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified by those skilled in the art, or some technical features can be replaced by equivalent features, and any modification, equivalent replacement, improvement, etc. within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A biomarker for monitoring the efficacy of PD-L1 Positive gastric cancer patient to PD-1 antibody, characterized in that, The biomarker is laminin γ2.

2. The biomarker according to claim 1 for use in the manufacture of a product for monitoring the efficacy of a PD-L1 Positive treatment antibody in a gastric cancer patient.

3. Use according to claim 2, characterized in that, The product comprises detection reagents for quantitatively detecting biomarkers.

4. Use according to claim 3, characterized in that, The detection reagents comprise reagents for detecting biomarkers at the gene level or protein level.

5. Use according to claim 2, characterized in that, The product comprises at least one of reagents, kits, test papers and chips.

6. A product for PD-L1 Positive A product for monitoring the efficacy of PD-1 therapeutic antibodies in gastric cancer patients, characterized in that, The reagents comprise reagents for detecting the biomarker of claim 1.

7. The product for PD-L1 according to claim 6, wherein the PD-L1 is PD-L1 Positive The product for monitoring the curative effect of PD-1 therapeutic antibody on gastric cancer patients, characterized in that, Further comprising sample collection devices, sample detection devices and monitoring devices; The sample collection device is a device for collecting a blood sample of a subject, wherein the subject is a gastric cancer patient; The sample detection device is a device for detecting the content / amount of expression of the biomarker laminin γ2 or its nucleic acid in the blood sample; The monitoring device comprises a data acquisition module and a monitoring module, wherein the data acquisition module is used to acquire data detected by the sample detection device.

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