Patent Description:
Programmed death protein-<NUM> (PD-<NUM>) is the main immune checkpoint receptor, and by binding to its ligand, Programmed Death Ligand-<NUM> (PD-L1), it can down-regulate the effector function of T cells, thereby helping to maintain tolerance to tumor cells. There are currently three main inhibitors for PD-<NUM> and PD-L1 on the market, namely pembrolizumab (trade name: Keytruda), Nibolumab (trade name: Opdivo) and Atezolizumab (trade name: Tecentriq), which can be used for the treatment of various cancers such as melanoma, non-small cells lung cancer and bladder cancer. However, not all patients can benefit from the treatment of PD-<NUM>/PD-L1 inhibitors, PD-<NUM>/PD-L1 inhibitors are currently only able to produce lasting tumor control effects in a small number of cancer patients. Therefore, detecting whether the patient has positive expression of PD-L1 can effectively help the patient choose the appropriate medicine for treatment. At present, the detection methods of PD-<NUM>/PD-L1 are mainly based on the detection of cellular protein levels, and in clinical practice, immunohistochemical methods are mainly used, and tumor tissues obtained after surgery or puncture are used for section staining. The results of immunohistochemistry are closely related to the experience of the pathologist. Therefore, a new non-invasive evaluation method and standard, and a relatively stable PD-L1 detection method are urgently needed.

Circulating tumor cells fall off from the primary tumor, enter the blood circulation, settle in remote organs or primary organs, and form metastasis foci. As a hot field of liquid biopsy, circulating tumor cell (CTC) detection has gradually emerged in the clinical manifestations such as tumor diagnosis, treatment and monitoring, and is currently the most promising non-invasive tumor diagnosis and real-time efficacy monitoring method, and has extremely significant clinical application value. Therefore, it will be of great significance if a method for detecting PD-L1 on the surface of tumor cells such as circulating tumor cells can be provided.

For example, Chinese patent <CIT> discloses a method for detecting circulating tumor cell surface marker molecule PD-L1, which comprises the following steps: (<NUM>) treating whole blood with the red cell lysing solution to separate nucleated cells and fixing them with formaldehyde; (<NUM>) first positive screening by tumor immunofluorescence marker cytokeratin antibody anti-CK, incubating all cells with PD-L1 antibody, then incubating all cells with PD-L1 secondary antibody labeled with FITC fluorophore, and then labeling all cells with nuclear fluorescent dye DAPI; (<NUM>) using high-throughput multicolor imaging analysis, selecting CY5, FITC and DAPI filters, observing the fluorescence color of the channel surface, to finally achieve the detection of the circulating tumor cell surface marker molecule PD-L1. This detection method uses whole blood to be processed with red cell lysing solution to separate nucleated cells, but fails to directly separate CTC cells, where the background cells are complex and it is difficult to ensure the accuracy of the detection.

Another example is the Chinese patent <CIT> discloses a method for detecting the PDL1 gene of circulating tumor cells in the peripheral blood of patients with non-small cell lung cancer, which comprises the following steps: (<NUM>) membrane filtering the peripheral blood samples from patients with non-small cell lung cancer, to obtain circulating tumor cells in the peripheral blood; (<NUM>) fixing the filter membrane obtained in step <NUM>; (<NUM>) detecting the filter membrane obtained in step <NUM> to determine the expression of PD-L1. This detection method cannot accurately obtain CTCs with PD-L1 expression only by relying on these three steps, and must be combined with HE staining and expert reading to accurately identify CTCs. On the one hand, such steps are cumbersome and complicated; on the other hand, expert reading not only has a strong subjectivity to affect the judgment result, but also has strong professionalism to make it difficult to be promoted and used.

Other reference can be found in <CIT>, <NPL>, <CIT>, <NPL>, <CIT> and <CIT>.

The present invention is aimed to provide a novel method for detecting a tumor cell surface marker molecule PD-L1, which improves detection accuracy and is easier in terms of detection operation.

Here, the term "room temperature" refers to <NUM> ~ <NUM>.

Due to the use of the above solutions, the present invention has the following advantages over the prior art:
using a capture screen to capture tumor cells in body fluids (such as blood, urine or peritoneal fluid) and then incubating and detecting through PD-L1 and other antibodies, effectively avoids the effects of various other cells, cytokines and proteins in body fluid on the incubation and detection of PD-L1 and other antibodies, which improves detection accuracy and reliability, and has better specificity; and the capture screen is directly used as the carrier for incubating and detecting the captured and separated tumor cells, which is simple and convenient.

For more clearly explaining the technical solutions in the embodiments of the present invention, the accompanying drawings used to describe the embodiments are simply introduced in the following. Apparently, the below described drawings merely show a part of the embodiments of the present invention, and those skilled in the art can obtain other drawings according to the accompanying drawings without creative work.

In the following, the preferable embodiments of the present invention are explained in detail combining with the accompanying drawings so that the advantages and features of the present invention can be easily understood by the skilled persons in the art. It should be noted that the explanation on these implementations is to help understanding of the present invention, and is not intended to limit the present invention.

This embodiment detected the tumor cell surface marker molecule PD-L1, which was specifically carried out as follows.

The capture screen comprises a stainless-steel body and a protective layer covering the surface of the stainless-steel body. The material of the protective layer is gold or gold alloy (such as, AuPd), and the EpCAM antibodies are arranged on the protective layer. The protective layer is an AuPd layer deposited on the stainless-steel body by magnetron sputtering or electrochemical methods. The EpCAM antibodies was attached to the protective layer via Traut's reagent, and thiolate molecules with biotin-avidin can be replace the Traut's reagent.

The specific preparation process of the above-mentioned capture screen was as follows.

Selection of screen: gold-coated stainless-steel screen: <NUM> holes were chosen and magnetron sputtering was used to coat AuPd, the size of the screen was <NUM> × <NUM><NUM>. Pre-functionalization: various cleaning methods were used to prepare the screen before functionalization, including high-pressure steam sterilization, oxygen plasma cleaning, and ultrasonic cleaning in a variety of solutions, including piranha solutions, ammonia-hydrogen peroxide mixing liquid. For example, the screen was ultrasonic treated in a detergent for <NUM> minutes, rinsed, ultrasonic treated in <NUM>% ~ <NUM>% ethanol solution for <NUM> minutes, and rinsed with high purity water for <NUM> minutes.

EpCAM antibodies: <NUM> ~ <NUM>µl of EpCAM antibodies were taken and freezed, and then used to prepare a reaction mixture (i.e., EpCAM antibodies + PBS with EDTA).

Traut's Reagent: it was freezed quickly after purchase. The volume ratio of Traut's reagent and EpCAM antibodies was <NUM> ~ <NUM>: <NUM>. Other methods, such as thiolate molecules with biotin-avidin may replace the Traut's reagent to connect to the screen to form capture screen.

Incubation time of Traut's reagent with antibody: the best reaction time is <NUM> hour.

Incubation of the screen in the above solution: the screen with the above incubated antibody-containing Traut's reagent was incubated at <NUM>, room temperature or <NUM> for <NUM> minutes to within <NUM> hours, so that the EpCAM antibodies were connected to the screen.

(<NUM>) A patient's peripheral blood was collected, and red blood cell lysing solution or lymphocyte separation solution was used to separate nucleated cells;.

(<NUM>) The capture screen bound with the tumor cells was placed in <NUM>µL of <NUM>% paraformaldehyde solution, fixed at room temperature for <NUM>, and washed with phosphate buffer for <NUM> times.

(<NUM>) <NUM>µL of PD-L1 primary antibody solution was added (the volume ratio of antibody stock solution and PBS was <NUM>: <NUM>), and the cells fixed on the capture screen were incubated at <NUM> for <NUM>, and then washed with <NUM>µL of phosphate buffer for <NUM> times.

(<NUM>) <NUM>µL of PD-L1 secondary antibody solution labeled with fluorophore AlexaFluor <NUM> was added (the volume ratio of antibody stock solution and PBS was <NUM>: <NUM>), and the cells were incubated at <NUM> for <NUM>, and then washed with phosphate buffer for <NUM> times.

(<NUM>) <NUM>µL of pan-CK-AlexaFluor <NUM> primary antibody solution was added (the volume ratio of antibody stock solution and PBS was <NUM>: <NUM>), and the cells fixed on the capture screen were incubated at <NUM> for <NUM>, and then washed with <NUM>µL of phosphate buffer for <NUM> times.

(<NUM>) <NUM>µL of CD45 primary antibody solution was added (the volume ratio of antibody stock solution and PBS was <NUM>: <NUM>), and the cells fixed on the capture screen were incubated at <NUM> for <NUM>, and then washed with <NUM>µL of phosphate buffer for <NUM> times.

(<NUM>) <NUM>µL of CD45 secondary antibody solution labeled with fluorophore AlexaFluor <NUM> was added (the volume ratio of antibody stock solution and PBS was <NUM>: <NUM>), and the cells were incubated at <NUM> for <NUM>, and then washed with phosphate buffer for <NUM> times.

(<NUM>) All the cells on the capture screen were labeled with <NUM>µL of nuclear fluorescent dye DAPI.

(<NUM>) Through high-throughput multicolor imaging analysis using filters of CY5, FITC, PE and DAPI, the fluorescence color of each channel was observed to detect the tumor cell surface marker molecule PD-L1.

The results of the detection of the positive and negative expression of the tumor cell surface marker molecule PD-L1 by a pan-CK filter (green), a PD-L1 (primary antibody)-Alexa Fluor <NUM> (secondary antibody) filter (red), a CD45 (primary antibody)-Alexa Fluor <NUM> (secondary antibody) filter (red ), a DAPI filter (blue) and the combination of the four are shown in <FIG>, respectively.

Test sample: collecting <NUM> of healthy human peripheral blood, adding about <NUM> cells of the transfected NCI-H226 reference cell line to it, using lymphocyte separation solution to separate nucleated cells PBMC, and using the nucleated cells PBMC as the test sample.

Experimental group: performing detection according to the detection method of this application (the incubation sequence is: PD-L1+ secondary antibody, pan-CK, CD45+ secondary antibody, DAPI staining), the specific process was as follows.

Control group <NUM>: On basis of the detection method of this application, the incubation sequence was changed (the sequence was CD45+ secondary antibody, PD-L1+ secondary antibody, pan-CK, DAPI staining), the specific process was as follows.

Control group <NUM>: On basis of the detection method of this application, the incubation sequence was changed (the sequence was CD45+ secondary antibody, pan-CK, PD-L1+ secondary antibody, DAPI staining), the specific process was as follows:.

CTC criterion: For the cells in the same position, keep the capture screen sample still, switch the filter of the microscope, observe the fluorescence effect of the cells after staining with different dyes, the sequence to be switches is green fluorescence (CK), blue fluorescence (DAPI), red fluorescence (CD45), and magenta fluorescence (PD-L1). If the cell fluorescence captured on the chip is CK-positive, DAPI-positive, and CD45-negative (which can effectively eliminate false positives), it can be identified as a CTC.

The criterion for PD-L1 expression: after confirming that the cell is a CTC, identify the PD-L1 stained fluorescence at the position with the CTC, and if the CTC has PD-L1 expression (i.e., magenta fluorescence), then the CTC has PD-L1 expression; if there is no magenta fluorescence, the CTC does not have PD-L1 expression.

According to the above criteria, according to the experimental conditions of each group, the detection results are summarized as shown in Table <NUM>.

From the results in the above table, it can be seen that the proportion of CTCs with PD-L1 expression in the control group <NUM> and the control group <NUM> is significantly lower than the result in the experimental group, indicating that some CTCs with PD-L1 expression in control group <NUM> and control group <NUM> were not detected. It shows that using the staining sequence in the control group <NUM> and the control group <NUM>, the fluorescence of PD-L1 of some CTCs is weak, and some do not even have expression of PD-L1, that is, cells with positive PD-L1 expression are not stained. The reason for the analysis to cause such results may be: using the staining sequence in the control group <NUM> and the control group <NUM>, the AlexaFluor <NUM>-labeled PD-L1 secondary antibody binds to the CD45 antibody which reduces the binding efficiency of the PD-L1 primary antibody and the AlexaFluor <NUM>-labeled secondary antibody, which leads to inaccurate detection of CTCs with PD-L1 expression, and reduces the expression efficiency of PD-L1.

In the embodiment of this application, a capture screen connected with EpCAM antibodies is used to specifically capture tumor cells in nucleated cells by controlling the flow rate of the system through the microfluidics; then to the capture screen that captures tumor cells, a PD-L1 antibody solution is used and combined with the use of three fluorescent antibodies, namely DAPI, CK and CD45, to identify the CTCs with PD-L1 expression. That is to say, the embodiment only needs to use the capture screen to specifically capture the cells, and only perform two steps of incubation and combined immunofluorescence analysis to accurately identify the CTCs with PD-L1 expression.

Through the technical solutions of the embodiments of this application, in addition to the excellent effects brought about by the above-mentioned staining sequence, it also has the following advantages:.

The detection method of the present application is mainly used for non-diagnostic purposes of PD-L1 detection, but can also be used for diagnostic purposes.

Claim 1:
A method for detecting a tumor cell surface marker molecule PD-L1, characterized in that, the method comprises the following steps:
providing a capture screen that has antibodies capable of specifically binding to tumor cells, wherein the capture screen comprises a mesh substrate and EpCAM antibodies arranged on the mesh substrate by incubation;
making a sample to be tested flow through the capture screen, such that tumor cells in the sample to be tested bind to the capture screen;
fixing captured tumor cells on the capture screen; and
successively using a PD-L1 primary antibody solution, a PD-L1 secondary antibody solution labeled with a fluorophore AlexaFluor <NUM>, a pan-CK-AlexaFluor <NUM> primary antibody solution, a CD45 primary antibody solution and a CD45 secondary antibody solution labeled with a fluorophore AlexaFluor <NUM>, to incubate the cells fixed on the capture screen, and then labeling all cells on the capture screen with a nuclear fluorescent dye.