Kit for detecting CTC resistant NK cell killing and application thereof

Abstract

The invention relates to a kit for detecting CTC resistant NK cell killing. The kit comprises an antibody combination I and an antibody combination II, the antibody combination I comprises an anti-EpCAM antibody, an anti-CD155 antibody, an anti-CD45 antibody, an anti-MICBA / B antibody, an anti-PD-L1 antibody and an anti-HLA-G antibody; and the antibody combination II comprises an anti-TGF-beta antibody and an anti-Pan-CK antibody. The CTC can be detected to resist the NK cell killing, and a specific expression antigen which specifically plays a role in resisting the NK cell killing can be detected, so that corresponding medicine taking or corresponding treatment can be facilitated. For example, a TGF-beta inhibitor and a PD-L1 inhibitor are taken in a targeted manner, or CAR-NK targeting HLA-G or MICB is designed.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor detection reagents, and particularly relates to a kit for detecting the resistance of CTCs to NK cell killing and its application. Background Art

[0002] CTC (circulating tumor cells) binds to inhibitory receptors on the surface of NK cells by highly expressing molecules such as PD-L1, HLA-G, and MICBA / B, thereby inhibiting their activation. CTCs bind to platelets to form a protective layer, hide surface antigens, and release inhibitory factors such as TGF-β.

[0003] To detect whether circulating tumor cells can resist NK cell killing, it can be achieved by co-culturing NK cells with tumor cells. However, this method is cumbersome and time-consuming for multiple steps. It can also be detected by detecting related surface antigens and platelet adhesion to determine whether circulating tumor cells can resist NK cell killing. However, the detection does not target the substances that inhibit NK cell activity, that is, symptomatic medication cannot be carried out. Summary of the Invention

[0004] The purpose of the present invention is to provide a kit for detecting the resistance of CTCs to NK cell killing, so as to detect the resistance of CTCs to NK cell killing.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A kit for detecting the resistance of CTCs to NK cell killing, comprising antibody combination one and antibody combination two; antibody combination one includes anti-EpCAM antibody, anti-CD155 antibody, anti-CD45 antibody, anti-MICBA / B antibody, anti-PD-L1 antibody, and anti-HLA-G antibody; antibody combination two includes anti-TGF-β antibody and anti-Pan-CK antibody.

[0007] Further, the fluorescence labeling sequences of the anti-EpCAM antibody, anti-CD155 antibody, anti-CD45 antibody, anti-MICBA / B antibody, anti-PD-L1 antibody, and anti-HLA-G antibody in antibody combination one are: BV421, APC, PerCP, BV570, PE-CF594, BB515, respectively.

[0008] Further, the labeling sequences of the anti-TGF-β antibody and anti-Pan-CK antibody in antibody combination two are: PE-Cy7, AF700, respectively.

[0009] Further, it also includes a 4% paraformaldehyde fixative, a permeabilizing agent containing 0.3% saponin and 0.5% bovine serum albumin.

[0010] Further, the antibodies in the antibody combination are monoclonal antibodies.

[0011] Furthermore, it includes the described antibody combination; when the antibody combination includes a first antibody combination and a second antibody combination, the first antibody combination and the second antibody combination are independently packaged.

[0012] Furthermore, the dosages of the fixative and the permeabilizer are 200 μL and 100 μL respectively.

[0013] Application of the kit for detecting CTC resistance to NK cell killing, which is used to distinguish the expressed antigens of CTC resistance to NK cell killing.

[0014] Furthermore, it includes a detection part and an analysis part; the detection part includes the described kit, which is used to detect the antigen expression level of the individual to be tested by flow cytometry; the analysis part is used to analyze the detection results of the detection part.

[0015] Advantages of the present invention:

[0016] The kit for detecting CTC resistance to NK cell killing of the present invention can detect CTC resistance to NK cell killing and can also detect the specific expressed antigens that specifically play a role in resisting NK cell killing, so as to facilitate taking corresponding drugs or performing corresponding treatments. For example, taking TGF-β inhibitors, PD-L1 inhibitors specifically, or designing CAR-NK targeting HLA-G or MICB, etc. Description of the drawings

[0017] Figure 1 It is the gating analysis result of FSC-A / SSC-A in Example 1;

[0018] Figure 2 It is the gating analysis result of CD45 in Example 1;

[0019] Figure 3 It is the gating analysis result of CD155 in Example 1;

[0020] Figure 4 It is the gating analysis result of EpCAM-A / Pan-CK-A in Example 1;

[0021] Figure 5 It is the gating analysis result of MICBA / B and TGF-β in Example 1;

[0022] Figure 6 It is the gating analysis result of PD-L1 and HLA-Gf in Example 1. Detailed implementation manners

[0023] The present invention will be further described below in combination with the embodiments of the present invention and the drawings.

[0024] Example 1

[0025] The kit for detecting CTC resistance to NK cell killing in this embodiment includes antibody combination one and antibody combination two; antibody combination one includes anti-EpCAM antibody, anti-CD155 antibody, anti-CD45 antibody, anti-MICBA / B antibody, anti-PD-L1 antibody and anti-HLA-G antibody; antibody combination two includes anti-TGF-β antibody and anti-Pan-CK antibody.

[0026] The order of fluorescein labeling for the anti-EpCAM, anti-CD155, anti-CD45, anti-MICBA / B, anti-PD-L1, and anti-HLA-G antibodies in antibody combination 1 is: BV421, APC, PerCP, BV570, PE-CF594, BB515, respectively. The order of labeling for the anti-TGF-β and anti-Pan-CK antibodies in antibody combination 2 is: PE-Cy7, AF700, respectively.

[0027] The solution also includes a 4% paraformaldehyde fixative and a 0.3% saponin and 0.5% bovine serum albumin permeabilization agent. The amounts of the fixative and permeabilization agent used are 200 μL and 100 μL, respectively.

[0028] 1. Preparation of reagents

[0029] The antibody composition of this example is fluorescently labeled with EpCAM-BV421, CD155-APC, CD45-PerCP, MICBA / B-BV570, PD-L1-CF594, and HLA-G-BB515. The above six monoclonal antibody reagents are mixed in a volume ratio of 1:1:2:1:1:2:2 and placed in a first container.

[0030] The second fluorescent label of the antibody composition of this embodiment is TGF-β-PE-Cy7 and Pan-CK-AF700. The above two monoclonal antibody reagents are mixed in a volume ratio of 1:1 and placed in a second container.

[0031] 2. Specimen processing

[0032] Peripheral blood samples:

[0033] Use EDTA and treat at room temperature for 12 hours. Red blood cell lysis: Use commercial lysis buffer BD PharmLyse TM The peripheral blood was lysed for 10 minutes and washed twice with PBS after lysis.

[0034] Antibody staining workflow

[0035] Surface antigen staining: EpCAM, CD45, CD155, MICBA / B, HLA-G, PD-L1

[0036] Antibody Incubation: Add antibodies at a ratio of 10 μg / 10 6 cells, and incubate for 15 - 30 minutes at room temperature in the dark.

[0037] Fluorescence Compensation Control: Since the spectra of PerCP (blue laser, 488 nm) and BV421 (violet laser, 405 nm) are similar, the concentration needs to be adjusted or single-stained control compensation should be used.

[0038] Washing: Wash twice with PBS (300 - 400×g, 5 minutes) to remove unbound antibodies.

[0039] Intracellular / Secreted Antigen Staining: PD-L1, TGF-β, Pan-CK

[0040] Fixation and Permeabilization: Fix with pre-cooled 4% paraformaldehyde for 10 minutes, and then treat with a permeabilization agent containing 0.3% saponin and 0.5% bovine serum albumin for 15 minutes.

[0041] TGF-β Activation: Acid treatment (1N HCl at room temperature for 10 minutes) is required to activate latent TGF-β, and then detect after neutralization.

[0042] Intracellular Antibody Incubation: Add TGF-β-PE-Cy7 and Pan-CK-AF700, incubate for 30 minutes at room temperature in the dark, wash, and resuspend.

[0043] 3. Sample Detection

[0044] Process the specimens according to the above specimen processing method, and detect them on a Beckman Coulter 3-laser 13-color DxFlex flow cytometer. After preferably obtaining 100,000 cells per tube (recommended at least 10,000), analyze the data using Kaluza software. When performing flow cytometry detection, set gates in the following manner:

[0045] Use FSC-A / FSC-H to set gate A to remove doublet cells, use SSC-A / SSC-H to continue setting gate B to remove adhesion, use FSC-A / SSC-A to set a gate to remove debris and dead cells, and set the live single cells as gate R1. Use SSC-A / CD45 to set a gate to circle tumor cells, detect the markers of CTC with CD155 / SSC-A, and further verify CTC with EpCAM-A / Pan-CK-A. Then, use MICBA / B-A / TGF-β-A to set a gate to analyze the expression of MICBA / B and TGF-β. Use PD-L1-A / HLA-G-A to set a gate to analyze the expression of PD-L1 and HLA-G.

[0046] 4. Analysis of Results of Typical Cases

[0047] As Figure 1 shown, use FSC-A / SSC-A to set a gate to remove debris and dead cells. As Figure 2And Figure 3 As shown, the SSC-A / CD45 gating circle outlines tumor cells. As Figure 4 shown, the high expression of EpCAM-A / Pan-CK-A further verifies CTC. As Figure 5 and Figure 6 shown, the MICBA / B-A / TGF-β-A gating analyzes the expression of MICBA / B and TGF-β. The PD-L1-A / HLA-G-A gating analyzes the expression of PD-L1 and HLA-G. In this case, both MICBA / B and TGF-β are highly expressed, and both PD-L1 and HLA-G are also highly expressed. Corresponding TGF-β inhibitors, PD-L1 inhibitors, or CAR-NK targeting HLA-G or MICB can be used for treatment.

Claims

1. A kit for detecting the resistance of CTC to NK cell killing, characterized in that, It includes Antibody Combination 1 and Antibody Combination 2; Antibody Combination 1 includes anti-EpCAM antibody, anti-CD155 antibody, anti-CD45 antibody, anti-MICA / B antibody, anti-PD-L1 antibody and anti-HLA-G antibody; Antibody Combination 2 includes anti-TGF-β antibody and anti-Pan-CK antibody.

2. The kit for detecting CTC resistance to NK cell killing according to claim 1, characterized in that, The fluorescence labeling sequences of anti-EpCAM antibody, anti-CD155 antibody, anti-CD45 antibody, anti-MICA / B antibody, anti-PD-L1 antibody and anti-HLA-G antibody in the Antibody Combination 1 are: BV421, APC, PerCP, BV570, PE-CF594, BB515 respectively.

3. The kit for detecting the resistance of CTCs to NK cell killing according to claim 1, wherein, The labeling sequences of anti-TGF-β antibody and anti-Pan-CK antibody in the Antibody Composition 2 are: PE-Cy7, AF700 respectively.

4. The kit for detecting the resistance of CTC to NK cell killing according to claim 1, wherein, It also includes a fixing agent containing 4% paraformaldehyde, and a permeabilizing agent containing 0.3% saponin and 0.5% bovine serum albumin.

5. The kit for detecting the resistance of CTC to NK cell killing according to claim 1, wherein The antibodies in the antibody combination are monoclonal antibodies.

6. The kit for detecting the resistance of CTC to NK cell killing according to claim 1, wherein It includes the antibody combination according to any one of claims 1-5; when the antibody combination includes a first antibody combination and a second antibody combination, the first antibody combination and the second antibody combination are independently packaged.

7. The kit for detecting the resistance of CTCs to NK cell killing according to claim 1, wherein, The dosages of the fixing agent and the permeabilizing agent are: 200 μL and 100 μL respectively.

8. Use of the kit for detecting CTC resistance to NK cell killing according to claim 1, characterized in that, It is used to distinguish the expressed antigens that resist NK cell killing by CTC.

9. Use of the kit for detecting the resistance of CTCs to NK cell killing according to claim 1, characterized in that, It includes a detection part and an analysis part; the detection part includes the kit according to any one of claims 1-8, and is used to detect the antigen expression level of a to-be-tested individual by flow cytometry; the analysis part is used to analyze the detection result of the detection part.

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