Method for predicting anti-tumor immune activation after HER2-ADC treatment based on metabolite MetO

Abstract

The invention belongs to the technical field of biology, discloses a method for predicting anti-tumor immune activation after HER2-ADC treatment based on metabolite MetO, and particularly discloses application of a substance for inhibiting methionine sulfoxide generation in preparation of a product for promoting the anti-tumor immune activation ability after antibody drug conjugate treatment. According to the present invention, the metabolite of the antibody drug conjugate (DS8201) after Her2 positive breast cancer treatment is analyzed for the first time to obtain the difference between the metabolite methionine sulfoxide in tumor cells with good DS8201 sensitivity and the metabolite methionine sulfoxide in tumor cells with poor DS8201 sensitivity, such that the content difference of the metabolite methionine sulfoxide in the tumor microenvironment is indicated; it is verified that methionine sulfoxide can significantly inhibit the killing function of CD8 + T cells after DS8201 treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for predicting anti-tumor immune activation after HER2-ADC treatment based on the metabolite MetO. Background Art

[0002] Breast cancer is the most common health problem and the second leading cause of cancer-related death in women. Its incidence has been on the rise since the mid-2000s, increasing at a rate of 5.2% per year. HER2-positive breast cancer is a subtype of breast cancer with a poor prognosis and strong invasiveness. HER2-targeted therapy is an important treatment for this type of breast cancer. However, as treatment progresses, some patients may develop resistance to HER2-targeted therapy, especially to single targeted drugs. The side effects of targeted drugs and chemotherapy remain a challenge in treatment, with common side effects including cardiac toxicity, gastrointestinal discomfort, and blood system suppression. Antibody-drug conjugate (ADC) drugs for the treatment of HER2-positive breast cancer are an important development in recent years, especially for patients who have developed resistance to traditional treatments (such as chemotherapy and targeted antibody drugs). ADC drugs can precisely deliver toxins to cancer cells and reduce damage to normal cells by connecting monoclonal antibodies targeting HER2 to cytotoxic drugs. Recent studies have shown that DS8201 (trastuzumab) not only kills cancer cells through direct cytotoxic effects, but may also enhance anti-tumor immune responses through immune activation mechanisms. It is mainly reflected in the following aspects: ① Antigen release and tumor antigen exposure. DS8201 may prompt the immune system to recognize and attack tumor cells. Through the cytotoxic effect of the drug, it can increase the exposure of these antigens, thereby activating the immune response. ② Infiltration of immune cells: Studies have found that in tumor models treated with DS8201, immune cells such as T cells, dendritic cells, and macrophages may migrate to the tumor site. This infiltration of immune cells can promote the immune response in the tumor microenvironment and enhance the attack on the tumor. ③ Regulation of immune checkpoints: Some studies are exploring the potential of DS8201 in combination with immune checkpoint inhibitors. Immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) enhance the immune system's recognition and killing of tumors by relieving the inhibitory effect of tumors on the immune system. DS8201 may further improve the treatment effect by improving the immune response and combining it with immune checkpoint inhibitors. The expression level of HER2 is usually one of the most important markers for evaluating HER2 targeted therapy (including ADC). Immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) are used to determine the expression intensity and gene amplification status of HER2. However, it still has great limitations. For example, the HER2 copy number in tumor cells may change during treatment, especially after treatment, the expression or amplification of HER2 may decrease, affecting the treatment effect. In addition, the copy number of the HER2 gene varies greatly among different tumors. Amplification of high copy number may be associated with a better treatment response, but some tumors with low copy number amplification may also have a certain response to ADC treatment.It can be seen that since the accuracy of current prediction methods is affected by multiple factors such as tumor heterogeneity, dynamic changes in HER2 expression, complexity of the immune microenvironment, drug transport and clearance mechanisms, there is a lack of methods that can better predict and evaluate the tumor-killing effect of HER2-ADC, and the existing methods do not involve the anti-tumor immune activation function of ADC. Summary of the invention

[0003] Based on the problems existing in the prior art, the present invention aims to develop a method for predicting anti-tumor immune activation after HER2-ADC treatment based on the metabolite MetO.

[0004] The purpose of the first aspect of the present invention is to provide the use of a substance that inhibits the formation of methionine sulfoxide in the preparation of a product that promotes the anti-tumor immune activation ability after antibody-drug conjugate treatment.

[0005] The second aspect of the present invention aims to provide methionine sulfoxide for the preparation of a method for inhibiting CD8 + Application of products with T cell killing function.

[0006] The third aspect of the present invention aims to provide a reagent for detecting the content of methionine sulfoxide for use in preparing a product for predicting the efficacy of antibody-drug conjugates in treating tumors.

[0007] The fourth aspect of the present invention aims to provide a reagent for detecting the content of methionine sulfoxide for use in the preparation of a method for predicting CD8 + Application of T in products with anti-tumor immune efficacy.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides the use of a substance that inhibits the formation of methionine sulfoxide in the preparation of a product that promotes the anti-tumor immune activation ability after antibody-drug conjugate treatment.

[0010] In some embodiments of the present invention, the antibody-drug conjugate comprises DS8201.

[0011] In some embodiments of the present invention, the tumor includes at least one of breast cancer, gastric cancer, lung cancer, and colorectal cancer.

[0012] In some embodiments of the invention, the tumor comprises HER2-positive breast cancer.

[0013] In some embodiments of the present invention, the product inhibits the generation of methionine sulfoxide after antibody drug conjugate treatment, thereby activating anti-tumor immunity.

[0014] In some embodiments of the invention, the product comprises a medicament.

[0015] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0016] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

[0017] In some embodiments of the present invention, the drug further comprises a combination drug, including but not limited to immune effector molecules, cells, cytotoxic substances, and multikinase inhibitors.

[0018] In a second aspect of the present invention, methionine sulfoxide is provided for preparing a method for inhibiting CD8 + Application of products with T cell killing function.

[0019] In some embodiments of the present invention, the effective dosage of methionine sulfoxide in the product is 0.1 mM to 2 mM.

[0020] In some embodiments of the present invention, the effective dosage of methionine sulfoxide in the product is 0.25 mM to 1 mM.

[0021] In some embodiments of the present invention, the product comprises a reagent or a drug.

[0022] The third aspect of the present invention provides the use of a reagent for detecting the content of methionine sulfoxide in the preparation of a product for predicting the efficacy of antibody-drug conjugates in treating tumors.

[0023] In some embodiments of the present invention, the antibody-drug conjugate comprises DS8201.

[0024] In some embodiments of the present invention, the tumor includes at least one of breast cancer, gastric cancer, lung cancer, and colorectal cancer.

[0025] In some embodiments of the invention, the tumor comprises HER2-positive breast cancer.

[0026] In some embodiments of the present invention, the product includes but is not limited to a reagent, a kit, a test paper, a system or a chip.

[0027] In some embodiments of the present invention, the test sample of the product is selected from at least one of the blood, tissues, cells, and excrement of the subject to be tested.

[0028] In a fourth aspect of the present invention, a reagent for detecting the content of methionine sulfoxide is provided for preparing a method for predicting CD8+ Application of T in products with anti-tumor immune efficacy.

[0029] In some embodiments of the present invention, the antibody-drug conjugate comprises DS8201.

[0030] In some embodiments of the present invention, the tumor includes at least one of breast cancer, gastric cancer, lung cancer, and colorectal cancer.

[0031] In some embodiments of the invention, the tumor comprises HER2-positive breast cancer.

[0032] In some embodiments of the present invention, the product includes but is not limited to a reagent, a kit, a test paper, a system or a chip.

[0033] In some embodiments of the present invention, the test sample of the product is selected from at least one of the blood, tissues, cells, and excrement of the subject to be tested.

[0034] The beneficial effects of the present invention are:

[0035] The present invention firstly analyzes the metabolites of antibody-drug conjugate (DS8201) after treatment of Her2-positive breast cancer, and obtains that the metabolite methionine sulfoxide is different in tumor cells with good sensitivity to DS8201 and poor sensitivity to DS8201, indicating the difference in the content of the metabolite methionine sulfoxide in the tumor microenvironment, and verifies that methionine sulfoxide can significantly inhibit CD8 + The killing function of T cells.

[0036] Methionine sulfoxide can not only affect the efficacy of DS8201, but also affect the anti-tumor immune activation efficiency after DS8201 treatment. By detecting the content of methionine sulfoxide, the efficacy of DS8201 treatment and the CD8 + T anti-tumor immune efficacy, providing the possibility for further personalized treatment or drug combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The metabolomics test results of DS8201 with good sensitivity vs. poor sensitivity in Example 1.

[0038] Figure 2 This is a flow chart of the experiment in Example 2 showing the effect of metabolites on anti-tumor immune activation after DS8201 treatment.

[0039] Figure 3 This is the flow cytometry result in Example 2.

[0040] Figure 4 The effects of different concentrations of methionine sulfoxide (0 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1.0 mM) on CD8+ Effect of T cell killing ability, in the figure, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below through specific examples.

[0042] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0043] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0044] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Example 1

[0046] This example uses a metabolomics method to investigate the differences in metabolomics in HER2-positive cells with good and poor sensitivity to DS8201, as follows:

[0047] DS8201 treatment (10 ng / mL) / no drug treatment was used to treat HER2-positive cells with good sensitivity to DS8201 (SKBR3 cells) and poor sensitivity (BT474 cells), namely, there were four treatment groups: ① good sensitivity NC group, ② good sensitivity + DS8201 group, ③ poor sensitivity NC group, and ④ poor sensitivity + DS8201 group. After 72 hours of treatment, the cell supernatants of the above four treatment groups were collected for non-targeted metabolomics detection.

[0048] Specific process of non-targeted metabolomics:

[0049] (1) Collect at least 5×10 6 cells, at least 6 replicate wells (Note: The cell volume is at least 5×10 6 );

[0050] (2) remove the cell culture medium;

[0051] (3) Add 1 mL of 0.9% saline to wash the cells. Wash twice until the cell culture medium becomes colorless and discard the waste liquid.

[0052] (4) Add 800 μL of pre-cooled methanol (pre-cooled in a -80°C refrigerator overnight) and 320 μL of ice water to the cells, and gently shake up and down and left and right for 20 seconds to mix them evenly; scrape the cell metabolites with a cell scraper, and transfer the cell metabolites to a 2 mL centrifuge tube, add 800 μL of pre-cooled chloroform overnight, and vortex for 10 minutes;

[0053] (5) Centrifugation at 14,000 rpm for 10 min at 4°C;

[0054] (6) Take 700 μL of supernatant and place it in a new 1.5 mL centrifuge tube. Freeze-dry at 4°C. The dried sample can be stored in a -80°C refrigerator or sent for sample delivery (polar small molecule layer).

[0055] (7) After separation, metabolites were detected using LC-MS (liquid chromatography tandem mass spectrometry).

[0056] Metabolomics test results Figure 1 As shown, there were 52 metabolites with no difference between the sensitive and poorly sensitive NC groups, but with difference after DS8201 treatment, among which the metabolite methionine sulfoxide was highly expressed in the poorly sensitive group.

[0057] Example 2

[0058] This example is used to investigate whether the metabolites in Example 1 affect the anti-tumor immune activation after DS8201 treatment. Figure 2 The model found that among the top 10 differential metabolites of 52 differential metabolites, methionine sulfoxide (MetO) had the ability to inhibit CD8 + The killing function of T cells.

[0059] The whole experiment was divided into two groups: DS8201 sensitive group (SKBR3) and DS8201 insensitive group (BT474). Each large group was divided into four subgroups, namely ① general culture medium group (DMEM + 10% FBS + 1% penicillin-streptomycin double antibody), ② tumor cell NC group, ③ tumor cell + DS8201 group and ④ general culture medium + MetO group. The experiment was carried out according to the above grouping (flow chart as shown in the figure). Figure 2 ), among which, the experimental process of the tumor cell + DS8201 group was as follows: take the tumor cell suspension in the logarithmic phase (8*10^6 tumor cells), add DS8201 (10ng / mL); the number of tumor cells in the tumor cell NC group was the same as that in the tumor cell + DS8201 group; the experimental process of the general culture medium + MetO group was as follows: add MetO (0.5mM) to the culture medium. After 72h of treatment, 1mL of the supernatant was taken from each treatment group and added to the CD8 +T cells (1*10^6) were treated in 2 mL culture medium (RPMI 1640+10% FBS+25U / mL IL-2) for 48 h. + T cells (5*10^5) were co-cultured with tumor cell antigens (8*10^6 SKBR3 / BT474 cells, digested and resuspended in PBS, frozen and thawed for 5 cycles at 42℃-80℃, 10min each time, centrifuged at 500g for 8min, and the supernatant was aspirated and resuspended in PBS (500ul, which is the tumor antigen)), DC cells (1*10^5) for 48h, and CD8+T cells (5*10^5) in the supernatant were taken, centrifuged and added to untreated tumor cells (5*10^4) for co-culture for 72h, and CD8 was detected by flow cytometry. + T cell cytotoxicity (CFSE / PI method).

[0060] The results of flow cytometry are as follows: MetO can inhibit CD8 + In the group with poor sensitivity to DS8201, the metabolites produced by the tumors treated with DS8201 had an effect on the killing function of CD8 + T cells had a significant inhibitory effect, but this phenomenon was not found in the group with good sensitivity (such as Figure 3 ).

[0061] Based on the above results, the inventors proposed a new possibility for the difference in sensitivity of DS8201 in treating HER2-positive breast cancer: the difference in the content of the metabolite methionine sulfoxide in the tumor microenvironment. Methionine sulfoxide can not only affect the efficacy of DS8201, but also affect the anti-tumor immune activation efficiency after DS8201 treatment. By detecting the content of methionine sulfoxide, the killing effect of DS8201 on HER2-positive breast cancer and the CD8 + T anti-tumor immune efficacy, providing the possibility for further personalized treatment or drug combination.

[0062] Furthermore, the effects of different concentrations of methionine sulfoxide (0 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1.0 mM) on CD8 + The effect of T cell killing ability, the results are as follows Figure 4 As shown, 0.5 mM methionine had an effect on CD8 + The strongest inhibition of T cell killing ability.

[0063] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of substances that inhibit the formation of methionine sulfoxide in the preparation of products that promote the anti-tumor immune activation ability after antibody-drug conjugate treatment.

2. The use according to claim 1, characterized in that: The antibody drug conjugate includes DS8201.

3. The use according to claim 1, characterized in that: The tumor includes at least one of breast cancer, gastric cancer, lung cancer, and colorectal cancer.

4. The use according to claim 3, characterized in that: The tumors include HER2-positive breast cancer.

5. The use according to any one of claims 1 to 4, characterized in that: The product inhibits the production of methionine sulfoxide after antibody-drug conjugate treatment, thereby activating anti-tumor immunity.

6. Preparation of Methionine Sulfoxide to Inhibit CD8 + Application of products with T cell killing function.

7. The use according to claim 6, characterized in that: The effective dosage of methionine sulfoxide in the product is 0.1 mM to 2 mM.

8. Application of reagents for detecting methionine sulfoxide content in the preparation of products for predicting the efficacy of antibody-drug conjugates in treating tumors.

9. The reagent for detecting methionine sulfoxide content is used in the preparation of CD8 + Application of T in products with anti-tumor immune efficacy.

10. The use according to claim 8 or 9, characterized in that: The antibody drug conjugate includes DS8201.

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