New target for predicting curative effect of PD-1 antibody on tumor and colitis

Abstract

The invention discloses a new target for predicting the curative effect of a PD-1 antibody on tumor and colitis, and belongs to the technical field of biomedicine. Based on experiments, the WD6305 is used independently to promote tumor growth, and the tumor growth speed when the WD6305 is combined with the PD-1 antibody is slower than that when the PD-1 antibody is used independently, so that the WD6305 and the PD-1 antibody have potential application value in preparation of the medicine for treating the tumor, and the application prospect is broad. Meanwhile, experiments show that METTL14 is a new target for predicting the curative effect of the PD-1 antibody on tumor and colitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a new target for predicting the efficacy of PD-1 antibody in treating tumors and colitis. Background Art

[0002] PD-1 is an immune checkpoint molecule expressed on the cell surface, mainly expressed on activated T cells, and plays an immunosuppressive function. The concept that blocking the activation of PD-1 can revive the anti-tumor function of immune cells has been widely confirmed and translated into the treatment of many types of cancers in clinical practice. Antibody drugs show obvious advantages, but the overall response rate is still not satisfactory. These challenges reflect the urgent need to comprehensively understand the potential regulatory mechanisms of immune checkpoints. Currently, the regulation of PD-1 mainly focuses on the transcriptional and post-translational levels. However, little is known about whether its encoding gene PDCD1 is regulated at the post-transcriptional level. N6-methyladenosine (m6A)-dependent gene regulation has emerged as a widespread regulatory mode of mRNA, which changes the molecular fate by regulating the splicing, translation, and stability of target genes. It has been reported that the m6A modification of tumor cells regulates tumor immune escape by targeting different genes and mediates various immune regulations. In addition, the m6A modification of tumor cells regulates the immune response to PD-1 antibody treatment. However, the target genes of m6A modification that regulate anti-tumor immunity in T cells still lack in-depth research.

[0003] Tumor immunotherapy can effectively identify and eliminate cancer cells by activating and enhancing the body's own immune system, and has the advantages of less side effects and better long-term effects. However, the current response rate of PD-1 antibody treatment is low. Clinically, there are various drugs that can be combined with PD-1 antibody to treat tumors. Among them, camrelizumab, nivolumab, and toripalimab are commonly used PD-1 antibody drugs, which can be combined with different chemotherapy drugs or other targeted drugs to enhance the treatment effect. The combination of PD-1 antibody and chemotherapy may cause adverse side effects in tumor patients, and there may be significant differences in the treatment response among different tumor individuals. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a new target for predicting the efficacy of PD-1 antibody in treating tumors and colitis.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] A kit for detecting METTL14, comprising reagents for detecting METTL14.

[0007] Further, the reagents include protein immunoblotting reagents.

[0008] A drug for treating tumors, comprising WD6305 combined with a PD-1 antibody.

[0009] The application of WD6305 combined with a PD-1 antibody in the preparation of a drug for treating tumors.

[0010] Furthermore, for the application of WD6305 combined with a PD-1 antibody in the preparation of a drug for treating tumors according to claim 4, the dosage form of the drug is tablets, granules, capsules or injections.

[0011] A method for constructing a mouse model, comprising the following steps:

[0012] Step 1: Establishing a subcutaneous tumor model in mice. The mice are divided into 4 groups:

[0013] PBS+IgG group: Inject IgG once every 3 days, 200 μg per mouse, for a total of 4 injections; inject PBS once every two days, 200 μg per mouse, for a total of 5 injections.

[0014] WD6305+IgG group: Inject IgG once every 3 days, 200 μg per mouse, for a total of 4 injections; inject WD6305 once every two days, 200 μg per mouse, for a total of 5 injections.

[0015] PBS+anti-PD-1 group: Inject anti-PD-1 once every 3 days, 200 μg per mouse, for a total of 4 injections; inject PBS once every two days, 200 μg per mouse, for a total of 5 injections.

[0016] WD6305+anti-PD-1 group: Inject anti-PD-1 once every 3 days, 200 μg per mouse, for a total of 4 injections; inject WD6305 once every two days, 200 μg per mouse, for a total of 5 injections.

[0017] Step 2: Through the establishment of the subcutaneous tumor model in mice, tumors are generated in the mice. Starting from when the tumors are visible to the naked eye, measure the tumor volume with a vernier caliper once every two days, make a growth curve, and compare the tumor growth rates of each group.

[0018] Furthermore, the establishment of the subcutaneous tumor model in mice includes pre-injecting the MC38 cell line subcutaneously into each C57B6J mouse.

[0019] Furthermore, WD6305 alone can promote tumor growth, and the tumor growth rate when WD6305 is combined with a PD-1 antibody is slower than that when the PD-1 antibody is used alone.

[0020] A method for constructing a mouse model for PD-1 antibody treatment of METTL14 low-expression enteritis, comprising the following steps:

[0021] Step 1: PD-1 antibody or IgG was injected into 4-week-old METTL14 conditional knockout mice and their littermate control mice. The littermate control mice were wild-type mice and were divided into 4 groups:

[0022] WT+IgG group: Each wild-type mouse was injected with IgG every three days, 200 μg per mouse, for a total of 10 times;

[0023] CKO+IgG: Each METTL14 conditional knockout mouse was injected with IgG every three days, 200 μg per mouse, for a total of 10 times;

[0024] WT+anti-PD-1: Each wild-type mouse was injected with anti-PD-1 every three days, 200 μg per mouse, for a total of 10 times;

[0025] CKO+anti-PD-1: Each METTL14 conditional knockout mouse was injected with anti-PD-1 every three days, 200 μg per mouse, for a total of 10 times;

[0026] Step 2: After 30 days, the colonic lamina propria lymphocytes of all mice were isolated.

[0027] Step 3: The isolated colonic lamina propria lymphocytes were stained with flow antibodies and finally flow cytometry was performed.

[0028] Furthermore, the isolation operation of the colonic lamina propria lymphocytes included dissecting and removing the mouse colon tissue, cutting the colon into small pieces, pre-digesting in Hanks balanced salt solution supplemented with 8% FCS, 10 mM Hepes and 10 mM EDTA for 1 hour, and then digesting 3 times in HBSS supplemented with 8% FCS, type IV collagenase, DNaseI and Liberase TM DH, separating lymphocytes using a mouse organ tissue lymphocyte separation kit. Flow cytometry included staining the extracted lymphocytes with LIVE / DEAD dye, followed by surface staining and intracellular staining, and then performing flow cytometry analysis. The results showed that PD-1 antibody treatment could rescue the decrease in RORγt+Foxp3+ Treg cells in colonic lamina propria lymphocytes.

[0029] Advantages of the present invention:

[0030] Through the application of WD6305 combined with PD-1 antibody in the preparation of drugs for treating tumors, a new treatment plan for enhancing the efficacy of PD-1 antibody was obtained through experiments. METTL14 is a new target for predicting the efficacy of PD-1 antibody in treating tumors and colitis, and WD6305 can increase the anti-tumor efficacy of PD-1 antibody. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 : Expression map based on TlSIDB software;

[0033] Figure 2 : Expression map of immunoblotting experiment;

[0034] Figure 3 : Tumor growth curve graph;

[0035] Figure 4 : Comparison graph of the proportion of RORγt+ Treg cells in the colonic lamina propria lymphocytes of mice. Detailed implementation manners

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0037] Example 1

[0038] First, based on the TIGER software, a group of glioblastomas treated with anti-PD-1 were analyzed, and the expression of METTL14 in the tumor tissues of responders was significantly lower than that of non-responders.

[0039] The verification object / sample was: clinical samples in the TIGER database.

[0040] The specific operation steps of the TIGER software were to open the website:

[0041] http: / / tiger.canceromics.org / # / immuneResponse, enter the gene name "METTL14" to view the correlation between the response of patients to tumor immunotherapy and the expression of METTL14 in the database. The results showed that in a group of glioblastomas treated with anti-PD-1, the expression of METTL14 in the tumor tissues of responders was significantly lower than that of non-responders.

[0042] Specifically refer to Figure 1 , Figure 1 where the abscissa is non-response and response respectively; the ordinate is the expression level of METTL14.

[0043] Example 2

[0044] After culturing cells in complete medium containing 500 nM WD6305 for 48 hours, the cells were collected, proteins were extracted, and immunoblotting experiments were performed. It was found that WD6305 could degrade METTL3 and METTL14 in human and mouse T cells and increase the expression of PD-1.

[0045] The verification objects / samples were: Jurkat cell line, EL-4 cell line, isolated human primary CD8+ T cells, and isolated mouse primary CD8+ T cells.

[0046] The pre-experiment treatment methods for the samples were as follows: Anti-CD3 / CD28 antibodies and IL-2 were added to the complete RPMI 1640 medium for human and mouse primary CD8+ T cells, and 500 nM WD6305 or an equal volume of DMSO was added simultaneously, and the cells were cultured in a 6-well plate; The Jurkat cell line and EL-4 cell line were cultured in complete RPMI 1640 medium containing 150 ng / ml phytohemagglutinin, and 500 nM WD6305 or an equal volume of DMSO was added simultaneously.

[0047] Experimental procedures and results: After culturing the cells according to the above culture methods for 48 hours, the cells were collected into a 15 ml centrifuge tube, centrifuged at 1000 rpm, the supernatant was discarded, washed 3 times with PBS solution, 100 μl of protein lysate was added, lysed on ice for 30 minutes, centrifuged at 12000 rpm at 4°C for 30 minutes, and the supernatant was taken, which was the whole cell lysate. After measuring the concentration, loading buffer was added, SDS polyacrylamide gel electrophoresis was performed, and after electrophoresis, membrane transfer, blocking, incubation with primary and secondary antibodies, and development were carried out. The results showed that WD6305 could remove METTL3 and METTL14 in 4 types of cells and up-regulate the expression of PD-1.

[0048] Specifically refer to Figure 2 , Figure 2 for the detection of the expression levels of METTL14, METTL3, and PD-1 in cells treated with WD6305 and cells treated with DMSO, and β-Actin was used as an internal reference.

[0049] Example 3

[0050] C57B6J mice were subcutaneously injected with the MC38 cell line and divided into 4 groups:

[0051] PBS + IgG, WD6305 + IgG, PBS + anti-PD-1, and WD6305 + anti-PD-1. The PD-1 antibody or IgG was injected once every 3 days, 200 μg per mouse, for a total of 4 times. WD6306 or PBS was injected once every two days, 200 μg per mouse, for a total of 5 times. Starting from when the tumor was visible to the naked eye, the tumor volume was measured every two days to generate a growth curve. It was found that WD6305 alone promoted tumor growth, but the combination with the PD-1 antibody had a better effect than using the PD-1 antibody alone.

[0052] The verification object / sample was: MC38 subcutaneous tumor tissue (the MC38 cell line is a mouse colon cancer cell line).

[0053] Grouping and treatment methods of the samples before the experiment: The experiment was divided into 4 groups, PBS + IgG, PBS + IgG, WD6305 + IgG, PBS + anti-PD-1, and WD6305 + anti-PD-1 (WD6305 is a potent and selective proteolysis-targeting chimera degrader of the METTL3-METTL14 complex, PBS was used as a control for WD6305; anti-PD-1 is a PD-1 therapeutic antibody, and IgG was used as a control for the PD-1 antibody).

[0054] Experimental procedures and results: 5 mice were in each group, and 5×105 MC38 cells were subcutaneously injected into each mouse. Starting from when the tumor was visible to the naked eye, generally on the 8th day when the tumor was visible to the naked eye, the tumor size was measured with a vernier caliper to plot a growth curve. The results showed that compared with the PBS + IgG group, the tumor in the WD6305 + IgG group grew faster, the tumor in the PBS + anti-PD-1 group grew slower, and the tumor in the WD6305 + anti-PD-1 group grew slower than the other three groups, indicating that WD6305 alone promoted tumor growth, but the tumor growth rate when WD6305 was combined with the PD-1 antibody was slower than that when the PD-1 antibody was used alone, that is, when WD6305 was combined with the PD-1 antibody, the tumor growth rate was the slowest.

[0055] Specific reference is made to Figure 3 , Figure 3 The four corresponding curve graphs corresponding to the 4 groups in the experiment, and the abscissa is the number of days after inoculating the tumor; the ordinate is the tumor volume. Tumor volume = 0.5 × the longest diameter of the tumor × the square of the shortest diameter of the tumor.

[0056] Example 4

[0057] Four-week-old METTL14fl / fl; Cd4Cre (METTL14 conditional knockout mice, CKO) and littermate control mice (wild-type mice, WT) were injected with anti-PD-1 antibody or IgG (200 μg per mouse, injected once every three days for a total of 10 times). The experiment was divided into 4 groups:

[0058] WT+IgG, CKO+IgG, WT+anti-PD-1, CKO+anti-PD-1. After 30 days, the colonic lamina propria lymphocytes of the 4 groups of mice were isolated, and the cells were stained with flow antibodies. Finally, flow cytometry detection found that the RORγt+ Tregs in the colonic lamina propria lymphocytes of CKO mice were significantly reduced, and the anti-PD-1 antibody could rescue this phenomenon.

[0059] The verification object / sample was: mouse colonic lamina propria lymphocytes.

[0060] Grouping and treatment methods for the sample before the experiment:

[0061] WT+IgG, CKO+IgG, WT+anti-PD-1, CKO+anti-PD-1 (WT represents wild-type mice, CKO represents mice with specific knockout of Mettl14 in T cells, anti-PD-1 is a PD-1 therapeutic antibody, and IgG is used as a control for the PD-1 antibody). There were 6 mice in each group. Starting from 4 weeks of age, the PD-1 antibody and IgG were intraperitoneally injected according to the groups (200 μg per mouse, injected once every three days for a total of 10 times).

[0062] Experimental procedures and results: After 10 injections, the mice were euthanized, the colon tissues were dissected, the colon was cut into small pieces, pre-digested in Hanks balanced salt solution (HBSS) supplemented with 8% FCS, 10 mM Hepes (pH 7.5) and 10 mM EDTA for 1 hour, and then digested 3 times in HBSS supplemented with 8% FCS, type IV collagenase (157 U / ml), DNaseI (0.2 mg / ml) and Liberase TM DH (0.125 mg / ml). Lymphocytes were isolated using a mouse organ tissue lymphocyte isolation kit. The purified lymphocytes were stained with LIVE / DEAD dye, and then surface staining and intracellular staining were performed for flow cytometry analysis. The results showed that anti-PD-1 antibody treatment could rescue the reduction of RORγt+Foxp3+ Treg cells in colonic lamina propria lymphocytes, that is, the anti-PD-1 antibody could treat colitis of the low-expression type of METTL14.

[0063] Construction of METTL14 conditional knockout mice:

[0064] Conditional knockout of mice is achieved by Cre-loxP.

[0065] 1. Genetically modify mouse germ cells: Insert loxP sites in the same direction twice in the key exons of Mettl14 to ensure that the exons are precisely deleted after Cre recombination.

[0066] 2. Design primers F1 / R1 at the 5'arm loxP site, with an amplified fragment size of 228bp. Design primers F2 / R2 at the 3'arm loxP site, with an amplified fragment size of 356bp. PCR system and program: 94°C for 3 min; 94°C for 30 s, 60°C for 35 s, 72°C for 35 s, for 35 cycles; 72°C for 5 min. Detect the results by agarose gel electrophoresis. If two positive bands are amplified, it indicates successful targeting.

[0067] 3. For heterozygous (flox / +) and homozygous (flox / flox) mice, genotype is identified with primers F1 / R1: homozygous is 228bp, heterozygous is 228bp and 161bp, wild type is 161bp.

[0068] 4. Cross Cd4-specific Cre tool mice with heterozygous or homozygous mice to obtain Mett14flox / +;Cd4Cre mice. The general primers for Cre tool mice are shown in Table 3-33, and the size of the Cre positive band is 413bp.

[0069] Specifically refer to Figure 4 , Figure 4 In it, the left side is the representative flow cytometry diagram, and the right side is the statistical chart. The abscissa is the group, and the ordinate is the percentage of RORγt and Foxp3 double-positive cells in CD4-positive cells.

[0070] Based on the above results, it is shown that METTL14 is a new target for predicting the efficacy of PD-1 antibody in treating tumors and colitis, and WD6305 increases the anti-tumor efficacy of PD-1 antibody.

[0071] Unless otherwise specified, the reagents or instruments used in the present invention are all commercially available.

[0072] In the description of this specification, the description referring to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0073] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification is only to illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. A kit for detecting METTL14, characterized in that, Comprising a reagent for detecting METTL14.

2. The kit for detecting METTL14 according to claim 1, wherein The reagent comprises a Western blot reagent.

3. A drug for treating tumors, characterized in that, Comprising WD6305 combined with a PD-1 antibody.

4. The application of WD6305 combined with a PD-1 antibody in the preparation of a drug for treating tumors.

5. Use of the WD6305 combined with a PD-1 antibody according to claim 4 in the preparation of a medicament for treating tumors, characterized in that, The dosage form of the drug is tablets, granules, capsules or injections.

6. A method for constructing a mouse model, characterized in that, Comprising the following steps: Step 1: Establishing a mouse subcutaneous tumor model, dividing the mice into 4 groups: PBS+IgG group: Injecting IgG once every 3 days, 200 μg per mouse, for a total of 4 injections; injecting PBS once every two days, 200 μg per mouse, for a total of 5 injections. WD6305+IgG group: Injecting IgG once every 3 days, 200 μg per mouse, for a total of 4 injections; injecting WD6305 once every two days, 200 μg per mouse, for a total of 5 injections. PBS+anti-PD-1 group: Injecting PD-1 once every 3 days, 200 μg per mouse, for a total of 4 injections; injecting PBS once every two days, 200 μg per mouse, for a total of 5 injections. WD6305+anti-PD-1 group: Injecting PD-1 once every 3 days, 200 μg per mouse, for a total of 4 injections; injecting WD6305 once every two days, 200 μg per mouse, for a total of 5 injections. Step 2: Through the establishment of the mouse subcutaneous tumor model, tumors are generated in the mice. Starting from when the tumors are visible to the naked eye, measure the tumor volume with a vernier caliper every two days, make a growth curve, and compare the tumor growth rates of each group.

7. A method for constructing a mouse model according to claim 6, characterized in that, The establishment of the mouse subcutaneous tumor model includes pre-injecting the MC38 cell line subcutaneously into each C57B6J mouse.

8. A method for constructing a mouse model according to claim 6, characterized in that, When used alone, WD6305 can promote tumor growth. The tumor growth rate effect when WD6305 is combined with a PD-1 antibody is slower than that when the PD-1 antibody is used alone.

9. A method for constructing a mouse model for treating METTL14 low-expression enteritis with a PD-1 antibody, characterized in that, Comprising the following steps: Step 1: Performing PD-1 antibody or IgG injection operations on 4-week-old METTL14 conditional knockout mice and littermate control mice. The littermate control mice are wild-type mice and are divided into 4 groups: WT+IgG group: Injecting IgG once every three days for each wild-type mouse, 200 μg per mouse, for a total of 10 injections. CKO+IgG: Injecting IgG once every three days for each METTL14 conditional knockout mouse, 200 μg per mouse, for a total of 10 injections. WT+anti-PD-1: Injecting PD-1 once every three days for each wild-type mouse, 200 μg per mouse, for a total of 10 injections. CKO+anti-PD-1: Injecting PD-1 once every three days for each METTL14 conditional knockout mouse, 200 μg per mouse, for a total of 10 injections. Step 2: Separating the colonic lamina propria lymphocytes of all mice after 30 days. Step 3: Performing flow antibody staining on the separated colonic lamina propria lymphocytes, and finally performing flow cytometry detection.

10. A method for constructing a mouse model for treating METTL14 low-expression enteritis with a PD-1 antibody according to claim 9, characterized in that, The separation operation of the lamina propria lymphocytes of the colon includes dissecting and removing the mouse colon tissue, cutting the colon into small pieces, pre-digesting in Hanks balanced salt solution supplemented with 8% FCS, 10 mM Hepes and 10 mM EDTA for 1 hour, and then digesting 3 times in HBSS supplemented with 8% FCS, type IV collagenase, DNase I and Liberase TM DH, separating lymphocytes using a mouse organ tissue lymphocyte separation kit. Flow cytometry detection includes staining the extracted lymphocytes with LIVE / DEAD dye, followed by surface staining and intracellular staining, and then performing flow cytometry analysis. The results show that PD-1 antibody treatment can rescue the decrease of RORγt+Foxp3+ Treg cells in the lamina propria lymphocytes of the colon.

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