Application of CTLA-4, TIM-3 and LAG-3 as immune checkpoints in screening medicines for treating oral squamous cell carcinoma

Abstract

The invention relates to the technical field of tumor molecular biology. The invention provides application of CTLA-4, TIM-3 and LAG-3 as immune checkpoints in screening of drugs for treating oral squamous cell carcinoma. According to the invention, expression differences of CTLA-4, TIM-3 and LAG-3 in oral squamous cell carcinoma tissues and normal tissues and correlation between expression of CTLA-4, TIM-3 and LAG-3 and CD8 + T cell infiltration are compared and analyzed, CTLA-4, TIM-3 and LAG-3 are screened as suitable immune checkpoints, and by inhibiting expression levels of CTLA-4, TIM-3 and LAG-3, activation and proliferation of T cells can be improved, positive regulation immune response can be increased, and the immunologic function of the oral squamous cell carcinoma is enhanced. Therefore, the oral squamous cell carcinoma treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor molecular biology, and particularly to the application of CTLA-4, TIM-3, and LAG-3 as screening targets in screening drugs for the treatment of oral squamous cell carcinoma. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is a common head and neck malignant tumor, which has a high prevalence rate and has attracted wide attention. Its occurrence and development are closely related to immune escape, and immune checkpoint molecules play an important role in the occurrence and development of OSCC. As inhibitory signal mediators, immune checkpoint molecules are usually used to maintain the tolerance of the immune system and prevent the immune system from attacking self-antigens through negative regulation of effector immune cells, and cancer cells take advantage of this mechanism to achieve immune escape. At present, inhibitors targeting these immune checkpoints have shown significant efficacy in the treatment of various cancers, but their application in OSCC has not been fully studied. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of CTLA-4, TIM-3, and LAG-3 as immune checkpoints in screening drugs for the treatment of oral squamous cell carcinoma. The inhibitors of immune checkpoints CTLA-4, TIM-3, and LAG-3 can enhance the activation and proliferation of T cells, increase the positive regulation of immune responses, and thus improve the treatment effect of oral squamous cell carcinoma.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides the application of CTLA-4, TIM-3, and LAG-3 as immune checkpoints in screening drugs for the treatment of oral squamous cell carcinoma, and screens drugs related to the expression levels of CTLA-4, TIM-3, and LAG-3 as drugs for the treatment of oral squamous cell carcinoma.

[0006] Preferably, inhibiting the expression levels of CTLA-4, TIM-3, and LAG-3 can enhance the activation and proliferation of T cells and improve the immune response.

[0007] The present invention also provides an immune checkpoint inhibitor, which inhibits the expression levels of CTLA-4, TIM-3, and LAG-3.

[0008] The present invention also provides the application of the immune checkpoint inhibitor in the preparation of drugs for the treatment of oral squamous cell carcinoma.

[0009] The present invention also provides the application of reagents for detecting CTLA-4, TIM-3, and LAG-3 in the preparation of oral squamous cell carcinoma diagnostic kits.

[0010] The present invention also provides the use of reagents for detecting CTLA-4, TIM-3 and LAG-3 in the preparation of an oral squamous cell carcinoma prognosis assessment kit.

[0011] By adopting the above technical solution, the present invention has the following beneficial effects:

[0012] The technical solution of the present invention provides the use of CTLA-4, TIM-3 and LAG-3 as immune checkpoints in screening drugs for treating oral squamous cell carcinoma. The expression differences of CTLA-4, TIM-3 and LAG-3 in oral squamous cell carcinoma tissues and normal tissues were comparatively analyzed, and the correlations between the expressions of CTLA-4, TIM-3 and LAG-3 and CD8 + T cell infiltration were screened out, and CTLA-4, TIM-3 and LAG-3 were selected as suitable immune checkpoints. By inhibiting the expression levels of CTLA-4, TIM-3 and LAG-3, the activation and proliferation of T cells can be improved, the positive regulation of immune response can be increased, and thus the treatment effect of oral squamous cell carcinoma can be improved. Description of the Drawings

[0013] Figure 1 For the immunohistochemical results of CTLA-4, TIM-3, LAG-3 ( Figure 1 in which A is the immunohistochemical result diagram of CTLA-4, with a strong positive expression degree; B is the immunohistochemical result diagram of TIM-3, with a positive expression intensity; C is the immunohistochemical result diagram of LAG-3, with a positive expression intensity);

[0014] Figure 2 For the statistical chart of fluorescence cell counting results of CTLA-4, TIM-3, LAG-3 ( Figure 2 in which A is the fluorescence result of CTLA-4, B is the fluorescence result of TIM-3, and C is the fluorescence result of LAG-3);

[0015] Figure 3 For the multi-color fluorescence labeling result diagram of CTLA-4, TIM-3, LAG-3 and CD8 ( Figure 3 in which A is the multi-color fluorescence labeling of CTLA-4 and CD8, B is the multi-color fluorescence labeling of TIM-3 and CD8, and C is the multi-color fluorescence labeling of LAG-3 and CD8; among them, blue is the cell nucleus, red fluorescence is CD8, and green is each immune checkpoint molecule respectively, and co-expression shows yellow). Detailed Embodiments

[0016] The present invention provides the use of CTLA-4, TIM-3 and LAG-3 as immune checkpoints in screening drugs for treating oral squamous cell carcinoma.

[0017] In the present invention, drugs related to the expression levels of CTLA-4, TIM-3, and LAG-3 are screened as drugs for treating oral squamous cell carcinoma.

[0018] In the present invention, the expression levels of CTLA-4, TIM-3, and LAG-3 are inhibited, T cell activation and proliferation are enhanced, and the immune response is enhanced.

[0019] The present invention also provides an immune checkpoint inhibitor that inhibits the expression levels of CTLA-4, TIM-3, and LAG-3.

[0020] The present invention also provides the application of the immune checkpoint inhibitor in the preparation of drugs for treating oral squamous cell carcinoma.

[0021] The present invention also provides the application of reagents for detecting CTLA-4, TIM-3, and LAG-3 in the preparation of diagnostic kits for oral squamous cell carcinoma.

[0022] The present invention also provides the application of reagents for detecting CTLA-4, TIM-3, and LAG-3 in the preparation of prognostic evaluation kits for oral squamous cell carcinoma.

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] The Donkey anti-Rabbit IgG Alexa Fluor TM 488 and Donkey anti-Mouse Alexa Fluor TM 546 used in the present invention are purchased from Invitrogen; TIM-3 Rabbit mAb is purchased from cellsignalling; the ready-to-use immunohistochemistry Ultra Sensitive TM SP (mouse / rabbit) kit used in the present invention is purchased from Maixin Biotechnology Development Co., Ltd.

[0025] Example 1

[0026] With the informed consent of the patients and approval by the hospital ethics committee, 40 cases of oral squamous cell carcinoma specimens were collected from the Affiliated Stomatological Hospital of China Medical University. Clinical and demographic data were obtained from the patient files, and all cases were independently evaluated by oral physicians. Among them, there were 7 female patients and 33 male patients. According to the clinical TNM stage, there were 19 cases of well-differentiated (T4), 14 cases of moderately differentiated (T2 and T3), and 7 cases of poorly differentiated (T1). The incidence sites of oral squamous cell carcinoma were 20 cases in the oral cavity, 14 cases in the oropharynx, and 6 cases in the gingiva.

[0027] (I) Immunohistochemistry

[0028] By using the ready-to-use immunohistochemistry Ultra Sensitive TM SP (mouse / rabbit) kit to perform immunohistochemical analysis on the above oral squamous cell carcinoma specimens, and observe the staining results under an optical microscope.

[0029] The oral squamous cell carcinoma specimens were routinely paraffin-embedded and sectioned. The slides to be stained were baked in an incubator at 56 °C for 2 h. Routine dewaxing was performed; then, antigen retrieval was carried out using pH = 9 EDTA (98 °C for 15 min); the endogenous peroxidase blocker was incubated at room temperature for 20 min, and the non-specific staining blocker was incubated at room temperature for 20 min. CTLA-4, TIM-3, and LAG-3 antibodies (all purchased from Abcam) were added respectively and incubated overnight at 4 °C; the next day, they were washed with PBS for 5 min, washed three times, biotinylated goat anti-mouse / rabbit IgG polymer was added, and after incubation at room temperature for 20 min, they were washed with PBS for 5 min, washed three times, streptavidin-peroxidase was added, and they were incubated at room temperature for 20 min again, washed with PBS for 5 min, washed three times; DAB chromogenic solution was prepared and added for chromogenic reaction; finally, hematoxylin was used for nuclear staining, dehydration, and mounting.

[0030] Observe the staining results, score the proportion of positive cells and the staining depth of each section. The integral score = percentage score of positive stained cells × staining intensity score. Measurement data are expressed as ; the t-test was used for comparison of means between two groups, and analysis of variance was used for comparison of means among multiple groups; Pearson correlation analysis was used for correlation analysis among variables. A P < 0.05 was considered statistically significant.

[0031] Determination criteria for the percentage of positive stained cells: For each specimen, 3 high-power fields (×200) were randomly selected, 100 keratinocytes were counted in each high-power field, and the average of the positive cell ratios in the 3 fields was calculated. When <10%, it was scored 0; when 10% - 25%, it was scored 1; when 26% - 50%, it was scored 2; when 51% - 75%, it was scored 3; when >75%, it was scored 4.

[0032] Determination criteria for staining intensity: When there was no staining or it showed light yellow uniform with the background, it was scored 0; when it showed light brownish yellow, it was scored 1; when it showed brownish yellow, it was scored 2; when it showed brownish black, it was scored 3.

[0033] Positive grade: When the score was 0, it was negative (-); when the score was 1 - 4, it was weakly positive (+); when the score was 5 - 8, it was positive (++); when the score was 9 - 12, it was strongly positive (+++).

[0034] 1. Histochemical results of CTLA-4, TIM-3, and LAG-3

[0035] Table 1 Immunohistochemical staining scores of CTLA-4, TIM-3, and LAG-3

[0036]

[0037] (Note: Compared with T1, *P < 0.05, **P < 0.01)

[0038] The expression levels of CTLA-4, TIM-3, and LAG-3 were weakly positive in both T1 and T2 stages, while they were positive and strongly positive in T3 and T4 stages. Moreover, most of these three immune checkpoints were expressed in the cytoplasm of epithelial tissues (as shown in Figure 1 ). In OSCC tissue samples, the expression of CTLA-4 in T3 and T4 stages was significantly higher than that in T1 stage. The expression of LAG-3 in T3 stage was significantly higher than that in T1 stage (P < 0.05), as shown in Table 1.

[0039] 2. Correlation of the expression levels of CTLA-4 and TIM-3 in OSCC

[0040] By using Pearson correlation analysis to analyze the correlation between T1 and T3, T4 of CTLA-4, the results showed that the expression of CTLA-4 was significantly positively correlated with the T stage. Similarly, the expression of LAG-3 was also positively correlated with the T stage (CTLA-4: r = 0.857, P < 0.05; TIM-3: r = 0.941, P < 0.01).

[0041] (II) Multicolor immunofluorescence

[0042] Select the corresponding paraffin sections for heating, dewaxing, antigen retrieval, and incubation with an endogenous peroxidase blocker at room temperature for 20 min; add CTLA-4 / CD8, TIM-3 / CD8, LAG-3 / CD8 antibodies (all diluted 1:100), and incubate overnight at 4°C. Subsequently, add fluorescently labeled secondary antibodies (Donkey anti-Rabbit IgG Alexa Fluor TM 488, Donkey anti-Mouse Alexa Fluor TM546, diluted evenly at 1:400), incubated in an incubator at 37°C for 1 h; then sealed with a mounting medium containing DAPI. After drying overnight, observation was performed using a confocal microscope (for the specific experimental method, refer to "Prosaposin expression in the regenerated muscles of mdx and cardiotoxin-treated mice" (Histol Histopathol, 2013 Jul; 28(7): 875-92. Prosaposin expression in the regenerated muscles of mdx and cardiotoxin-treated mice. PMID: 23325523)). The fluorescence results were analyzed for cell counting using ImageJ, and the number of co-expressing cells of CTLA-4, TIM-3, LAG-3 and CD8 and the number of CD8 + cells were counted.

[0043] 1. Results of the expression correlation of CTLA-4, TIM-3, LAG-3 and CD8 + with T cells

[0044] Table 2 Results of fluorescence cell counting of co-expression of CTLA-4, TIM-3, LAG-3 and CD8 + with T cells (x±s)

[0045]

[0046] (Note: Compared with T1, *P < 0.05)

[0047] Cell counting was performed on the multi-color fluorescence results of CTLA-4, LAG-3, TIM-3 and CD8 using ImageJ. The results showed that the expression of the three immune checkpoints was higher on CD8 + T cells in stages T3 and T4 (as Figure 2 shown), and all three immune checkpoints were expressed on the surface of T cells (as Figure 3 shown). In the OSCC tissue samples, the co-expression of the three immune checkpoints in stages T3 and T4 was mostly significantly different from that in stage T1 (P < 0.05), as shown in Table 2.

[0048] 2. Correlation analysis of CTLA-4, LAG-3, TIM-3 and CD8

[0049] Pearson correlation analysis showed that LAG-3 and TIM-3 were in CD8 +The expression on T cells was positively correlated with the T stage of OSCC (LAG-3: r = 0.999, P < 0.05; TIM-3: r = 0.997, P < 0.05).

[0050] In summary, it can be seen that the expressions of CTLA-4 and LAG-3 are abnormally increased in oral squamous cell carcinoma at T3 and T4 stages and are related to the T stage; the expressions of CTLA-4, TIM-3, and LAG-3 on CD8 + T cells are abnormally increased in oral squamous cell carcinoma at T3 and T4 stages and are related to the T stage. In OSCC, CTLA-4, TIM-3, and LAG-3 are distributed on different subtypes of T cells, and tumor immune escape is achieved by inhibiting the activities of corresponding T cells, promoting the occurrence and development of OSCC. That is to say, CTLA-4, TIM-3, and LAG-3 are suitable immune checkpoints. By inhibiting the expression levels of CTLA-4, TIM-3, and LAG-3, the activation and proliferation of T cells can be improved, the positive regulation of immune response can be increased, and thus the treatment effect of oral squamous cell carcinoma can be improved.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of CTLA-4, TIM-3 and LAG-3 as immune checkpoints in screening drugs for treating oral squamous cell carcinoma, characterized in that: Screening of drugs associated with CTLA-4, TIM-3, and LAG-3 expression levels as therapeutic agents for oral squamous cell carcinoma.

2. The use according to claim 1, characterized in that: Inhibit the expression levels of CTLA-4, TIM-3 and LAG-3, enhance T cell activation and proliferation, and improve immune response.

3. An immune checkpoint inhibitor, characterized in that Inhibit the expression levels of CTLA-4, TIM-3 and LAG-3 described in claim 1.

4. Use of the immune checkpoint inhibitor according to claim 3 in the preparation of a drug for treating oral squamous cell carcinoma.

5. Application of reagents for detecting CTLA-4, TIM-3 and LAG-3 in the preparation of diagnostic kits for oral squamous cell carcinoma.

6. Application of reagents for detecting CTLA-4, TIM-3 and LAG-3 in the preparation of a prognosis evaluation kit for oral squamous cell carcinoma.

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