Application of CCR6 antagonist in treatment of oral squamous cell carcinoma

Abstract

The invention discloses an application of a CCR6 antagonist in treatment of oral squamous cell carcinoma. The invention belongs to the field of biomedicine, and particularly relates to application of a CCR6 antagonist in treatment of oral squamous cell carcinoma. The invention provides an application of a substance for inhibiting CCR6 gene expression or a substance for inhibiting CCR6 protein activity or a substance for reducing CCR6 protein content in preparation of products of drugs for treating oral squamous cell carcinoma or / and preventing oral squamous cell carcinoma or / and preparing drugs for inhibiting tumor cell activity or anti-tumor activity. By adopting the CCR6 antagonist HY-151435, the normal high expression of CCR6 in OSCC can be blocked, and the recruitment of immunosuppressive cells of tumor-related regulatory T cells (Tregs) is reduced, so that the immunosuppressive microenvironment is reversed.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of CCR6 antagonists in the treatment of oral squamous cell carcinoma. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck. Although current treatments (such as surgery, radiotherapy, chemotherapy, and targeted therapy) have some effect, the 5-year survival rate is still not ideal (approximately 50-60%), and problems such as recurrence, metastasis, and treatment resistance exist. It is widely distributed in the oral cavity. P. gingivalis , P. gingivalis It can worsen the malignancy of OSCC patients. Surgical resection: Advanced tumors require extensive resection, leading to facial deformities and functional losses (such as difficulty chewing and speaking); micrometastases cannot be completely eliminated. Radiotherapy and chemotherapy: Radiotherapy easily causes side effects such as oral mucositis and dry mouth; chemotherapy drugs (such as cisplatin and 5-FU) have high systemic toxicity and are prone to drug resistance. Targeted therapy (such as cetuximab): It only targets a few targets such as EGFR, with limited efficacy; some patients do not respond or develop rapid drug resistance. Immunotherapy (such as PD-1 inhibitors): The response rate is only 15-30%, and there is a lack of reliable predictive biomarkers for efficacy; it may cause immune-related adverse reactions.

[0003] CCR6 is a 7-transmembrane domain G protein-coupled receptor expressed in the intestinal mucosa, lung mucosa, and lymph nodes, and highly expressed in epithelial tumors. At the cellular level, CCR6 is mainly expressed on immune cells such as regulatory T cells, macrophages, and myeloid-derived suppressor cells. Upregulation of CCL20 during inflammation may increase the migration of CCR6-positive regulatory T cells into tissues, promoting the formation of an immunosuppressive microenvironment and contributing to the malignant biological behavior of oral squamous cell carcinoma. Antagonizing CCR6 expression could reverse this trend, providing a new therapeutic approach for oral squamous cell carcinoma. Summary of the Invention

[0004] The main problem this invention aims to solve is how to treat oral squamous cell carcinoma.

[0005] To address the above problems, the present invention provides the following applications: 1. Inhibition CCR6 The use of gene-expressing substances in the preparation of products for the treatment and / or prevention of oral squamous cell carcinoma.

[0006] 2. Inhibition CCR6 Application of gene-expressing substances in the preparation of drugs that inhibit tumor cell activity or have anti-tumor activity.

[0007] 3. Application of substances that inhibit CCR6 protein activity in the preparation of products for the treatment and / or prevention of oral squamous cell carcinoma.

[0008] 4. Application of substances that inhibit CCR6 protein activity in the preparation of drugs that inhibit tumor cell activity or have anti-tumor activity.

[0009] 5. Application of substances that reduce CCR6 protein content in the preparation of drugs that inhibit tumor cell activity or have anti-tumor activity.

[0010] 6. Application of substances that reduce CCR6 protein content in the preparation of products for the treatment and / or prevention of oral squamous cell carcinoma.

[0011] The amino acid sequence of the CCR6 protein is Genbank: NP_001381511.1 CCR6 [organism=Homosapiens] [GeneID=1235], updated on July 5, 2025.

[0012] The CCR6 The nucleotide sequence of the gene is a DNA molecule with the following GenBank address: NC_000006.12:167111795-167139141CCR6 [organism=Homo sapiens] [GeneID=1235][chromosome=6], updated July 5, 2025.

[0013] Furthermore, the inhibition CCR6 The substance that expresses the gene or inhibits the activity or content of CCR6 protein is the CCR6 antagonist HY-151435 (MCE Company; catalog number: 588674-64-0).

[0014] Furthermore, the dosage of the CCR6 antagonist HY-151435 is 1 mg / kg.

[0015] Furthermore, the tumor cells are P. gingivalis Induced oral squamous cell carcinoma cells.

[0016] In this article, the inhibition of tumor cell activity can be manifested in: reducing tumor volume, reducing tumor weight, and reducing CD4 count. + T cells, reduced Treg cells, reduced immune checkpoint PD1 + Cells and / or CTLA4 + Cells and / or TIGIT + Cells and / or TIM3 + cell.

[0017] This invention demonstrates that CCR6 antagonists can treat oral squamous cell carcinoma, reducing tumor volume and weight in animal experiments. CCR6 antagonists inhibit oral squamous cell carcinoma by suppressing the expression of Treg cells and immune checkpoints.

[0018] Compared with the prior art, the beneficial effects of the present invention are: CCR6 is frequently overexpressed in OSCC and is associated with tumor progression, lymph node metastasis, and poor prognosis. CCR6 antagonists can block this pathway, reducing the recruitment of tumor-associated regulatory T cells (Tregs) and immunosuppressive cells, thereby reversing the immunosuppressive microenvironment. Compared to other immunosuppressants, which have significant adverse reactions and unclear efficacy, CCR6 antagonists have shown stable anti-tumor effects in animal studies. Compared to traditional chemotherapy, targeting CCR6 may reduce systemic toxicity and improve patients' quality of life. Furthermore, it can be combined with immunotherapy; in combination with inhibitors such as PD-1, it may overcome immunotherapy resistance, especially at CCR6 levels. P. gingivalis In highly expressed OSCC subtypes, CCR6 or CCL20 is highly expressed or P. gingivalis Patients with high OSCC expression may benefit more from CCR6 antagonist therapy, enabling personalized precision medicine. Attached Figure Description

[0019] Figure 1 Reversal of CCR6 antagonist P.gingivalis The induced increase in tumor volume and weight in the oral squamous cell carcinoma experimental animal model was statistically significant. The following data are presented: A) Chemical structure of the CCR6 antagonist; B) Gross image of the mouse model of oral squamous cell carcinoma; C) Gross image of the tumor in the animal model of oral squamous cell carcinoma; D) Statistical graph of tumor volume in the animal model of oral squamous cell carcinoma; E) Statistical graph of tumor weight in the animal model of oral squamous cell carcinoma.

[0020] Figure 2 Reversal of CCR6 antagonist P.gingivalis Changes in malignant immune markers in an induced oral squamous cell carcinoma experimental animal model, including a decrease in CD4 levels by A and CCR6 antagonists. + A) T cell percentage; B) CCR6 antagonists reduce Treg cell percentage; C) CCR6 antagonists reduce PD-1. + Cell percentage; D, CCR6 antagonists reduce CTLA-4 + Cell percentage; E and CCR6 antagonists reduce TIGIT + Cell percentage; F and CCR6 antagonists reduce TIM3 + Cell percentage. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0024] Porphyromonas gingivalis in the following examples ( P.gingivalis Purchased from Beijing Beina Innovation Biotechnology Research Institute (item number: BNCC353909).

[0025] The following examples used SPSS 26 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. ns represents no statistical difference, P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0026] Example 1: Application of CCR6 antagonists in the treatment of oral squamous cell carcinoma 1. Take an appropriate amount of SCC7 mouse cells (Yimo Life Science Co., Ltd., catalog number: IM-M102) in the logarithmic growth phase, digest, resuspend, and count.

[0027] 2. Cultivation P.gingivalis Anaerobic incubator, 37℃ constant temperature incubator, Columbia solid and liquid culture media. Columbia broth liquid culture medium was purchased from Solarbio, catalog number: LA8080; Columbia blood agar plate solid culture medium was purchased from Nanjing Quanlong Biotechnology Co., Ltd., catalog number: glby-001.

[0028] Take an appropriate amount P.gingivalis Centrifuge the bacterial culture at 5000 rpm for 10 min, resuspend in PBS, centrifuge again, discard the supernatant, and resuspend in cell culture medium.

[0029] 3. In accordance with the experimental design requirements, the bacteria were diluted to a multiplicity of infection (MOI) of 50. The diluted bacteria were then added to cell culture plates to construct a bacterial-cell co-culture model.

[0030] 4. Select 6-8 week old female C57BL / 6 mice with standard weight (18-20g) (purchased from the Animal Experiment Center of Xinjiang Medical University) and divide them into three groups, with 4 mice in each group; Group a: Injection of SCC7 mouse cells (Yimo Life Science Co., Ltd., catalog number: IM-M102), 200,000 SCC7 mouse cells / mL, 100μL injected into the cheek; Group b: Injection of a suspension of SCC7 murine cells and Porphyromonas gingivalis, and buccal injection of 200,000 SCC7 murine cells / mL, 100 μL. Group C: Injected with a suspension of SCC7 murine cells and *Porphyromonas gingivalis*, and buccally injected with 200,000 SCC7 murine cells / mL, 100 μL. Additionally, every 4 days, the mice received an in situ buccal injection of 1 mg / kg of CCR6 antagonist (MCE; catalog number: 588674-64-0, structural formula see [link to MCE product]). Figure 1 (A)

[0031] 100 μL was injected into the left cheek of each mouse.

[0032] Sixteen days later, mice were sacrificed, fluid tissues were dissected, and their volume and weight were recorded. The tissues were fixed with paraformaldehyde. Immunohistochemistry was performed to stain for CD4, FOXP3, PD-1, CTLA-4, TIGIT, and TIM3, which are immunosuppressive markers involved in OSCC.

[0033] 5. Immunological marker staining Paraffin-embedded tissue sections (4 μm) were cut for immunohistochemical staining: sections of the above specimens were treated with peroxidase inhibitors, then blocked with 5% goat serum, and incubated overnight at 4°C with anti-CD4 (Abcam, 1:400), anti-FOXP3 (BIOSS, 1:400), anti-PD-1 (Abcam, 1:400), anti-CTLA4 (BOSTER, 1:400), anti-TIGIT (BOSTER, 1:400), and anti-TIM3 (BIOSS, 1:400). After incubation with the corresponding secondary antibodies and anti-biotin-biotin peroxidase, visualization was performed using a DAB kit (Mxb Bio), and hematoxylin was used for nuclear staining. IHC expression levels were assessed using Image-Pro Plus version 6.0 software (MediaCybernetics, Inc., Bethesda, MD, USA).

[0034] The results are as follows Figure 1The results from the BE study show that: group a received injections of SCC7 murine cells, group b received injections of a suspension of SCC7 murine cells and *Porphyromonas gingivalis*, and group c received injections of a suspension of SCC7 murine cells and *Porphyromonas gingivalis* plus a CCR6 antagonist. Group b showed increased tumor volume and weight compared to group a. This demonstrates... P. gingivalis Under induction, the volume and weight of oral squamous cell carcinoma tumors increased, while the volume and weight of tumors in group C decreased compared to group B, demonstrating that the CCR6 antagonist reversed the tumor's growth. P. gingivalis The induced increase in tumor volume and weight in an experimental animal model of oral squamous cell carcinoma reduced the malignant phenotype of oral squamous cell carcinoma.

[0035] Immunohistochemistry revealed that CCR6 antagonists reversed [the disease]. P. gingivalis Changes in malignant immune markers in an induced oral squamous cell carcinoma experimental animal model: P. gingivalis It can promote the expression of CD4, FOXP3, and immune checkpoints PD-1, CTLA4, TIGIT, and TIM3 in OSCC, but CCR6 inhibition can reverse this trend, and the difference is statistically significant. Figure 2 (AF). Therefore, it can be concluded that... P. gingivalis via CD4 + FOXP3 + Treg cells promote the expression of immune checkpoints, which in turn promotes the formation of an immunosuppressive microenvironment and the malignant biological behavior of OSCC. CCR6 antagonism can relieve the immunosuppression of OSCC and reduce its malignant progression, providing a theoretical basis for the prevention and treatment of OSCC.

[0036] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Inhibition CCR6 The use of gene-expressing substances in the preparation of products for the treatment and / or prevention of oral squamous cell carcinoma.

2. Inhibition CCR6 Application of gene-expressing substances in the preparation of drugs that inhibit tumor cell activity or have anti-tumor activity.

3. Application of substances that inhibit CCR6 protein activity in the preparation of products for the treatment and / or prevention of oral squamous cell carcinoma.

4. Application of substances that inhibit CCR6 protein activity in the preparation of drugs that inhibit tumor cell activity or have anti-tumor activity.

5. Application of substances that reduce CCR6 protein content in the preparation of drugs that inhibit tumor cell activity or have anti-tumor activity.

6. Application of substances that reduce CCR6 protein content in the preparation of products for the treatment and / or prevention of oral squamous cell carcinoma.

7. The application according to claim 2, 4 or 6, characterized in that... The tumor cells are P. gingivalis Induced oral squamous cell carcinoma cells.

8. The application according to any one of claims 1-7, characterized in that: The inhibition CCR6 The substance that expresses the gene or inhibits the activity or content of CCR6 protein is the CCR6 antagonist HY-151435.

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