Method for enhancing tumor radiotherapy sensitivity by using affinity peptide of DC (dendritic cell) alone or in combination

Abstract

The invention belongs to the technical field of tumor radiotherapy related medicines. CCK8 and other experiments find that the DC and the affinity peptide thereof can enhance the radiosensitivity of tumor cells, so that candidate drugs can be provided for clinical tumor radiotherapy sensitization.

Description

Technical fields:

[0001] This invention belongs to the field of drug technology related to tumor radiotherapy. Background technology:

[0002] Malignant tumors are a major disease posing a serious threat to human life and health worldwide. According to the latest statistics, in 2022, there were 19.96 million new cancer cases and approximately 9.74 million cancer deaths globally, with both incidence and mortality rates continuing to rise. Therefore, exploring effective treatment strategies to reduce cancer mortality has become an important goal for improving patients' quality of life and alleviating the global disease burden. Against this backdrop, radiotherapy, as one of the three core methods of cancer treatment, plays an irreplaceable role in clinical practice due to its advantages such as precision, broad indications, and non-invasiveness and repeatability. Radiotherapy directly destroys the DNA structure of tumor cells through high-energy rays (such as X-rays, gamma rays, or particle beams), inhibiting their proliferation and inducing apoptosis. Statistics show that approximately 50%-60% of cancer patients require radiotherapy during treatment, and its application spans the entire course of cancer management: from radical treatment of early-stage tumors to palliative care for advanced-stage patients; from neoadjuvant therapy to reduce tumor volume before surgery to adjuvant therapy to remove residual lesions after surgery. Radiotherapy has become an indispensable part of a multidisciplinary comprehensive treatment system.

[0003] However, radioresistance is a major bottleneck limiting its efficacy, potentially leading to tumor recurrence, poor treatment outcomes, adverse prognosis, decreased quality of life, and increased disease burden. Some tumor cells exhibit enhanced DNA damage repair, abnormal cell cycle regulation, or increased anti-apoptotic capabilities after radiotherapy, resulting in local recurrence or distant metastasis.

[0004] Dendritic cells (DCs) are among the most important antigen-presenting cells involved in T lymphocyte responses. In humans and mice, they can be classified into at least three types: plasmacytoid DCs (pDCs), blood-derived conventional DCs (cDCs), and tissue-derived cDCs. cDCs include cDC1, cDC2, and 33 subgroups, with cDC1 exhibiting the strongest antigen cross-presentation capacity. Currently, the most widely studied DC membrane receptors are C-type lectin receptors, such as Clec9a (C-type Lectin domain family 9A). Following radiation-induced cell death, damage-associated molecular patterns (DAMPs) are exposed or released. These signals are recognized by specific innate immune receptors on the surface of myeloid cells, triggering a cascade of inflammatory responses and promoting the recruitment of myeloid cells to damaged tissues. As a key node in innate immune regulation, tissue damage repair is precisely regulated to prevent secondary damage. The applicant has researched and patented numerous Clec9a affinity peptides, one of which is CBP-12. Clec9a + Whether cDC1 and CBP-12 can significantly improve the sensitivity of tumor cells to radiotherapy and enhance the killing effect of radiation on tumor cells has not yet been studied. Summary of the Invention:

[0005] In a first aspect, the inventors have discovered that dendritic cells (DC cells, sometimes referred to as DCs herein) can serve as tumor radiosensitizers, where sensitization refers to the ability to enhance the anti-tumor effect during radiotherapy; DCs can be cells expressing Clec9a (denoted as Clec9a). + DC), conventional DC (cDC), such as the cDC1 subgroup, preferably Clec9a + cDC1.

[0006] Optionally, the tumor may be a solid tumor, such as esophageal cancer.

[0007] Optionally, the radiotherapy uses X-rays.

[0008] Furthermore, the radiotherapy is also combined with a DC-specific affinity peptide, such as an affinity peptide targeting Clec9a, whose amino acid sequence may be as shown in SEQ ID NO.1 (specifically: Trp-Pro-Arg-Phe-His-Ser-Ser-Val-Arg-His-Thr-His), wherein the conformation of each amino acid of the peptide is independently selected from L-type or D-type, such that all amino acids of the peptide are L-type or all are D-type. As an example, the peptide is CBP-12 (all amino acids are L-type).

[0009] Secondly, the present invention provides a method for radiotherapy based on the peptide described in the first aspect, combined with DC, to enhance the radiosensitivity of tumor cells.

[0010] Thirdly, based on the foregoing aspects, the present invention provides a corresponding radiosensitizer containing any of the previously described DCs, which has a synergistic therapeutic effect with radiotherapy. This sensitizer may contain any of the previously described peptides, and may also contain pharmaceutically usable excipients.

[0011] Beneficial effects of this invention:

[0012] This invention explores the effects of DC and peptides (such as CBP-12) on improving radiotherapy resistance, specifically Clec9a. + The receptor Clec9a of dendritic cells (DCs) specifically recognizes F-actin exposed to radiation-damaged / dead cells, thus enhancing tumor radiosensitivity. Combined with its affinity peptide CBP-12, this further enhances tumor radiosensitivity. Based on these properties, peptide CBP-12 and similar drugs provide candidate drugs for overcoming radioresistance. Attached image description:

[0013] Figure 1 To experimentally investigate the effects of Clec9a + The effect of cDC1 on the radiosensitivity of AKR cells;

[0014] Figure 2 To further investigate the effect of combining CBP-12 (all amino acids are L-form) on the radiosensitivity of AKR cells, CBP-12 in the figure represents the combination regimen group;

[0015] In each figure, the significance indicators * indicate P<0.05, ** indicate P<0.01, and *** indicate P<0.001. Detailed implementation method:

[0017] The embodiments of the present invention will be described in detail below with reference to the examples. However, the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0018] Unless otherwise specified, the experimental methods used below are commonly used in the field, and the reagents, biological materials, culture media, and solutions are all commonly used, publicly available, or commercially available items. For example, AKR refers to esophageal cancer cells. The CCK8 assay is a cell proliferation and toxicity detection method based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt), widely used in cell biology, pharmacology, and toxicology. Its principle is that WST-8 is reduced to water-soluble formazan dye by dehydrogenases in the cell mitochondria. The amount of formazan produced is directly proportional to the number of viable cells. The absorbance value at a wavelength of 450 nm is measured using an ELISA reader, which indirectly reflects the number of viable cells.

[0019] I. Colony Formation Experiment

[0020] (1) Plating: 5000 AKR cells were seeded in a well plate and allowed to grow overnight to adhere to the plate.

[0021] (2) Irradiation: The cells were irradiated with X-rays at total doses of 0, 1, 2, and 4 Gy, respectively, in the experimental groups. After irradiation, 5000 Clec9a cells were added to the experimental group. + cDC1 (not added to the Control group), culture for 7-10 days;

[0022] (3) Staining: When the cell clones in the 0Gy group reached confluence, the cells were harvested, fixed, and stained with crystal violet; (4) The number of colonies in each group was counted, and the differences in cell viability were analyzed. The results are as follows: Figure 1 As shown.

[0023] II. CCK8 Experiment: Sensitization by Combination of DC and CBP-12

[0024] (1) Plating: Select AKR cells in good growth condition, prepare a single cell suspension and place it on a flat-bottomed 96-well plate. Inoculate 1000 cells in each well and let them adhere and grow overnight.

[0025] (2) Synchronization: On the second day, carefully remove the old culture medium, add serum-free culture medium, and starve the cells for 6-8 hours to synchronize their growth.

[0026] (3) Irradiation: Four hours after cell synchronization, the cells were irradiated with X-rays at a total dose of 2 Gy and 4 Gy, respectively, in groups.

[0027] (4) Incubation: After synchronization, carefully remove the old culture medium. Add 100 μL of fresh culture medium to each well, followed by 100 μL of peptide CBP-12 and Clec9a. +cDC1 (Control group does not add DC and peptide); (5) Color development: After culturing cells for 48h, add 20μL CCK8 to each well 4h before the end of culture;

[0028] (6) Reading: At the end of the culture, the OD value of the sample at 450 nm wavelength was read using a microplate reader. The data was saved and analyzed, and the results are as follows: Figure 2 As shown.

[0029] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is meant that all variations and modifications that include the scope of the invention in the claims are within the protection scope of the invention.

Claims

1. Application of Clec9a-expressing DC cells in the preparation of tumor radiosensitizers.

2. The application as described in claim 1, characterized in that, The DC cells were Clec9a. + cDC1 cells.

3. The application as described in claim 1, characterized in that, The tumor in question is a solid tumor, such as esophageal cancer.

4. The application as described in claim 1, characterized in that, The radiotherapy used X-rays.

5. The application as described in any of the prior claims, characterized in that, The radiotherapy is also combined with the affinity peptide of the DC cells, the amino acid sequence of which may be as shown in SEQ ID NO.

1.

6. The application as described in the preceding claim, characterized in that, The configuration of each amino acid in the affinity peptide is independently selected from L-type or D-type, such that all amino acids in the affinity peptide are either L-type or D-type.

7. Tumor radiosensitizers containing DC cells expressing Clec9a.

8. The tumor radiosensitizer as described in the preceding claim, characterized in that, The DC cells were Clec9a. + cDC1 cells.

9. The tumor radiosensitizer as claimed in any of the prior claims, characterized in that, It also contains the affinity peptide described in any of the prior claims.

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