APPLICATION OF CAERIN 1.1/1.9 COMBINED WITH ANTI-CD47 ANTIBODY IN PREPARING DRUG FOR TREATING MELANOMA

The present invention belongs to the field of biotechnology, and particularly relates to an application of Caerin 1.1/1.9 combined with an anti-CD47 antibody in preparing a drug for treating melanoma. In the present invention, an amino acid sequence of the Caerin 1.1 is shown in SEQ ID NO.1; and an amino acid sequence of the Caerin 1.9 is shown in SEQ ID NO.2. The Caerin 1.1/1.9 of the present invention may promote B16 cells to highly express CD47 while leading to apoptosis of the B16 cells, promote macrophages to phagocytose tumor cells in the presence of anti-CD47 antibodies, improve the curative effect of the Caerin 1.1/1.9, which is also superior to that of a single anti-CD47 antibody, and lays a foundation for expanding the application of CD47 inhibition/blocking drugs.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application claims priority to Chinese patent application No. 202211465805.4, filed on Nov. 22, 2022, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The sequence listing xml file submitted herewith, named “SeqList.xml”, created on May 14, 2025, and having a file size of 2,728 bytes, is incorporated by reference herein.

TECHNICAL FIELD

The present invention belongs to the field of biotechnology, and particularly relates to an application of Caerin 1.1/1.9 combined with an anti-CD47 antibody in preparing a drug for treating melanoma.

BACKGROUND

Malignant melanoma is a tumor produced by melanocytes of skin and other organs. Cutaneous melanoma is characterized by obvious changes in pigmented skin lesions over months or years. Although the incidence of the disease is low, it has high malignancy, early metastasis, and high mortality, so that early diagnosis and treatment are very important. The malignant melanoma mostly occurs in adults, and cases of secondary carcinogenesis of giant congenital melanocytic nevus are more common in children. Because melanoma is extremely harmful to human bodies, its treatment has always been concerned by all walks of life.

Immunotherapy is a new method for tumor treatment. Immune checkpoint inhibitors such as anti-PD1 and anti-CTLA4 have been widely used in clinic. With the continuous development and progress of scientific research, new immune checkpoints are continuously discovered, such as anti-CD47. But there is a common problem in the actual use of these immune checkpoints. Because these immune checkpoints can induce apoptosis of tumor cells, macrophages are affected and cannot phagocytose the tumor cells, and finally a tumor immune microenvironment is destroyed, so that the efficiency of the immunotherapy is limited.

Caerin 1 polypeptide family is an antibacterial polypeptide secreted by skin of Litoria caerulea, including Caerin 1.1, Caerin 1.9, etc., and has antibacterial and anti-tumor cell growth activity. Researches show that Caerin 1.1 and Caerin 1.9 may inhibit the growth of human and mouse cervical cancer cells, human thyroid cancer cells, and human breast cancer cells in vitro, and induce Hela cell apoptosis through a TNFalpha signal conduction path. Coupling nuclide may improve its anti-tumor efficiency. The growth of TC-1 cells may be inhibited in vivo and blood cells such as T cells, NK cells, and the like are attracted to tumor tissues. However, these only prove that Caerin 1.1 and Caerin 1.9 may inhibit tumors and do not change the above-mentioned problems. Furthermore, there is no report about the application of Caerin 1.1 and Caerin 1.9 in treating melanoma in the prior art.

In conclusion, the prior art generally has the technical problems that the tumor immune microenvironment is destroyed, the immunotherapy efficiency is influenced, the application range of Caerin 1.1 and Caerin 1.9 is narrow, and the like in the treatment process.

SUMMARY

In view of the problems generally existing in the prior art, the present invention provides an application of Caerin 1.1/1.9 combined with an anti-CD47 antibody in preparation of a drug for treating melanoma. The three components are combined to prepare a drug for treating melanoma, so that the immunotherapy effect is improved.

To achieve the above objectives, a technical effect adopted by the present invention is:

The dosage of the anti-CD47 antibody added to a mouse is 200 to 500 μg/mouse, and the adding dosage of Caerin 1.1/1.9 is 60 μg/mouse, where a mass ratio of Caerin 1.1 to Caerin 1.9 is 1:1, i.e., adding 30 μg/mouse each.

Preferably, an amino acid sequence of Caerin 1.1 is shown in SEQ ID NO.1; and an amino acid sequence of Caerin 1.9 is shown in SEQ ID NO.2.

The present invention further provides an application of Caerin 1.1/1.9 in inducing melanoma cells B16 to express CD47 in a large quantity.

Preferably, the drug further includes a pharmaceutically acceptable carrier.

The present invention further provides an application of Caerin 1.1/1.9 in preparing a pharmaceutical composition for treating tumors.

Preferably, the pharmaceutical composition further includes an anti-CD47 antibody and a therapeutic vaccine.

Preferably, the therapeutic vaccine consists of a melanoma therapeutic vaccine, MPLA, and α-IL10r.

Preferably, the tumor is melanoma or cervical cancer.

A mechanism research shows that Caerin 1.1/1.9 improves the tumor immune microenvironment, and re-polarizes tumor infiltrating macrophages, so as to convert the tumor infiltrating macrophages from an M2 type to an M1 type. More interleukin 12 and less interleukin 10 are secreted. If the therapeutic vaccine contains a melanoma therapeutic vaccine and an interleukin 10 inhibitory antibody, combination use of Caerin 1.1/1.9 and them can inhibit the growth of TC-1 cells more effectively. The combination use of Caerin 1.1/1.9, the anti-CD47 antibody, and the therapeutic vaccine may eliminate tumor completely in 20-50% of TC-1 tumor-bearing mice.

The inventors found that Caerin 1.1/1.9 inhibited the growth of melanoma B16 cells in vitro, induced apoptosis of melanoma B16 cells, and could enter into B16 cells, so as to inhibit the growth of B16 cells in vivo. The high expression of CD47 by the B16 cells improves the efficiency of the anti-CD47 antibody in a B16 model, attracts blood cells to tumors, particularly macrophages and CD4+T cells to the tumors, so that the macrophages phagocytose the tumor cells. When combined with the anti-CD47 antibody and the therapeutic vaccine, tumors of 50% of tumor-bearing mice may be completely eliminated.

Compared with the prior art, the technical solution of the present invention has the following advantages: the present invention solves the curative effect problem of the anti-CD47 in melanoma, induces B16 cells to highly express CD47, and improves the curative effect of treating the B16 tumor by combined use of the anti-CD47 and Caerin 1.1/1.9. The triple therapy of the anti-CD47, the therapeutic vaccine, and the Caerin 1.1/Caerin 1.9 may achieve better effect of treating the B16 tumor than that when an α-PD-1 is used.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention is further explained below in conjunction with embodiments, but it should be noted that the following embodiments are only used to explain rather than limit the present invention, and all technical solutions identical or similar to the present invention fall within the protection scope of the present invention. Where no specific techniques or conditions are specified in the embodiments, operations are performed according to conventional technical methods and content of instrument specifications in the art; and where no manufacturers are specified for reagents or instruments used herein, they are all conventional products that are commercially available.

Experiment Example 1 In Vitro Detection Experiment

Experiment Example 2 In Vivo Detection Experiment

Experiment Example 3 Detection of Tumor Smearing Therapy

Experiment Example 4 F1/F3 For Combined Treatment of Conversion of MDSC Into Macrophages

Experiment Example 5 F1/F3 Combined with α-PD-1 for Treatment of Melanoma

Experiment Example 6 Changes Of Increase In CD47 Expression In F1/F3 Combined Treatment Of Model

Experiment Example 7 F1/F3 Combined With CD47 for Treatment of Melanoma

Experiment Example 8 F1/F3 Combined With α-PD-1 and Therapeutic Vaccine for Treatment of Melanoma

Experiment Example 9 Study on Changes of Immune Microenvironment in Triple Therapy

Experiment Example 10 F1/F3 for Treatment of Nude Mice Bearing Tumor Model

Experiment Example 11 F1/F3 Combined with Anti-CD47 and Therapeutic Vaccine for Treatment of Melanoma

Finally, it should be noted that the above embodiments are only intended to exemplarily illustrate the principle, performance, and effect of the present invention but not intended to limit the present invention. Any person of ordinary skill in the art can modify or change the above examples without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those of ordinary skill in the art without departing from the spirit and technical ideal disclosed by the present invention should still fall within the claims of the present invention.