A polypeptide capable of orientable enhancement of thymic naive t cell killing of breast cancer and applications thereof

Abstract

The application discloses a polypeptide capable of directionally enhancing thymus newborn T cells to kill breast cancer and application thereof, and a peptide segment sequence is NH2-KLLSLIKGVIVHRLEGVE-GPSL-KDMQLGR-CONH2. A Th cell epitope sequence (KLLSLIKGVIVHRLEGVE) is from a measles virus fusion protein 288-302 peptide segment (MVF), and a four-residue linker (GPSL) is behind the T cell epitope. The linker is integrated into the fusion peptide to help independent folding of the T cell epitope and a B cell epitope. A B cell epitope sequence is a C segment fragment (KDMQLGR) of C5a. The polypeptide of the application can inhibit breast cancer tumor growth, enhance thymus T cell function output, and enhance T cell directional killing function on breast cancer, has low toxic side effects, and can be applied to breast cancer treatment, thereby providing a new scheme and a new idea for breast cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and provides a polypeptide capable of directionally enhancing thymus newly born T cells to kill breast cancer and application thereof. BACKGROUND

[0002] Breast cancer is a common malignant tumor in women worldwide, and its incidence continues to rise, seriously threatening women's lives and health. Existing breast cancer treatment methods include surgical treatment, radiotherapy, chemotherapy, etc. However, these treatment methods have defects and deficiencies. For example, surgery can only remove cancer cells that have formed solid lesions, and cannot completely remove cancer cells, while radiotherapy and chemotherapy have large side effects, including hair loss, nausea, fatigue, immune system suppression, etc. These side effects affect the quality of life of patients. In addition, the physiological structure of the tumor site is complex, and the local drug concentration is often difficult to reach the ideal level, and abnormal tumor blood vessels also cause interstitial pressure to rise and blood flow to stagnate, making it difficult for drugs to effectively penetrate into tumor tissue, affecting the overall treatment effect. Therefore, there is an urgent need to develop new treatment methods to overcome the defects of existing methods, improve the accuracy and effectiveness of treatment, reduce side effects, and ultimately achieve the purpose of improving the survival rate and quality of life of breast cancer patients. In recent years, immunotherapy has made significant progress in the field of treating malignant tumors, providing a new choice for breast cancer treatment.

[0003] Studies have shown that B cell epitope peptides against C5a can effectively alleviate the symptoms of Alzheimer's disease by activating plasma cells to produce specific antibodies against C5a, reducing the interaction of C5a and C5aR, but its anti-tumor biological activity in vivo has not been reported. The binding of complement C5a to its receptor C5aR can promote the development of breast cancer, therefore, targeting C5a / C5aR may be an effective strategy for tumor immunotherapy.

[0004] At present, there is no related report on the polypeptide of the present application. SUMMARY

[0005] In view of this, the purpose of the present application is to provide a polypeptide capable of directionally enhancing thymus newly born T cells to kill breast cancer and application thereof, so as to realize breast cancer immunotherapy.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] 1. A polypeptide capable of directionally enhancing thymus newly born T cells to kill breast cancer, which is assembled by sequentially connecting the following peptide segments:

[0008] (1) Th cell epitope sequence: KLLSLIKGVIVHRLEGVE;

[0009] (2) Four-residue linker: GPSL;

[0010] (3) B cell epitope sequence: KDMQLGR.

[0011] As one of the preferred technical solutions, the assembled peptide segment sequence is:

[0012] KLLSLIKGVIVHRLEGVEGPSLKDMQLGR, as shown in SEQ ID NO. 1.

[0013] 2. The preparation method of the aforementioned polypeptide capable of orienting and enhancing the killing of breast cancer by thymus newborn T cells, which is obtained by connecting and assembling according to the following amino acid sequence: KLLSLIKGVIVHRLEGVEGPSLKDMQLGR, as shown in SEQ ID NO. 1.

[0014] As one of the preferred technical solutions, the Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO. 1.

[0015] As one of the further preferred technical solutions, the Fmoc solid-phase synthesis method is used to connect the first amino acid at the C-terminus of the polypeptide to the PS resin carrier, and then according to the sequence of the polypeptide, the amino acids are sequentially condensed from the C-terminus to the N-terminus. The amino acid is protected by a Fmoc (9-fluorenylmethyloxycarbonyl) group at its amino terminus, and after activation at the carboxylic acid terminus, it is coupled with the growing chain (the polypeptide chain gradually constructed on the resin carrier; the amino acid residues are gradually added from the C-terminus (carboxylic acid terminus) to the N-terminus (amino terminus) until the desired sequence length is reached). Then the Fmoc group is removed by piperidine treatment and the process is repeated to complete the peptide assembly.

[0016] As one of the further preferred technical solutions, after the completion of the peptide assembly, it can be removed from the resin carrier by treatment with trifluoroacetic acid (TFA); at the same time, the protecting group on the side chain of the amino acid is also removed, obtaining a crude linear peptide; the polypeptide is purified by high performance liquid chromatography (HPLC) to remove by-products and unreacted substances.

[0017] HPLC chromatographic conditions:

[0018] Chromatographic column: kromasil C18-5 column (4.6*150mm); mobile phase A is 0.1% trifluoroacetic acid acetonitrile solution by volume concentration, B is 0.1% trifluoroacetic acid aqueous solution by volume concentration, gradient elution (0.01 min, 5% A 95% B, 25.0 min, 75% A 25% B, 30 min, 90% A 10% B; the change of mobile phase refers to the volume percentage); flow rate 1.0 mL / min; detection wavelength 214 nm; injection volume 20 ul.

[0019] MS conditions: expected MS value 3185.88; flow rate 0.2 mL / min; run time 1 min; buffer A was 0.1% formic acid in water, and buffer B was 0.1% formic acid in acetonitrile.

[0020] 3. Use of the aforementioned polypeptide capable of directionally enhancing thymic newly generated T cells to kill breast cancer in the preparation of anti-breast cancer drugs.

[0021] The beneficial effects of the present invention are:

[0022] The present invention has prepared a polypeptide that can specifically enhance the killing of breast cancer cells by thymic nascent T cells. The peptide sequence is NH2-KLLSLIKGVIVHRLEGVE-GPSL-KDMQLGR-CONH2. The Th cell epitope sequence (KLLSLIKGVIVHRLEGVE) is derived from the 288-302 peptide segment of the measles virus fusion protein (MVF). The T cell epitope is followed by a four-residue linker (GPSL). This linker is incorporated into the fusion peptide to facilitate the independent folding of the T cell and B cell epitopes. The B cell epitope sequence is the C segment of C5a (KDMQLGR).

[0023] The polypeptide of the present invention can inhibit the growth of breast cancer tumors, enhance the functional output of thymic T cells and enhance the directed killing function of T cells against breast cancer, reduce toxic side effects, and can be applied to the treatment of breast cancer, providing new solutions and new ideas for the treatment of breast cancer.

[0024] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0026] Figure 1 This is the result of HPLC analysis of Max449 peptide;

[0027] Figure 2 This is the result of Max449 peptide MS analysis;

[0028] Figure 3 This is the result of the anti-breast cancer peptide acting on the in vitro transplanted tumor model of Balbc mice;

[0029] Figure 4 The tumor volume of the in vitro transplanted tumor model of Balbc mice was measured by the effect of anti-breast cancer peptides;

[0030] Figure 5 The anti-breast cancer polypeptide's effect on the weight of tumor in the Balbc mouse xenograft model.

[0031] Figure 6 The anti-breast cancer polypeptide's effect on the change of thymus new T cells in the Balbc mouse xenograft model. DETAILED DESCRIPTION

[0032] The application will be further described in conjunction with the specific embodiments.

[0033] Unless otherwise specified, the techniques or conditions in the examples were carried out according to the techniques or conditions described in the literature or according to the product manual. Unless otherwise specified, the reagents or instruments used were conventional products that can be purchased through regular channels.

[0034] Example 1:

[0035] Preparation of the anti-breast cancer polypeptide (Max449 peptide)

[0036] The sequence of the anti-breast cancer polypeptide is NH2-KLLSLIKGVIVHRLEGVE-GPSL-KDMQLGR-CONH2. The sequence of the Th cell epitope (KLLSLIKGVIVHRLEGVE) is from the 288-302 peptide segment of the fusion protein of the measles virus (MVF), and the T cell epitope is followed by a four-residue linker (GPSL). The linker is integrated into the fusion peptide to help the independent folding of the T cell epitope and the B cell epitope. The sequence of the B cell epitope is the C segment fragment of C5a (KDMQLGR)

[0037] Preparation method: the following peptide segments are sequentially connected and assembled: (1) the sequence of the Th cell epitope:

[0038] KLLSLIKGVIVHRLEGVE; (2) the four-residue linker: GPSL; (3) the sequence of the B cell epitope: KDMQLGR.

[0039] Fmoc solid phase synthesis method, the first amino acid of the C-terminal of the polypeptide is connected to the PS resin carrier, and then according to the sequence of the polypeptide, the amino acids are condensed in order from C-terminal to N-terminal; the amino acid is protected by Fmoc (9-fluorenylmethyloxy carbonyl) group at its amino terminal, and after the carboxylic acid terminal is activated, it is coupled with the growing chain; then the Fmoc group is removed by piperidine treatment and the process is repeated to complete the peptide assembly. After the completion of the peptide assembly, it can be removed from the resin by treating with trifluoroacetic acid (TFA); at the same time, the protecting group on the side chain of the amino acid is also removed, and the crude linear peptide is obtained; the polypeptide is purified by high performance liquid chromatography (HPLC) to remove by-products and unreacted substances (such as Figure 1

[0040] HPLC chromatographic conditions:

[0041] Chromatographic column: kromasil C18-5 column (4.6*150mm); mobile phase A is 0.1% trifluoroacetic acid acetonitrile solution, B is 0.1% trifluoroacetic acid aqueous solution, gradient elution (0.01 min, 5% A 95% B, 25.0 min, 75% A 25% B, 30 min, 90% A 10% B; the change of mobile phase refers to volume percentage); flow rate 1.0 mL / min; detection wavelength 214 nm; injection volume 20ul.

[0042] Structure identification: the molecular weight and composition of the polypeptide are determined by mass spectrometry (MS) (such as Figure 2

[0043] Example 2:

[0044] Polypeptide treatment test in mice

[0045] 6 to 8 weeks old Balbc female mice (Chongqing Tengxin Bi'er Experimental Animal Sales Co., Ltd.), the mice were subcutaneously injected with 4T-1 cells (Wuhan Punuo Sai Life Science and Technology Co., Ltd.) (3*10 5 100ul) in the right axillary, and the tumor was formed 4 to 6 days later. The length and width of the tumor were measured every 2 days with a vernier caliper. The tumor volume was calculated as length x width 2 / 2 (mm 3 ​​). Mice were randomly assigned to three treatment groups (5 mice per group): PBS treatment group (Ctrl), anti-tumor polypeptide (SEQ ID NO: 7, patent CN103269711B) treatment group and anti-tumor polypeptide (Max449) treatment group. The second day after tumor-bearing, mice were injected with 1 mg / ml polypeptide (control group injected with PBS), every other day for a total of four doses, and the weight and volume of tumors were measured after 17 days of treatment, while the number of CD8 TM A1, BD Biosciences) in tumors was detected by flow cytometry (FACSymphony + T cells was analyzed, and the results are shown in Figures 3-6 .

[0046] Figure 3 Balbc female mice were subcutaneously injected with 4T-1 cells (3*10 5 100 μL) on the right axillary side (n=5 per group), group 1 was treated with PBS, group 2 was treated with anti-tumor polypeptide (SEQ ID NO: 7, patent CN103269711B), and group 3 was treated with anti-tumor polypeptide (Max449). Mice were injected with 1 mg / ml polypeptide (control group injected with PBS), every other day for a total of four doses, and tumors were removed after 17 days of treatment.

[0047] Figure 4 Balbc female mice were subcutaneously injected with 4T-1 cells (3*10 5 100 μL) on the right axillary side (n=5 per group), group 1 was treated with PBS, group 2 was treated with anti-tumor polypeptide (SEQ ID NO: 7, patent CN103269711B), and group 3 was treated with anti-tumor polypeptide (Max449). Mice were injected with 1 mg / ml polypeptide (control group injected with PBS), every other day for a total of four doses, and tumors were removed after 17 days of treatment. The length and width of the tumor were measured with a caliper, and the tumor volume was calculated as length x width 2 / 2 (mm 3 ) (n=5 per group).

[0048] Figure 5 Balbc female mice were subcutaneously injected with 4T-1 cells (3*10 5 100 μL) on the right axillary side (n=5 per group), group 1 was treated with PBS, group 2 was treated with anti-tumor polypeptide (SEQ ID NO: 7, patent CN103269711B), and group 3 was treated with anti-tumor polypeptide (Max449). Mice were injected with 1 mg / ml polypeptide (control group injected with PBS), every other day for a total of four doses, and tumors were removed after 17 days of treatment. The length and width of the tumor were measured with a caliper, and the tumor volume was calculated as length x width

[0049] Figure 6Balbc female mice were injected subcutaneously with 4T-1 cells (3*10 5 100μL) in the right axillary of the mice (n=3 in each group). Group 1 was treated with PBS, group 2 was treated with anti-tumor polypeptide (SEQ ID NO: 7, patent CN103269711B), and group 3 was treated with anti-tumor polypeptide Max449. The mice were injected with 1 mg / ml polypeptide (the control group was injected with PBS), and the injection was given every other day for a total of four times. The tumors were removed after 17 days of treatment, and the CD8 + T cells in the tumors were analyzed by flow cytometry.

[0050] Compared with the Ctrl group and the anti-tumor polypeptide (SEQ ID NO: 7, patent CN103269711B) group, the tumor volume and weight of the anti-tumor polypeptide Max449 group were smaller, indicating that the anti-tumor polypeptide Max449 could significantly inhibit tumor growth. Compared with the Ctrl group and the anti-tumor polypeptide (SEQ ID NO: 7, patent CN103269711B) group, the CD8 + T cells in the anti-tumor polypeptide Max449 group increased, indicating that the thymus newly generated T cells increased. The above experiments showed that the anti-tumor polypeptide Max449 could effectively treat breast cancer.

[0051] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present technical solutions, and all of them should be covered in the scope of the claims of the present application.

Claims

1. A polypeptide that can direct enhanced thymic ne na T cell killing of breast cancer, characterized in that, is assembled by sequentially connecting the following peptide segments: (1) Th cell epitope sequence: KLLSLIKGVIVHRLEGVE; (2) Four-residue linker: GPSL; (3) B cell epitope sequence: KDMQLGR; The assembled peptide segment sequence is: KLLSLIKGVIVHRLEGVEGPSLKDMQLGR, as shown in SEQ ID NO.

1.

2. The method of producing a polypeptide that directionally enhances thymic neona- tal T cell killing of breast cancer of claim 1, wherein, is assembled by sequentially connecting the following peptide segments: KLLSLIKGVIVHRLEGVEGPSLKDMQLGR, as shown in SEQ ID NO.

1.

3. The preparation method according to claim 2, characterized in that The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1.

4. The production method according to claim 3, characterized by, The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1.

5. The production method according to claim 4, characterized by, The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO.

1. The Fmoc solid-phase synthesis method is used to connect and assemble according to the amino acid sequence shown in SEQ ID NO. 1

Images