A polypeptide capable of orientable enhancement of thymic neotrans T cells in inhibiting melanoma and application thereof

Abstract

The application discloses a polypeptide capable of directionally enhancing thymus newborn T cells to inhibit melanoma and application thereof, and a peptide segment sequence of the polypeptide is NH2-FNNFTVSFWLRVPKVSASHL-GPSL-KDMQLGR-CONH2. A Th cell epitope sequence (FNNFTVSFWLRVPKVSASHL) is from a tetanus toxoid 947-967 peptide segment (TT3), and a four-residue linker (GPSL) is arranged after the T cell epitope. The linker is integrated into a 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 melanoma growth, enhance thymus newborn T cell function, has low toxicity and side effects, and can be applied to melanoma treatment, thereby providing a new scheme and a new idea for melanoma treatment.

Description

TECHNICAL FIELD

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

[0002] Melanoma is a highly malignant skin tumor, and its incidence is increasing worldwide year by year. Therefore, it is urgent to find an effective method for treating melanoma. In recent years, with the progress of immunotherapy technology, tumor immunotherapy has become a hot spot in the field of melanoma treatment.

[0003] Melanoma, as a highly malignant skin cancer, its treatment methods mainly include surgical resection, radiotherapy, chemotherapy and immunotherapy. However, these treatment methods have defects and deficiencies. First, surgical resection can directly remove the tumor, but it is not suitable for all patients, especially for advanced melanoma, the risk and complexity of surgery for patients will greatly increase. Secondly, radiotherapy and chemotherapy can control the development of tumors to a certain extent, but at the same time, they will also kill a large number of normal cells, causing skin damage, nausea, vomiting and other serious side effects, and the effect is not ideal for the treatment of drug-resistant tumors.

[0004] Therefore, it has become an urgent problem to develop new safe and effective melanoma treatment strategies. Polypeptide therapy has gradually become a research hotspot due to its high selectivity and low toxicity. Polypeptides can specifically bind to the anionic cell membrane molecules of cancer cells, inhibit their growth and spread by interfering with specific pathways of tumor cell life activities, and have no effect on normal cells, so they can selectively kill cancer cells, and their distribution and removal in the body are more convenient and safe. Through further research and development, anti-melanoma polypeptides are expected to bring new treatment hope to more patients.

[0005] 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 and reducing the interaction between C5a and C5aR, but its anti-tumor biological activity in vivo has not been reported. The receptor C5aR1 signal of complement C5a supports the growth of melanoma by promoting the infiltration of immunosuppressive leukocyte populations into the tumor microenvironment, so targeting C5a / C5aR may be an effective strategy for tumor immunotherapy.

[0006] At present, there is no related report on the polypeptide of the application. SUMMARY

[0007] Therefore, the purpose of the present application is to provide a polypeptide capable of directionally enhancing thymus newly born T cells to inhibit melanoma and application thereof, so as to realize melanoma immunotherapy.

[0008] To achieve the above object, the present application provides the following technical solutions.

[0009] 1. A polypeptide capable of orienting and enhancing thymus newly-born T cells to inhibit melanoma, which is assembled by sequentially connecting the following peptide segments:

[0010] (1) Th cell epitope sequence: FNNFTVSFWLRVPKVSASHL;

[0011] (2) Four-residue linker: GPSL;

[0012] (3) B cell epitope sequence: KDMQLGR.

[0013] As one of the preferred technical solutions, the assembled peptide segment sequence is:

[0014] FNNFTVSFWLRVPKVSASHLGPSLKDMQLGR, as shown in SEQ ID NO. 1.

[0015] 2. A preparation method of the polypeptide capable of orienting and enhancing thymus newly-born T cells to inhibit melanoma, which is assembled by sequentially connecting the following amino acid sequence: FNNFTVSFWLRVPKVSASHLGPSLKDMQLGR, as shown in SEQ ID NO. 1.

[0016] As one of the preferred technical solutions, the Fmoc solid-phase synthesis method is used to connect and assemble the amino acid sequence shown in SEQ ID NO. 1.

[0017] As one of the further preferred technical solutions, the Fmoc solid-phase synthesis method is used to connect and assemble the amino acid sequence shown in SEQ ID NO. 1.

[0018] As one of the further preferred technical solutions, after the peptide assembly is completed, it can be removed from the resin by treating it 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.

[0019] HPLC chromatographic conditions:

[0020] 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 is volume percent); flow rate 1.0 mL / min; detection wavelength 214 nm; injection volume 20ul.

[0021] MS mass spectrum conditions: expected MS is 3532.15; flow rate 0.2 mL / min; running time 1 min; buffer A is 0.1% formic acid aqueous solution, buffer B is 0.1% formic acid acetonitrile solution.

[0022] 3. The use of the aforementioned polypeptide capable of directionally enhancing thymus neonatal T cells to inhibit melanoma in the preparation of anti-melanoma drugs.

[0023] The beneficial effects of the present application are:

[0024] The present application prepares a polypeptide capable of directionally enhancing thymus neonatal T cells to inhibit melanoma, and the peptide sequence is NH2-FNNFTVSFWLRVPKVSASHL-GPSL-KDMQLGR-CONH2. The Th cell epitope sequence (FNNFTVSFWLRVPKVSASHL) is from tetanus toxoid 947-967 peptide (TT3), and a four-residue linker (GPSL) is after the T cell epitope. The linker is integrated into the fusion peptide to help the independent folding of the T cell epitope and the B cell epitope. The B cell epitope sequence is the C segment fragment (KDMQLGR) of C5a.

[0025] The polypeptide of the present application can inhibit the growth of melanoma, enhance the function of thymus neonatal T cells, has low toxicity and side effects, and can be applied to the treatment of melanoma, thereby providing a new scheme and new idea for the treatment of melanoma.

[0026] Other advantages, objects, and features of the present application will be apparent to those skilled in the art from the following specification, in conjunction with the accompanying drawings, and to a certain extent, will be apparent to those skilled in the art based on the study of the following, or can be taught from the practice of the present application. The objects and other advantages of the present application can be achieved and obtained by the following specification. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to make the objects, technical solutions and advantages of the present application clearer, the preferred detailed description of the present application will be made below in conjunction with the drawings, wherein:

[0028] Figure 1HPLC analysis results of Max450 peptide

[0029] Figure 2 MS analysis results of Max450 peptide

[0030] Figure 3 Figure of anti-melanoma polypeptide acting on C57BL / 6 mouse in vitro transplanted tumor model results;

[0031] Figure 4 Anti-melanoma polypeptide acting on C57BL / 6 mouse in vitro transplanted tumor model tumor volume;

[0032] Figure 5 Anti-melanoma polypeptide acting on C57BL / 6 mouse in vitro transplanted tumor model tumor weight;

[0033] Figure 6 Anti-melanoma polypeptide acting on C57BL / 6 mouse in vitro transplanted tumor model thymus new T cell changes. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with the specific embodiments.

[0035] Unless otherwise specified, the specific techniques or conditions in the examples are in accordance with the techniques or conditions described in the literature or in accordance with the product specifications. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0036] Example 1:

[0037] Preparation of anti-melanoma polypeptide (Max450 peptide)

[0038] The sequence of the anti-tumor polypeptide peptide segment is NH2-FNNFTVSFWLRVPKVSASHL-GPSL-KDMQLGR-CONH2. The sequence of the Th cell epitope (FNNFTVSFWLRVPKVSASHL) is from the tetanus toxoid 947-967 peptide segment (TT3). After the T cell epitope is 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).

[0039] The preparation method is to sequentially connect and assemble the following peptide segments: (1) Th cell epitope sequence: FNNFTVSFWLRVPKVSASHL; (2) four-residue linker: GPSL; (3) B cell epitope sequence: KDMQLGR.

[0040] 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-fluorenylmethyloxycarbonyl) 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 as shown).

[0041] HPLC chromatographic conditions:

[0042] 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 (0.01min, 5% A 95% B, 25.0min, 75% A 25% B, 30min, 90% A 10% B; the change of mobile phase is volume percentage); flow rate 1.0mL / min; detection wavelength 214nm; injection volume 20ul.

[0043] The molecular weight and composition of the polypeptide are determined by mass spectrometry (MS) (such as Figure 2 as shown), MS mass spectrometry conditions: expected MS is 3532.15; flow rate 0.2mL / min; running time 1min; buffer A is 0.1% formic acid aqueous solution, buffer B is 0.1% formic acid acetonitrile solution.

[0044] Example 2:

[0045] Polypeptide treatment test in mice

[0046] C57BL / 6 mice (Chongqing Tengxin Bi'er Experimental Animal Sales Co., Ltd.) of 6 to 8 weeks old, the mice were subcutaneously injected with B16F10 cells (Wuhan Punuo Sai Life Science and Technology Co., Ltd.) (3*105 100μL) on the right back, and the tumors were formed 4 to 6 days later. The length and width of the tumor were measured every 2 days using a vernier caliper. The tumor volume was calculated as length x width2 / 2 (mm 3 ).

[0047] Mice were randomly assigned to three treatment groups (4 mice per group): PBS treatment group (Ctrl), anti-tumor peptide treatment group (SEQ ID NO: 7, patent CN103269711B), and anti-tumor peptide (Max450) treatment group. On the second day after tumor loading, mice were injected with 1 mg / ml peptide (control group received PBS injection). Dosing was repeated every other day for a total of four doses. Tumor volume was measured 13 days after treatment. The results are shown in Figure 2. Figures 3-5 shown.

[0048] Figure 3 B16F10 cells (3*10 5 Group 1 was treated with PBS, Group 2 was treated with an anti-tumor peptide (SEQ ID NO: 7, patent CN103269711B), and Group 3 was treated with an anti-tumor peptide (Max450). Mice were injected with 1 mg / ml peptide (the control group was injected with PBS) and dosed every other day for a total of four times. Tumors were harvested 13 days after treatment.

[0049] Figure 4 B16F10 cells (3*10 5 100 μL) after administration (n = 4 per group), Group 1 was treated with PBS, Group 2 was treated with anti-tumor peptide (SEQ ID NO: 7, patent CN103269711B), and Group 3 was treated with anti-tumor peptide (Max450). Mice were injected with 1 mg / ml peptide (control group was injected with PBS) and administered every other day for a total of four doses. Tumors were harvested 13 days after treatment, and the length and width of the tumors were measured with calipers. Tumor volume was calculated as length × width. 2 / 2(mm 3 )(n=4 per group).

[0050] Figure 5 B16F10 cells (3*10 5 Group 1 was treated with PBS, Group 2 was treated with an anti-tumor peptide (SEQ ID NO: 7, patent CN103269711B), and Group 3 was treated with an anti-tumor peptide (Max450). Mice were injected with 1 mg / ml peptide (the control group was injected with PBS) and dosed every other day for a total of four doses. Tumors were weighed after 13 days of treatment.

[0051] Compared with the Ctrl group, the anti-tumor polypeptide Max450 can significantly inhibit tumor growth; compared with the anti-tumor polypeptide SEQ ID NO: 7 (patent CN103269711B), the anti-tumor polypeptide Max450 can also significantly inhibit tumor growth, indicating that the anti-tumor polypeptide Max450 has better treatment effect than the anti-tumor polypeptide SEQ ID NO: 7 (patent CN103269711B).

[0052] Figure 6 C57BL / 6 mice were injected subcutaneously with B16F10 cells (3*10 5 100μL) after (n=3 in each group) administration, 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 (Max450). The mice were injected with 1mg / ml polypeptide (the control group was injected with PBS), and the administration was performed every other day, a total of four times. The tumors were taken after 14 days of treatment, and the CD8 + T cells in the tumors were analyzed by flow cytometry.

[0053] 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 Max450 can 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 Max450 group increased, indicating that the thymus newly born T cells increased. The above experiments show that the anti-tumor polypeptide Max450 can effectively treat melanoma.

[0054] 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 equivalently without departing from the purpose and scope of the technical solutions, which should be covered in the scope of the claims of the present application.

Claims

1. A polypeptide that can direct the enhancement of thymic neocortical T cells to suppress melanoma, characterized in that, is assembled by sequentially connecting the following peptide segments: (1) Th cell epitope sequence: FNNFTVSFWLRVPKVSASHL; (2) Four-residue linker: GPSL; (3) B cell epitope sequence: KDMQLGR; The assembled peptide segment sequence is: FNNFTVSFWLRVPKVSASHLGPSLKDMQLGR, as shown in SEQ ID NO.

1.

2. The method of producing a polypeptide that can directionally enhance the thymic neotype T cell suppression of melanoma according to claim 1, characterized in that, is assembled by sequentially connecting the following peptide segments: FNNFTVSFWLRVPKVSASHLGPSLKDMQLGR, 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. The Fmoc solid

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