Immunogenic peptide group with function of activating anti-tumor immune response as well as preparation method and application of immunogenic peptide group

Abstract

The invention discloses a group of immunogenic peptides with an anti-tumor immune response activating function as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The amino acid sequences of the immunogenic peptide are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and the immunogenic peptide is obtained by the following steps: extracting an enterovirus-like particle (VLPs) preparation from excrement of a healthy mouse, giving the preparation to a tumor model mouse through intragastric administration, and separating and screening from ileum tissues of the mouse by using a liquid chromatography-mass spectrometry technology. The VLPs can be migrated to tumor tissues and presented as the immunogenic peptide through tumor cell MHC-I molecules, so that CD8 + T cells are efficiently activated, a tumor immune microenvironment is remodeled, and tumor growth is inhibited. Experiments show that the immunogenic peptide shows a remarkable anti-tumor effect when being used alone or combined with a PD-1 inhibitor, and the tumor inhibition rate of drug combination can reach 89.4% and is remarkably superior to that of single treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a group of immunogenic peptides with the function of activating anti-tumor immune responses, their preparation methods, and applications. Background Technology

[0002] Malignant tumors pose a serious threat to human health, and immune checkpoint blockade therapy, represented by PD-1 / PD-L1 inhibitors, has brought revolutionary breakthroughs to cancer treatment. However, in clinical practice, a significant proportion of patients do not respond to this therapy initially or develop secondary resistance, limiting its efficacy. Studies have shown that one of the core reasons for this is that some patients lack a sufficient number of effector CD8 receptors in their tumor microenvironment that can specifically recognize tumor antigens. + T cells, thus forming a so-called "immune desert" or "immune rejection" phenotype. In this situation, even if PD-1 / PD-L1 inhibitors are used to relieve T cell suppression signals, it is difficult to generate an effective anti-tumor immune response due to the lack of "soldiers" that can be activated.

[0003] To improve the response rate of immunotherapy, clinicians often attempt to combine PD-1 / PD-L1 inhibitors with chemotherapy, radiotherapy, or other immunotherapies. However, traditional radiotherapy and chemotherapy have significant toxic side effects, affecting patients' quality of life and tolerability; the synergistic effects of existing combination strategies are still not ideal, and may bring additive toxic side effects.

[0004] Immunogenic peptide-based tumor therapeutic vaccines are theoretically an ideal solution. They aim to activate and expand tumor-specific T cells by delivering specific antigenic epitopes that can be recognized by T cells, thereby providing mobilized effector cells for immune checkpoint therapy. However, existing peptide vaccine technologies face two major bottlenecks: first, weak immunogenicity—short peptides are easily and rapidly degraded and cleared in vivo, making them difficult to be effectively captured and presented by antigen-presenting cells, resulting in limited immune response strength; second, a lack of clear synergistic effects with PD-1 / PD-L1 inhibitors—currently, there are few reports of screening for peptides that can efficiently activate CD8+. + T cells, and immunogenic peptides that can significantly enhance the efficacy of PD-1 / PD-L1 inhibitors.

[0005] Therefore, there is an urgent need in this field to develop novel, efficient immunogenic peptides that can synergize with existing immune checkpoint therapies to address the treatment challenges of "immune desert" tumors and improve the overall response rate and efficacy of immunotherapy. Summary of the Invention

[0006] This invention involves extracting enteric virus-like particles (VLPs) from the feces of healthy mice, administering them via gavage to tumor model mice, and then screening a group of immunogenic peptides from their ileum tissue using liquid chromatography-mass spectrometry (LC-MS / MS). The amino acid sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. These peptides can efficiently activate CD8+. + T cells, and when used in combination with PD-1 inhibitors, exhibit a significant synergistic anti-tumor effect.

[0007] On the one hand, the present invention provides a group of immunogenic peptides with the function of activating anti-tumor immune responses, using the following technical solution: A group of immunogenic peptides that activate anti-tumor immune responses, wherein the amino acid sequence is selected from at least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0008] Preferably, the amino acid sequences of the immunogenic peptides are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0009] On the other hand, the present invention also provides a method for preparing a group of immunogenic peptides with the function of activating anti-tumor immune responses, using the following technical solution: A method for preparing a group of immunogenic peptides with the function of activating anti-tumor immune responses includes the following steps: S1. Extracting VLPs preparations from the feces of healthy mammals; S2. Administer VLPs formulation to tumor-bearing animal models; S3. Immunogenic peptides are isolated and screened from the ileum tissue of the animal described in step S2.

[0010] Preferably, step S1 specifically includes: collecting feces from healthy mammals, homogenizing and centrifuging to obtain the supernatant, filtering it through a filter membrane, adding polyethylene glycol to the filtrate to incubate and form a precipitate, collecting the precipitate and resuspending it to obtain the VLPs formulation.

[0011] Preferably, the membrane filtration includes sequentially passing through 0.45 μm and 0.22 μm filter membranes; the polyethylene glycol is PEG 6000.

[0012] Preferably, in step S3, the MHC-peptide complex is obtained from the tumor tissue by immunoprecipitation, the peptide fragments are eluted, and then the immunogenic peptides are identified and screened by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0013] Preferably, the immunogenic peptide is synthesized using a solid-phase peptide synthesis method or a gene engineering expression method.

[0014] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an immunogenic peptide and a pharmaceutically acceptable carrier.

[0015] Preferably, it also contains a PD-1 inhibitor and / or an immune adjuvant.

[0016] Preferably, the use of an immunogenic peptide in the preparation of a medicament for the treatment and / or prevention of tumors.

[0017] In summary, the beneficial effects of the present invention are as follows: This invention provides a group of immunogenic peptides with the function of activating anti-tumor immune responses. Its core beneficial effect lies in the efficient activation and amplification of tumor-specific CD8. + T cells effectively address the "immune desert" problem that leads to the ineffectiveness of PD-1 / PD-L1 therapy. Experiments have demonstrated that these peptides can significantly reshape the tumor immune microenvironment and directly induce a potent anti-tumor T cell immune response.

[0018] Furthermore, the immunogenic peptides of this invention, when combined with PD-1 inhibitors, exhibit remarkable synergistic effects. In animal models, the combination therapy achieved a tumor inhibition rate as high as 89.4%, significantly superior to any single therapy. This provides a promising new combination therapy strategy for improving the response rate and efficacy of immune checkpoint blockade therapy in clinical practice. Attached Figure Description

[0019] Figure 1 This is a comparison of PyMT-MMTV tumor changes in the control group, CFVT group, and TFVT group in Example 1; Figure 2 This is a comparison of B16F10 tumor changes in the IgG group, PD-1 group, and PD-1+CFVT group in Example 2; Figure 3 Experimental procedures for preparing immunogenic peptides and validating their functions; Figure 4 This is a graph showing the results of the de novo immunogenicity test. Figure 5 This is a graph showing the results of an in vitro immunogenicity test. Figure 6 This is a schematic diagram of the experimental scheme for Example 4; Figure 7 Figure showing experimental results of the PyMT-MMTV tumor model; Figure 8 Figure showing experimental results of the KPC tumor model; Figure 9 The figure shows the experimental results of the B16F10 tumor model. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example

[0022] Example 1

[0023] The preparation and verification of the antitumor effect of FVT are carried out through the following steps: S1. Five healthy SPF-grade C57BL / 6 female mice aged 6-8 weeks were housed individually in sterile metabolic cages. Fresh fecal particles produced by these mice were collected and rapidly stored at -80°C. 1 g of mixed fecal sample was weighed and 10 mL of pre-cooled SM buffer (50 mM Tris-HCl, 100 mM NaCl, 8.5 mM MgSO4, pH 7.5) was added, and the mixture was vortexed for 5 minutes to homogenize thoroughly. The mixture was centrifuged at 4000×g for 5 minutes at 4°C, and the supernatant was collected. The supernatant was then centrifuged at 13500×g for 10 minutes to remove bacteria and debris, and filtered through a 0.45 μm filter membrane for sterilization. Four volumes of 20% PEG 6000 (w / v) were added to the filtrate, and the mixture was incubated at 4°C for 2 hours to precipitate virus particles. The precipitate was then collected by centrifugation at 21000×g for 1 hour. The precipitate was resuspended in 1 mL of SM buffer and 50 μL of 1M NaHCO3 was added to neutralize gastric acid, yielding a fecal virus graft (FVT) preparation, designated as healthy mouse-derived FVT (CFVT). Absolute counting of virus-like particles (VLPs) was performed using SYBR Green I staining and flow cytometry, and the concentration was adjusted to 1 × 10⁻⁶. 11 VLPs / mL, aliquoted and stored at -80℃.

[0024] S2, 5×10 5 One PyMT-MMTV breast cancer cell was mixed with matrix gel at a 1:1 volume ratio and injected orally into the fourth mammary fat pad of female mice. The tumor volume was approximately 100 mm². 3 At times, such as Figure 1 As shown, mice were randomly divided into three groups (n=8): (1) SM buffer control group: each mouse was gavaged with 200 μL of SM buffer once every 3 days; (2) TFVT group: an equal volume of tumor-bearing mouse-derived FVT was gavaged (prepared in the same way as healthy FVT); (3) CFVT group: an equal volume of healthy mouse-derived FVT (1×10⁻⁶) was gavaged. 11 VLPs / session, once every 3 days). Treatment continued for 21 days, with tumor volume measured every 3 days.

[0025] Experimental results of the PyMT-MMTV tumor model are as follows: Figure 1As shown, compared with the SM control group and the TFVT group, tumor growth in the CFVT-treated group was significantly inhibited ( P The tumor inhibition rate reached 62.3% (<0.05), indicating that the prepared CFVT preparation can significantly inhibit breast cancer growth through oral gavage. Its effect may be related to promoting cytotoxic T cell infiltration and inhibiting immunosuppressive cells.

[0026] Example 2

[0027] Analysis of the synergistic effect of CFVT combined with anti-PD-1 antibody in the treatment of breast cancer, pancreatic cancer, and melanoma, with specific steps as follows: C57BL / 6 mice were used to establish PyMT-MMTV breast cancer, KPC pancreatic cancer, and B16F10 melanoma-bearing mouse models. Five × 10⁵ PyMT-MMTV breast cancer cells were mixed with matrix gel (1:1 volume ratio) and injected orally into the right fourth mammary fat pad of 6-8 week old female C57BL / 6 mice at a total volume of 100 µL. Alternatively, 3 × 10⁵ KPC cells or 5 × 10⁵ B16F10 melanoma cells were subcutaneously seeded in the right posterior ventral region of the mice. Tumors were allowed to develop until the tumor volume reached approximately 80 mm². 3 At that time, the mice were randomly divided into the following three treatment groups (n=10): (1) treated with IgG isotype control antibody (BioXcell, BE0089) and SM buffer; (2) anti-PD-1 monotherapy group: treated with anti-PD-1 antibody and SM buffer; (3) combination therapy group: treated with anti-PD-1 antibody and CFVT. CFVT was administered by gavage at a dose of 200 μL (containing 2×10¹⁰ VLPs) every 3 days; anti-PD-1 antibody and isotype control antibody were administered by intraperitoneal injection at a dose of 100 μg / mouse on days 8, 11 and 14. Tumor growth and mouse survival were monitored.

[0028] Experimental results are as follows Figure 2 As shown, the left-hand figure compares tumor weight in the control group, the anti-PD-1 monotherapy group, and the combination therapy group. The combination therapy group showed a highly significant tumor inhibition rate (e.g., P <0.05). Secondly, physical photographs of the three types of tumors clearly show that the tumors in the combination therapy group have almost disappeared or are the smallest in size, while the tumors in the anti-PD-1 monotherapy group, although somewhat inhibited, are far less than those in the combination group.

[0029] In summary, the combination of CFVT and anti-PD-1 antibody shows a significant synergistic anti-tumor effect and can greatly prolong survival. The mechanism may be related to viral translocation and T cell activation.

[0030] Example 3

[0031] The specific steps for preparing immunogenic peptides and validating their functions are as follows: S1. To further investigate the mechanism of action of the prepared CFVT, MHC molecules were immunoprecipitated from mouse tumor tissues in the CFVT group using MHC immunopeptidomics analysis. The presented peptides were eluted and identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) as a group of 11 high-affinity immunogenic peptides, named P1-P11. These 11 peptides were then synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. using a solid-phase synthesis method.

[0032] S2. In the in vitro peptide immunogenicity analysis, naïve CD8 was isolated from the spleen of C57BL / 6 mice. + T cells were co-cultured for 6 days with bone marrow-derived dendritic cells (BMDCs) treated with 20 μg / mL peptide at a ratio of 10:1 (containing 25 U / mL interleukin-2 (IL-2)). IFNγ-secreting T cells were detected using the IFNγ ELISpot assay kit (Mabtech, 3321-4HST-2) according to the manufacturer's instructions. The stained spots were counted using an ELISpot reader (CTL, S6 ULTRA).

[0033] In S3, in vitro peptide immunogenicity analysis, C57BL / 6 mice were inoculated with a peptide vaccine containing Poly(I:C) adjuvant (50 μg each time) (100 μg each time) on days 1 and 6, respectively. CD8 was used. + T-cell isolation kit (Yimo Biotechnology, IMP-MK017) for isolating CD8 cells from the spleen of pre-immunized mice. + T cells. Obtained CD8 + T cells were used in a 5:1 ratio with antigen-specific dendritic cells (10 per well). 4 Cells were restimulated for 24 hours in RPMI 1640 medium containing 25 U / mL IL-2 (Novoprotein, CK24). IFNγ-secreting T cells were detected using the IFNγ ELISpot assay kit (Mabtech, 3321-4HST-2) according to the manufacturer's instructions. The stained spots were counted using an ELISpot reader (CTL, S6 ULTRA). The experimental procedure is as follows: Figure 3 As shown.

[0034] The result of step S1 is as follows Figure 4 As shown, in the in vitro peptide immunogenicity analysis, obvious colored spots appeared in the wells stimulated by P1, P2, P3, P4, P6, P7, P8, P9, and P11. The results of step S2 are as follows. Figure 5As shown, in the in vitro peptide immunogenicity analysis, obvious stained spots appeared in the wells stimulated by P2, P3, P4, P8, and P9. Among them, the number of cells forming the spot secreted by P8 for IFN-γ reached (285±21) / 10. 5 CD8 + T cells were significantly higher than those in the control group (32±5) / 10 5 Cells ( P The value <0.001 indicates that P8 has strong immunogenicity and can effectively activate CD8. + T cells.

[0035] Example 4

[0036] The specific steps for verifying the in vivo antitumor activity of immunogenic peptides are as follows: C57BL / 6 mice were selected, such as Figure 6 As shown, PyMT-MMTV breast cancer, KPC pancreatic cancer, and B16F10 melanoma-bearing mouse models were established on day 0, respectively. 5 × 10 5 One PyMT-MMTV breast cancer cell was mixed with matrix gel (1:1 volume ratio) and injected orally into the right fourth mammary fat pad of 6-8 week old female C57BL / 6 mice at a total volume of 100 µL. Alternatively, 3 × 10⁶ cells were subcutaneously injected into the right posterior ventral side of the mouse. 5 One KPC cell or 5 × 10 5 B16F10 melanoma cells were administered subcutaneously on days 6, 9, and 12. The cells were treated with a combination of P2, P3, P4, P8, and P9 peptides, Poly(I:C) adjuvant, and anti-PD-1 antibody. The peptide dose was 100 μg / cell, and the Poly(I:C) adjuvant dose was 50 μg / cell. Tumor growth was monitored during these treatments.

[0037] The results are as follows Figure 7 , Figure 8 , Figure 9 Immunogenic peptides P2, P4, P8, and P9 significantly delayed the growth of two or more mouse tumor models. Among them, the combined treatment with the P2 immunogenic peptide achieved a tumor inhibition rate of (54.6±4.8)% in PyMT-MMTV breast cancer (compared to the blank control group). P <0.01). Among the immunogenic peptides presented by CFVT to tumor cells, four peptides showed inhibitory effects on two or more tumor models in both in vitro and ex vivo experiments, demonstrating high immunogenicity. The amino acid sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0038] Table 1. Amino acid sequences of the four peptides

[0039] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A group of immunogenic peptides with the function of activating anti-tumor immune responses, characterized in that, Its amino acid sequence is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4.

2. The immunogenic peptide according to claim 1, characterized in that, The amino acid sequences of the immunogenic peptides are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4.

3. A method for preparing the immunogenic peptide according to claim 1 or 2, characterized in that, Includes the following steps: S1. Extracting VLPs preparations from the feces of healthy mammals; S2. The VLPs formulation is administered to tumor-bearing animal models; S3. Immunogenic peptides are isolated and screened from the ileum tissue of the animal described in step S2.

4. The preparation method according to claim 3, characterized in that, Step S1 specifically includes: collecting feces from healthy mammals, homogenizing the feces, centrifuging to obtain the supernatant, filtering the supernatant through a filter membrane, adding polyethylene glycol to the filtrate to incubate and form a precipitate, collecting the precipitate and resuspending it to obtain the VLPs formulation.

5. The preparation method according to claim 4, characterized in that, The membrane filtration process involves sequentially passing through 0.45 μm and 0.22 μm membranes; the polyethylene glycol is PEG 6000.

6. The preparation method according to claim 3, characterized in that, In step S3, MHC-peptide complexes are obtained from ileal tissue by immunoprecipitation, peptide fragments are eluted, and then the immunogenic peptides are identified and screened by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

7. A method for preparing the immunogenic peptide of claim 1 or 2, characterized in that, The immunogenic peptides are synthesized using solid-phase peptide synthesis or genetic engineering expression.

8. A pharmaceutical composition, characterized in that, It comprises a therapeutically effective amount of the immunogenic peptide as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, It also contains PD-1 inhibitors and / or immune adjuvants.

10. The use of an immunogenic peptide as described in claim 1 or 2 in the preparation of a medicament for treating and / or preventing tumors.

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