Polypeptide combined vaccine as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide combination vaccine as well as a preparation method and application thereof. The polypeptide combination vaccine comprises A02 and A24 polypeptides, a vaccine adjuvant cyclic diguanylate (CDGSF) and a linker L for coupling the polypeptides with the CDGSF. The polypeptide vaccine has good tumor inhibition activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a polypeptide combination vaccine, its preparation method, and its application. Background Technology

[0002] GPC3, or Glypican-3, is a member of the heparan sulfate (HS) proteoglycan family. GPC3 is an extracellular glycoprotein with a relative molecular weight of 66 kDa, anchored to the cell membrane via phosphatidylinositol. It is a multifunctional common receptor on the cell surface, playing a crucial role in mediating signal transduction. GPC3 is also a secretory protein, influencing signal transduction by binding to the extracellular matrix, growth factors, and proteases, thus playing a vital role in regulating tumor cell proliferation, differentiation, adhesion, and metastasis.

[0003] GPC3 is expressed in the liver and kidneys of healthy fetuses, but is almost not expressed in adult tissues except for placental tissue. Furthermore, GPC3 is differentially expressed in various tumor cells, showing high expression in hepatocellular carcinoma (HCC), non-small cell lung cancer, ovarian clear cell carcinoma (OCCA), and gastric cancer (GC), while it is lowly expressed or absent in uterine cancer, malignant mesothelioma, breast cancer, and gastric cancer. Studies have found that GPC3 participates in multiple signaling pathways and plays an important biological role in tumor cell growth. However, the mechanism of action of GPC3 on cancer cell growth is not yet clear, and currently there are three main theories: a) Hedgehog (Hh) signaling pathway: GPC3 can bind to Hh, causing Hh to lose its ability to bind to its receptor Ptc. Ptc inhibits the activity of Smo protein, thereby inhibiting downstream pathways; b) Wnt signaling pathway: GPC3 overexpression can upregulate c-Myc expression, a protein in the classic Wnt signaling pathway, and c-Myc can also increase GPC3 expression at the transcriptional level. Studies have shown that mutated GPC3 can block the Wnt signaling pathway and inhibit Wnt-dependent tumor growth; c) FGF2 signaling pathway: through immunoprecipitation, studies have found that GPC3 can bind to FGF2, and it is believed that GPC3 can inhibit the activity of FGF2 and BMP-7, thereby inhibiting the growth of liver cancer. Therapeutic liver cancer peptide vaccines based on GPC3 protein are under development. Among them, the GPC3 vaccine developed based on the HLA-A24-restricted CTL epitope GPC3 298-306 peptide and the HLA-A2-restricted epitope GPC3 144-152 peptide has entered clinical trials. Experimental results have demonstrated that the GPC3 peptide vaccine can prolong the time to recurrence and improve overall survival (OS), and may have an adjuvant therapeutic effect for hepatocellular carcinoma patients who have undergone surgical resection or radiofrequency ablation. Therefore, the research and development of safe, reliable, and effective GPC3 peptide vaccines has profound significance for the treatment of liver cancer. However, the GPC3-A2 and GPC3-A24 epitopes lack strong immunogenicity and cannot activate the immune system to produce a strong anti-tumor immunotherapy effect. Adjuvants enhance the adaptive immunity of vaccines by activating innate immune cells, thereby improving the immunotherapeutic effect of anti-tumor vaccines. While simply mixing antigens and adjuvants can achieve some immune enhancement, covalently linking antigens and adjuvants to construct vaccine molecules allows for the simultaneous delivery of both antigens and adjuvants, maximizing their combined effect.

[0004] The interferon-stimulated gene (STING) is an important cytoplasmic sensor expressed in the endoplasmic reticulum of various immune cells and other cells. Upon binding to its stimulators, STING induces the expression of immune molecules such as type I interferon, initiating the body's immune response. Cyclic dinucleotides (CDNs) are natural agonists of STING, interacting with it and activating immune cells. Some CDN derivatives can also activate the STING pathway and induce immune cell activation. Activated immune cells produce type I interferon, which promotes the rapid clearance of pathogens and facilitates antigen presentation by APCs, sensitizing effector T cells and thus killing tumor cells. Therefore, STING stimulators play a crucial role in anti-tumor therapy.

[0005] Designing synthetic antigen-adjuvant covalent vaccines can deliver both antigens and adjuvants simultaneously, eliciting a more effective immune response, thereby promoting tumor killing and enhancing the efficacy of tumor immunotherapy. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide combination vaccine, its preparation method, and its application.

[0007] To achieve the objectives of this invention, the following implementation scheme is provided.

[0008] In one embodiment, a polypeptide combination vaccine of the present invention comprises A02-L-CDG SF or / and A24-L-CDG SF ,in, A02 is a polypeptide with the following amino acid sequence: SEQ ID NO:1 Cys-Phe-Val-Gly-Glu-Phe-Phe-Thr-Asp-Val, A24 is a polypeptide with the following amino acid sequence: SEQ ID NO:2 Cys-Glu-Tyr-Ile-Leu-Ser-Leu-Glu-Glu-Leu, CDG SF As a vaccine adjuvant, L is a linker connecting the polypeptide and the adjuvant.

[0009] Terminology: CDG is an abbreviation for c-di-GMP (cyclic diguanosine monophosphate), which belongs to the class of cyclic dinucleotides (CDNs) and is used as a vaccine adjuvant. Cyclic dinucleotides are natural agonists of STING, which can interact with STING and cause activation of immune cells.

[0010] CDG SFThis refers to the substitution of one hydroxyl group on the sugar of cyclic diguanosine monophosphate by F, and the substitution of one -OH group on the phosphate group by -SH. Its chemical structural formula is as follows: .

[0011] Preferably, the polypeptide combination vaccine of the present invention comprises A02-L-CDG. SF and A24-L-CDG SF .

[0012] Preferably, in the polypeptide combination vaccine of the present invention, L is selected from compounds shown in L1, L2, and L3.

[0013] Preferably, in the polypeptide combination vaccine of the present invention, L is the compound shown in L3.

[0014] In some embodiments, the polypeptide combination vaccine of the present invention, wherein the A02-L-CDG SF The compounds are selected from the following: , and

[0015] In some embodiments, the above-described polypeptide combination vaccine of the present invention, wherein the A24-L-CDG SF The compounds are selected from the following: , and .

[0016] In another embodiment, the present invention also provides a method for preparing a polypeptide vaccine, comprising the following steps: 1) Prepare peptides A02 or A24 using the Fmoc solid-phase synthesis method; 2) Linker L is connected to adjuvant CDG via a thioether bond. SF L-CDG preparation SF ; 3) Relate A02 or A24 to L-CDG via a thioether bond. SF Connect to obtain A02-L-CDG SF Or A24-L-CDG SF , The definitions of A02, A24 and L are the same as those described above.

[0017] In yet another embodiment, the use of the polypeptide combination vaccine of the present invention in the preparation of a tumor treatment vaccine is also provided. Preferably, the tumor is hepatocellular carcinoma (HCC), non-small cell lung cancer, ovarian clear cell carcinoma (OCCA), or a special type of gastric cancer (GC).

[0018] A compound or its medicinal salt, selected from: , , , , and .

[0019] The polypeptide combination vaccine of the present invention combines polypeptides A02 and A24 with adjuvant CDG via a linker. SF After conjugation, its tumor-suppressive activity was significantly higher than that of a physical mixture of peptide and adjuvant. In particular, the combined use of peptides A02 and A24 produced a synergistic effect, exhibiting surprisingly significant anti-tumor activity, especially against liver cancer. Attached Figure Description

[0020] Figure 1 The bar chart shows the in vitro cell activation capacity of the peptide adjuvant conjugate in Example 2. Figure 2 The bar chart for the activity of antigen-specific TNFα+ T cells induced by the peptide adjuvant conjugate in Example 3 is shown. Figure 3 The graph shows the effective prolongation of survival of H22 tumor-bearing mice by subcutaneous injection of peptides and peptide adjuvant conjugates in each experimental group of Example 4. Figure 4 The graph shows the time curves of the effective inhibition of tumor growth in H22 tumor-bearing mice by subcutaneous injection of peptides and peptide adjuvant conjugates in each experimental group of Example 4. Figure 5 This is a time curve of mouse body weight change during subcutaneous injection of peptides and peptide adjuvant conjugates in each experimental group of Example 4. Figure 6 The bar graph shows the activation of antigen-presenting cells and T cells in mouse lymph nodes by the polypeptide adjuvant conjugate combination of Example 5. Detailed Implementation

[0021] The following examples are typical and are used to further understand the essence of the present invention, but are not intended to limit the scope of the present invention.

[0022] Example 1 A02-L-CDG SF and A24-L-CDG SF Preparation A02 and A24 polypeptide compounds with purities greater than 95% were prepared and purified by the Fmoc solid-phase synthesis method. The amino acid sequence of A02 is: Cys-Phe-Val-Gly-Glu-Phe-Phe-Thr-Asp-Val(CFVGEFFTDV); The amino acid sequence of A24 is: Cys-Glu-Tyr-Ile-Leu-Ser-Leu-Glu-Glu-Leu (CEYILSLEEL).

[0023] The compounds shown by linkers L1, L2, and L3 were respectively coupled with the STING agonist-CDG. SF L1-CDG was prepared by linking thiol groups to the thiol group (prepared according to the method in CN111592570). SF L2-CDG SF L3-CDG SF ; Mix A02 or A24 with L1-CDG respectively SF L2-CDG SF L3-CDG SF The target compound A02-L1-CDG was obtained by sulfhydryl linkage of cysteine ​​(Cys). SF A02-L2-CDG SF A02-L3-CDG SF A24-L1-CDG SF A24-L2-CDG SF A24-L3-CDG SF The specific preparation process is as follows: 1. L1-CDG SF L2-CDG SF Preparation of compounds Linker L1 or L2 (2.0 eq) was dissolved in DMF, and adjuvant CDG was added dropwise under ice bath cooling. SF The DMF solution was reacted in an oil bath at 40°C for 3 hours, quenched with water, and then purified by Shimadzu Semiconductor to obtain a white solid L1-CDG. SF or L2-CDG SF .

[0024] 2. L3-CDG SF Preparation of compounds Linker L3 (2.0 eq) was dissolved in DMF, and adjuvant CDG was added dropwise under ice bath cooling. SF The DMF solution was mixed with PBS buffer to make a DMF:PBS ratio of 3:1 (V:V). The mixture was reacted in an oil bath at 50°C for 1 hour. After quenching with water, the mixture was purified semi-preparatively to obtain a white solid, L3-CDG. SF .

[0025] 3. A02-L1-CDGSF A02-L2-CDG SF Preparation of compounds Peptide A02 (2.0 eq) was dissolved in DMF, and L1-CDG was added dropwise while cooling in an ice bath. SF or L2-CDG SF The reaction was carried out overnight in a DMF solution at 40°C in an oil bath. After quenching with water, the mixture was purified to obtain a white solid. The purified target compound was designated A02-L1-CDG. SF Or A02-L2-CDG SF .

[0026] 4. A24-L1-CDG SF A24-L2-CDG SF Preparation of compounds Peptide A24 (2.0 eq) was dissolved in DMF, and L1-CDG was added dropwise while cooling in an ice bath. SF or L2-CDG SF The reaction was carried out overnight in a DMF solution at 40°C in an oil bath. After quenching with water, the mixture was purified to obtain a white solid. The purified target compound was designated A24-L1-CDG. SF A24-L2-CDG SF .

[0027] 5. A02-L3-CDG SF Or A24-L3-CDG SF Preparation of compounds Peptide A24 or A02 (2.0 eq) was dissolved in DMF, and compound L3-CDG was added dropwise under ice bath cooling. SF The reaction was carried out in DMF solution at 40°C in an oil bath for 2 hours. After quenching with water, the mixture was purified by semi-preparative methods to obtain a white solid, A02-L3-CDG. SF Or A24-L3-CDG SF .

[0028] L-CDG prepared by the methods described in 1-5 above SF A02-L-CDG SF A24-L-CDG SF Purification was performed using a column chromatography method. The chromatographic conditions were mobile phase A (0.06% TFA-water) and mobile phase B (0.06% TFA-ACN), with gradient elution. The column type, chromatographic conditions, and gradient program are shown in Table 1.

[0029] Table 1. Column Types and Gradient Programs

[0030] The purity of the obtained compounds was determined by HPLC and the results were analyzed by mass spectrometry. A02-L1-CDG SF : Purity 95.46%, ESI-MS of A02-L1-CDG SF . Calculated for C 85 H 113 FN 22 O 30 P2S2[M+H] + : 2067.70; ESI found: [M+2H] 2+ : 1029.55; A24-L1-CDG SF : Purity 95.10%, ESI-MS of A24-L1-CDG SF . Calculated for C 84 H 125 FN 22 O 33 P2S2[M+H] + : 2115.78; ESI found: [M+2H] 2+ : 1058.65, [M+3H] 3+ : 706.25; A02-L2-CDG SF : Purity 96.62%, ESI-MS of A02-L2-CDG SF . Calculated for C 83 H 107 FN 20 O 33 P2S2[M+H] + : 2058.63; ESI found: [M+2H] 2+ : 1034.60, [M+3H] 3+ : 690.20; A24-L2-CDG SF : Purity 96.15%, ESI-MS of A24-L2-CDG SF . Calculated for C 82 H 119 FN 20 O 36 P2S2[M+H] + : 2105.71; ESI found: [M+2H] 2+: 1053.60, [M+3H] 3+ 703.00; A02-L3-CDG SF Purity: 95.46% ESI-MS of A02-L3-CDG SF Calculated for C 100 H 129 FN 24 O 33 P2S2[M+H] + : 2340.82; ESI found: [M+2H] 2+ : 1171.00, [M+3H] 3+ 780.95; A24-L3-CDG SF Purity 97.24%, ESI-MS of A24-L3-CDG SF Calculated for C 99 H 141 FN 24 O 36 P2S2[M+H] + : 2388.90; ESI found: [M+2H] 2+ : 1195.25, [M+3H] 3+ : 797.00.

[0031] Example 2: Peptide vaccine activates immune cell activity Two antigen-presenting cell lines (APCs), RAW264.7 (mouse macrophages) and J774A.1 (mouse macrophages), were selected to investigate the immune activation capacity of the vaccine. J774A.1 or RAW264.7 cells were seeded at a density of 400,000 per well in 24-well plates. After cell attachment, 500 μL of complete culture medium containing the following different forms of polypeptide compounds was added via medium replacement. 2.5μM A02 + 2.5μM A24 5μM CDG SF , 2.5μM A02 + 2.5μM A24 + 5μM CDG SF , 2.5μM A02-L1-CDG SF +2.5 μM A24-L1-CDG SF , 2.5μM A02-L2-CDG SF +2.5 μM A24-L2-CDG SF , 2.5 μM A02-L3-CDG SF +2.5 μM A24-L3-CDG SF .

[0032] Cells were cultured at a constant temperature of 37°C and in an incubator containing 5% CO2 for 24 hours. Cells were then collected using Accutase cell digestion solution, stained with antibodies for 45 min, and the expression levels of cell surface activation markers (such as CD86, CD80, and CD40) were measured by flow cytometry. Results are shown below. Figure 1 .

[0033] The results showed that the immune activation capacity of different conjugates was evaluated using two types of antigen-presenting cells: RAW264.7 cells and J774A.1 cells. Overall, the peptide adjuvant conjugates significantly improved the activation level of cells compared with direct physical mixing of peptides and adjuvants, and significantly improved the activation level of the intracellular STING pathway and the expression level of cell surface co-stimulatory molecules.

[0034] Example 3: Liver cancer vaccine activates memory T cell activity in mice Set up a PBS blank group, The peptide group consisted of 5 nmol A02 + 5 nmol A24. The peptide aluminum adjuvant group consists of 5 nmol A02 + 5 nmol A24 + 25 μL aluminum hydroxide adjuvant. Peptide CDG mixture: 5 nmol A02 + 5 nmol A24 + 10 nmol CDG SF , Peptide CDG conjugate 5 nmol A02-L3-CDG SF +5 nmol A24-L3-CDG SF .

[0035] Five groups of samples were used to immunize 6-week-old BALB / c mice, with immunization occurring every two weeks for a total of two immunizations. Seven days after the second administration, the mice were sacrificed, and their spleens were harvested. Splenic lymphocyte suspensions were obtained by grinding, filtering, cleavage, and centrifugation in a clean bench. Subsequently, the cells were cultured at 1 × 10⁻⁶ cells / mL. 7Cells were dispersed into 24-well plates at varying densities. Blank and antigen peptide groups were added to each well. Additionally, the protein secretion inhibitors Brefeldin (1:1000 dilution) and Monensin (1:1000 dilution) were added to each well. The plates were incubated for 8 hours, followed by antibody staining and flow cytometry analysis to detect TNFα (tumor necrosis factor α) expression levels in CD4+ T cells and CD8+ T cells. Results are shown below. Figure 2 .

[0036] Experimental results showed that after two doses of the peptide adjuvant conjugate, compared with other experimental groups, the spleen tissue of mice in the peptide CDG conjugate group showed a higher intensity of antigen-specific CD4+ T cells and CD8+ T cells. These results demonstrate that the peptide adjuvant conjugate combination (5 nmol A02-L3-CDG) effectively inhibited the formation of antigen-specific CD4+ T cells and CD8+ T cells. SF +5 nmol A24-L3-CDG SF This drug elicited a strong antigen-specific TNFα+ T cell response in mice, demonstrating excellent ability to induce cellular immune responses. T cells play a crucial role in tumor killing, and the combination of peptide adjuvant CDG conjugates effectively induces antigen-specific CD4+ T cells and CD8+ T cells. Antigen-specific T cells can directly kill tumors by secreting TNFα, which is essential for establishing an anti-tumor immune cycle.

[0037] Example 4 Evaluation of the anti-tumor effect of the vaccine The H22 mouse liver cancer model was selected as the experimental tumor model. Initially, tumor cells cultured in 2 million ascites fluid samples were subcutaneously implanted into the backs of BALB / c mice. When the tumor reached a suitable size, the mice were randomly divided into 5 groups: a blank control group, a peptide group, a peptide-aluminum group, a peptide-CDG mixed group, and a peptide-CDG conjugate group (treatment group). The mice were administered PBS, 5 nmol A02 + 5 nmol A24, 5 nmol A02 + 5 nmol A24 + 25 μL aluminum hydroxide adjuvant, and 5 nmol A02 + 5 nmol A24 + 10 nmol CDG, respectively, each time. SF and 5 nmol A02-L3-CDG SF +5 nmol A24-L3-CDG SF The drug was administered subcutaneously every three days for a total of three administrations. During treatment, tumor volume, body weight, and survival rate of the mice were monitored every two days. Results are shown below. Figure 3-5 .

[0038] Experimental results show that the combination of peptide adjuvant conjugates (5 nmol A02-L3-CDG) is effective. SF +5 nmol A24-L3-CDGSF The combination significantly inhibited the tumor growth rate of H22 hepatocellular carcinoma mice and effectively prolonged the survival of the mice, demonstrating excellent anti-tumor activity.

[0039] Example 5: Analysis of in vivo lymph node immune cell activation Lymph nodes are important immune organs, serving as crucial sites for antigen presentation and T cell activation. To analyze the immune response of tumor-bearing mice after treatment with antigen adjuvant conjugates, we analyzed the activation status of lymph node cells in mice after three intratumoral administrations. The following adjuvants were used: PBS, 5 nmol A02 + 5 nmol A24, 5 nmol A02 + 5 nmol A24 + 25 μL aluminum hydroxide adjuvant, and 5 nmol A02 + 5 nmol A24 + 10 nmol CDG. SF and 5 nmol A02-L3-CDG SF +5 nmol A24-L3-CDG SF Five groups of samples were used to immunize H22 tumor-bearing mice, with immunization occurring every three days for a total of three immunizations. Twenty-four hours after the third administration, mice were sacrificed, and tumor drainage lymph nodes were harvested. Lymphocyte single-cell suspensions were obtained through grinding, filtration, and centrifugation. After antibody staining and washing with PBS, flow cytometry was performed. Results are shown below. Figure 6 .

[0040] Flow cytometry results showed that after administration of the antigen adjuvant conjugate, CD69, an early activation marker on the surface of T cells (CD4+ T cells and CD8+ T cells), was significantly upregulated in the tumor draining lymph nodes (proximal to the tumor). The levels of co-stimulatory molecules CD80, CD86, and CD40 on the surface of dendritic cells (CD11c cells) and giant cells (F4 / 80* cells) were also significantly upregulated. Significant activation was observed in T cells, dendritic cells, and other cells, indicating that treatment with the antigen adjuvant conjugate may have elicited a dual response of innate and adaptive immunity, effectively leading to highly efficient activation of antigen-presenting cells and T cells in mouse lymph nodes.

Claims

1. A polypeptide combination vaccine comprising A02-L-CDG SF or / and A24-L-CDG SF ,in, A02 is a polypeptide, and its amino acid sequence is shown in SEQ ID NO:

1. A24 is a polypeptide, and its amino acid sequence is shown in SEQ ID NO:

2. CDG SF As a vaccine adjuvant, L is a linker connecting the polypeptide and the adjuvant.

2. The polypeptide combination vaccine as described in claim 1, comprising A02-L-CDG SF and A24-L-CDG SF .

3. The polypeptide combination vaccine of claim 1, wherein L is selected from L1, L2, and L3: 。 4. The polypeptide combination vaccine as described in claim 3, wherein L is L3.

5. The polypeptide combination vaccine as described in claim 1, wherein the A02-L-CDG SF Selected from the following compounds: , and .

6. The polypeptide combination vaccine of claim 1, wherein the A24-L-CDG SF Selected from the following compounds: , and .

7. A method for preparing a polypeptide vaccine, comprising the following steps: 1) Prepare peptides A02 or A24 using the Fmoc solid-phase synthesis method; 2) Linker L is connected to adjuvant CDG via a thioether bond. SF L-CDG preparation SF ; 3) Relate A02 or A24 to L-CDG via a thioether bond. SF Connect to obtain A02-L-CDG SF Or A24-L-CDG SF , The definitions of A02, A24 and L are the same as those in claims 1 and 3.

8. Use of the polypeptide combination vaccine according to any one of claims 1-6 in the preparation of a cancer treatment vaccine.

9. The use as described in claim 8, wherein the tumor is liver cancer, non-small cell lung cancer, clear cell ovarian cancer, or a special type of gastric cancer.

10. A compound or a pharmaceutical salt thereof, selected from: , , , , and .

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