A tumor vaccine containing a polysaccharide adjuvant and use thereof

Abstract

The application relates to the field of biological medicines, and discloses a tumor vaccine containing a polysaccharide adjuvant and application thereof. The tumor vaccine formula comprises, in terms of mass fractions, 100 parts of WT1-CM-betaG NPs, 10 parts of astragalus polysaccharide, 5 parts of quillaja saponin and 1 part of a TLR agonist. The WT1-CM-betaG NPs comprise, in terms of mass fractions, 10 parts of a WT1 polypeptide combination, 50 parts of carboxymethylated beta-glucan nanoparticles CM-betaG NPs, 10 parts of EDC and 6 parts of NHS. The WT1 polypeptide is covalently connected to the carboxymethylated beta-glucan nanoparticles through chemical coupling, and then cooperates with the adjuvant, has a synergistic effect on improving the ability of stimulating DC maturation (high expression of CD80 / CD86 / MHC-II) and improving the secretion of key Th1 type cytokine IL-12p70, can improve the antitumor effect, and has a wide application in treating WT1 positive tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a tumor vaccine containing a polysaccharide adjuvant and its application. Background Technology

[0002] Cancer therapeutic vaccines aim to activate a patient's own immune system to recognize and attack tumor cells. Their core components include tumor antigens and immune adjuvants. In recent years, natural polysaccharides have become a hot topic in novel adjuvant research due to their unique immunomodulatory properties and good biocompatibility. However, current research and applications often use polysaccharide adjuvants in their natural form (such as lentinan and astragalus polysaccharide injections) after simple physical mixing with antigens. This approach has significant drawbacks: antigens and adjuvants may not be distributed synchronously in the body, making it impossible to ensure uptake by the same antigen-presenting cells (APCs), severely limiting the efficiency of immune response initiation. Soluble polysaccharides and antigens are easily and rapidly cleared by the body, lacking targeting specificity, resulting in low lymph node accumulation and requiring frequent, high-dose injections, which not only increases costs but may also introduce systemic inflammation risks. Many studies on the mechanisms of action of polysaccharide adjuvants remain at the level of holistic observation (such as enhancing lymphocyte proliferation), lacking in-depth understanding and engineered utilization of how they precisely regulate key aspects such as dendritic cell (DC) maturation and antigen cross-presentation. For tumor-associated antigens (TAAs) such as WT1, the challenge lies in breaking immune tolerance. Existing adjuvant systems are often insufficient to achieve this efficiently, resulting in weak vaccine immunogenicity and limited clinical efficacy.

[0003] The market needs an "integrated intelligent immune activation platform" that combines targeted delivery, synergistic activation, and process optimization. The WT1-CM-βG NPs of this invention address this problem. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a tumor vaccine containing a polysaccharide adjuvant and its application, which can achieve targeted delivery, synergistic activation, and process optimization.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A tumor vaccine containing a polysaccharide adjuvant comprises, by weight parts: 100 parts WT1-CM-βG NPs, 10 parts Astragalus polysaccharide, 5 parts saponins, and 1 part TLR agonist; wherein the WT1-CM-βG NPs comprise, by weight parts: 10 parts WT1 polypeptide combination, 50 parts carboxymethylated β-glucan nano-CM-βG NPs, 10 parts EDC, and 6 parts NHS.

[0007] The aforementioned tumor vaccine containing a polysaccharide adjuvant comprises, by weight parts: peptide 1: 2.5 parts, peptide 2: 2.5 parts, and peptide 3: 5.0 parts;

[0008] The peptide 1 is: RMFPNAPYL SEQ ID No: 01;

[0009] The peptide 2 is: CMTWNQMNL SEQ ID No: 02;

[0010] Peptide 3 is: PGCNKRYFKLSHLQMHSRK SEQ ID No: 03.

[0011] The aforementioned tumor vaccine containing a polysaccharide adjuvant, the carboxymethylated β-glucan nanoparticles CM-βG NPs, comprises, by weight, 10 parts yeast β-glucan CM-βG and 12 parts sodium chloroacetate.

[0012] The aforementioned tumor vaccine containing polysaccharide adjuvant, the preparation method of carboxymethylated β-glucan nanoparticles CM-βG NPs is as follows: 10 parts of yeast β-glucan are placed in a container, isopropanol is added as a dispersion medium, and the mixture is stirred and suspended; an alkaline solution is added dropwise for alkalization; 12 parts of sodium chloroacetate are dissolved in ultrapure water and added dropwise to the reaction system; after heating and reaction, the mixture is purified to obtain yeast β-glucan CM-βG; then, after treatment with an ultrasonic cell disruptor and a high-pressure homogenizer, CM-βG nanoparticles CM-βG NPs are obtained by filtration.

[0013] The aforementioned tumor vaccine containing polysaccharide adjuvant, WT1-CM-βG NPs, is prepared as follows: 50 parts of the prepared CM-βG NPs are taken, 10 parts of EDC and 6 parts of NHS are added, and the reaction activates the carboxyl groups on the surface of CM-βG NPs; then WT1 polypeptide combination is added, the pH is adjusted to 6.5-7.5, and after the reaction, WT1-CM-βG NPs are obtained by purification.

[0014] The aforementioned tumor vaccine containing a polysaccharide adjuvant includes TLR agonists comprising one or more of the following: imidazoquinoline (IMDQ), CpGOligodeoxynucleotides (CpG-ODN), Poly(I:C), LPS (lipopolysaccharide), or MPL (monophosphoryl lipid A).

[0015] The aforementioned tumor vaccine containing a polysaccharide adjuvant has a TLR agonist of Poly(I:C) (polyinosinic acid).

[0016] The aforementioned tumor vaccine containing polysaccharide adjuvant has WT1-CM-βG NPs with a particle size of 100-200 nm.

[0017] The aforementioned tumor vaccine containing a polysaccharide adjuvant has a particle size of 150 ± 20 nm and a PDI < 0.2.

[0018] The aforementioned application of a tumor vaccine containing a polysaccharide adjuvant is as follows: it is used as a carrier for therapeutic vaccines for treating WT1-positive tumors, personalized neoantigen vaccines, in combination with PD-1 / PD-L1 antibodies, or for immune monitoring and diagnosis.

[0019] The advantages of this invention are:

[0020] This invention fundamentally solves the defects of physical mixing by chemically coupling WT1 peptides to carboxymethylated β-glucan nanoparticles. This design ensures that the antigen and adjuvant are engulfed by APCs as a whole, achieving precise "co-delivery" and greatly improving the efficiency of antigen processing and presentation, which is key to inducing a strong T cell response.

[0021] This invention enhances lymph node targeting and accumulation: Polysaccharides are made into nanoparticles of ~150 nm, which perfectly utilizes the physiological characteristics of the body's lymphatic system; particles of this size can be efficiently drained from the injection site to the lymph nodes and captured in large quantities by the dendritic cells (DCs) therein, thereby significantly reducing the dosage and reducing systemic exposure side effects.

[0022] Carboxymethylated β-glucan nanoparticles and saponins have a synergistic effect on enhancing the ability to stimulate DC maturation (high expression of CD80 / CD86 / MHC-II) and enhancing the secretion of the key Th1 cytokine IL-12p70, thereby improving the anti-tumor effect of the formulation of the present invention.

[0023] Carboxymethylation modification not only introduces carboxyl groups that facilitate chemical coupling, but also significantly improves the water solubility and formulation stability of β-glucan, making it more in line with the basic requirements of industrial pharmaceuticals for active pharmaceutical ingredients.

[0024] Definition of the noun:

[0025] EDC (Ethyldimethylaminopropyl Carbodiimide), also known as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, is an organic compound with the chemical formula C8H10H2O. 17 N 3。

[0026] NHS (N-Hydroxy succinimide) is a white to off-white crystalline solid.

[0027] Poly I:C is a synthetic double-stranded ribonucleic acid, an interferon inducer, and has antiviral and immunomodulatory functions. Detailed Implementation

[0028] The present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1:

[0030] Formula: 100 parts WT1-CM-βG NPs, 10 parts Astragalus polysaccharide, 5 parts Saponins, 1 part TLR agonist; WT1-CM-βG NPs by weight include: 10 parts WT1 polypeptide combination, 50 parts carboxymethylated β-glucan nano-CM-βG NPs, 10 parts EDC, 6 parts NHS. Carboxymethylated β-glucan nano-CM-βG NPs by weight include: 10 parts yeast β-glucan CM-βG, 12 parts sodium chloroacetate.

[0031] Samples were prepared according to the following method:

[0032] Step 1: Carboxymethylation of yeast β-glucan;

[0033] Principle: βG-OH + NaOH + ClCH2COONa → βG-O-CH2COONa + NaCl + H2O;

[0034] Reaction system;

[0035] Weigh 1.0 g of yeast β-glucan (YBG) and place it in a 100 mL round-bottom flask;

[0036] Add 20 mL of isopropanol as a dispersion medium and stir to suspend;

[0037] Add 5 mL of 8 M NaOH solution dropwise and alkalize at 30°C for 1 hour;

[0038] Weigh 1.2 g of sodium chloroacetate (molar ratio: YBG repeating unit: ClCH2COONa ≈ 1:1.2), dissolve it in 5 mL of ultrapure water, and slowly add it dropwise to the reaction system;

[0039] The reaction temperature was raised to 60°C and the reaction was continued for 4 hours.

[0040] Purification and post-processing;

[0041] After the reaction was complete, neutralize with 0.5 M HCl to pH ~7.0;

[0042] The residual salts, isopropanol, and byproducts were removed by thorough dialyzing with ultrapure water at MWCO 3.5 kDa for 72 hours.

[0043] The dialyzed product was freeze-dried to obtain a white flocculent solid CM-βG, which was stored at -20°C.

[0044] Step 2: Preparation of CM-βG nanoparticles;

[0045] Solution preparation: Dissolve 100 mg of CM-βG prepared in step one in 20 mL of ultrapure water to prepare a 5 mg / mL solution. Slight heating or sonication may be used to aid dissolution.

[0046] Pretreatment: First, use an ultrasonic cell disruptor (300W power, 5s on, 5s off, total time 5min) for initial dispersion;

[0047] High-pressure homogenization: The solution was transferred to a high-pressure homogenizer and homogenized 10 times at a pressure of 15,000 psi under ice bath conditions;

[0048] Filtration: The homogenized suspension was filtered through a 0.45 μm filter membrane to remove any small amount of large particles that may be present.

[0049] Characterization: Immediately take a small sample and measure its hydration kinetic diameter and polydispersity index (PDI) using DLS. Target: Particle size 150 ± 20 nm, PDI < 0.2.

[0050] Step 3: Covalent coupling of WT1 peptide;

[0051] Principle: EDC / NHS catalyzes the formation of amide bonds between carboxyl and amino groups;

[0052] CM-βG-COOH + Pep-NH2 → CM-βG-CONH-Pep;

[0053] Activated carboxyl group:

[0054] Take 10 mL of the CM-βG NPs solution prepared in step two (5 mg / mL, total 50 mg CM-βG).

[0055] Add 10 mg EDC and 6 mg NHS (molar ratio EDC:NHS:COOH≈ 2:1:1).

[0056] The carboxyl groups on the surface of CM-βG NPs were activated by gently stirring the reaction at room temperature for 15 minutes.

[0057] Coupled peptides:

[0058] Weigh out three WT1 peptides (Pep1: 2.5 mg, Pep2: 2.5 mg, Pep3: 5.0 mg) and dissolve them in 1 mL of PBS (pH 7.4). (Approximately 5-10 times molar excess).

[0059] Peptide 1: RMFPNAPYL SEQ ID No: 01; Peptide 2: CMTWNQMNL SEQ ID No: 02;

[0060] Peptide 3: PGCNKRYFKLSHLQMHSRK SEQ ID No: 03;

[0061] The peptide solution was added dropwise to the activated CM-βG NPs suspension;

[0062] Carefully adjust the pH of the reaction system to 6.5-7.5 using 0.1 M NaOH;

[0063] Mild, light-protected reaction at room temperature for 4 hours;

[0064] purification;

[0065] Transfer the reaction solution to a dialysis bag (MWCO 50 kDa) and dialyze with PBS (pH 7.4) for 24 hours, changing the solution every 4 hours to completely remove unreacted EDC / NHS and free peptides. The purified product is WT1-CM-βG NPs. Store at 4°C for short periods or freeze-dry for long-term storage.

[0066] Step 4: Preparation of the final vaccine formulation;

[0067] Preparation of adjuvant solution: Dissolve 100 mg WT1-CM-βG NPs, 10 mg Astragalus polysaccharide, 5 mg saponin QS-21, and 1 mg Poly(I:C) in 1 mL sterile PBS. Gently vortex to mix; no filtration is required. Store the final preparation at 4°C protected from light.

[0068] Step 5, Verification:

[0069] Bioactivity was verified by in vitro DC activation assay: DCs were co-incubated with mouse bone marrow-derived DCs for 24 h, CD80 / CD86 / MHC-II expression was detected by flow cytometry, and IL-12p70 secretion was detected by ELISA.

[0070] Comparative Example 1:

[0071] Formula: 100 parts WT1-CM-βG NPs, 5 parts saponins, 1 part TLR agonist; WT1-CM-βG NPs by weight include: 10 parts WT1 polypeptide combination, 50 parts carboxymethylated β-glucan nano-CM-βG NPs, 10 parts EDC, 6 parts NHS. Carboxymethylated β-glucan nano-CM-βG NPs by weight include: 10 parts yeast β-glucan CM-βG, 12 parts sodium chloroacetate. Astragalus polysaccharide is missing compared to Example 1.

[0072] Comparative Example 2:

[0073] Formula: 100 parts WT1-CM-βG NPs, 10 parts Astragalus polysaccharide, 1 part TLR agonist; WT1-CM-βG NPs by weight include: 10 parts WT1 polypeptide combination, 50 parts carboxymethylated β-glucan nano-CM-βG NPs, 10 parts EDC, 6 parts NHS. Carboxymethylated β-glucan nano-CM-βG NPs by weight include: 10 parts yeast β-glucan CM-βG, 12 parts sodium chloroacetate. Compared to Example 1, it lacks saponins.

[0074] Comparative Example 3

[0075] Formulation: 100 parts WT1 peptide combination, 10 parts Astragalus polysaccharide, 1 part TLR agonist. Compared with Example 1, this formulation lacks β-glucan carboxymethylated nanoparticles and steps one to three, directly mixing the WT1 peptide combination with the excipients.

[0076] Comparative Example 4:

[0077] Formulation: 100 parts WT1-CM-βG NPs, 5 parts saponins, 1 part TLR agonist; WT1-CM-βG NPs by weight include: 10 parts WT1 polypeptide combination, 50 parts yeast β-glucan CM-βG, 10 parts EDC, and 6 parts NHS. Compared to Example 1, this formulation lacks carboxymethylation of β-glucan nanoparticles and also lacks astragalus polysaccharides.

[0078] Comparative Example 5:

[0079] Formulation: 100 parts WT1-CM-βG NPs, 10 parts Astragalus polysaccharide, 1 part TLR agonist; WT1-CM-βG NPs by weight include: 10 parts WT1 polypeptide combination, 50 parts β-glucan nano-βG NPs, 10 parts EDC, and 6 parts NHS. Compared to Example 1, the β-glucan nano-NPs lack carboxymethylation and saponins.

[0080] Comparative Example 6:

[0081] Formulation: 100 parts WT1-CM-βG NPs, 10 parts Astragalus polysaccharide, 5 parts Saponins, 1 part TLR agonist; WT1-CM-βG NPs by weight include: 10 parts WT1 polypeptide combination, 50 parts β-glucan nano-βG NPs, 10 parts EDC, and 6 parts NHS. Compared to Example 1, the β-glucan nano-NPs lack carboxyl methylation.

[0082] Effect verification experiment:

[0083] Experiment: Evaluation of the in vitro maturation and activation efficacy of WT1-CM-βG NPs nanovaccine in dendritic cells (DCs).

[0084] I. Experimental Materials and Reagents

[0085] Cell source: femur and tibia of 6-8 week old female C57BL / 6 mice; Cytokines: recombinant mouse GM-CSF (rmGM-CSF) and recombinant mouse IL-4 (rmIL-4).

[0086] Experimental Groups:

[0087] Blank control group: Complete RPMI-1640 medium;

[0088] Positive control group: 1 μg / mL lipopolysaccharide (LPS, a classic strong DC activator);

[0089] Experimental Group 1: Formulation of Example 1 (containing APS, QS-21, TLR agonist, and nanoparticles);

[0090] Experimental Group 2: Comparative with the formulation of Example 1 (lacking Astragalus polysaccharides);

[0091] Experimental Group 3: Comparative formulation of Example 2 (lacking saponins);

[0092] Experimental Group 4: Comparative Example 3 formulation (physical mixing group);

[0093] Experimental Group 5: Comparative formulation of Example 4 (using β-glucan nanoparticles lacking carboxymethylation and also lacking astragalus polysaccharide);

[0094] Experimental Group 6: Comparative formulation of Example 5 (using β-glucan nanoparticles lacking carboxymethylation and saponins);

[0095] Experimental Group 7: Comparative formulation of Example 6 (using β-glucan nanoparticles lacking carboxymethylation);

[0096] Adjuvant control group: Blank CM-βG NPs (uncoupled peptides containing TLR agonists);

[0097] Flow cytometry antibodies: Anti-mouse CD11c-APC, CD80-FITC, CD86-PE, MHC-II-PerCP-Cy5.5;

[0098] ELISA kit: Mouse IL-12p70 ELISA detection kit.

[0099] Instruments and equipment: CO2 cell culture incubator, biosafety cabinet, flow cytometer, enzyme-linked immunosorbent assay (ELISA) reader, centrifuge.

[0100] II. Experimental Procedure

[0101] Mouse bone marrow-derived dendritic cells (BMDCs) were induced and cultured for 6 days. The femur and tibia of mice were aseptically harvested, and the bone marrow cavity was washed with culture medium to obtain a bone marrow cell suspension. Red blood cells were removed with erythrocyte lysis buffer, and the cells were resuspended in PBS. The suspension was then incubated at 2 × 10⁻⁶ cells / day. 6 Cells were seeded at a density of 1 / well in 6-well plates using complete medium containing 20 ng / mL rmGM-CSF and 10 ng / mL rmIL-4. The plates were incubated at 37°C in a 5% CO2 incubator, with half the medium replaced every two days, replenished with fresh medium containing cytokines.

[0102] On day six, group stimulation was performed: immature BMDCs were collected, counted, and resuspended. (1 × 10⁻⁶) 6 Cells were seeded at a density of 5 μg / mL in 24-well plates. Following the grouping described above, the appropriate stimulant was added to each group (all experimental groups were added at a final WT1 peptide concentration of 5 μg / mL to ensure comparable stimulation intensity). Cells were then incubated in an incubator for 24 hours.

[0103] Sample collection and testing on day 7: Flow cytometry detection of surface molecules: Cells from each well were collected and washed twice with pre-cooled PBS. Fc blocking agent (anti-CD16 / 32 antibody) was added and incubated for 15 minutes to block non-specific binding. A mixture of CD11c, CD80, CD86, and MHC-II fluorescent antibodies was added according to the manufacturer's instructions and incubated in the dark for 30 minutes. The cells were washed twice with PBS and resuspended in flow cytometry loading buffer. For flow cytometry detection, the CD11c-positive DC cell population was first identified, and then the mean fluorescence intensity (MFI) of CD80, CD86, and MHC-II in this population and the percentage of positive cells were analyzed. The data results are shown in Table 1.

[0104] ELISA detection of cytokine secretion: While collecting cells, the cell culture supernatant from each well was collected and stored at -80°C. Following the instructions of the mouse IL-12p70 ELISA kit, the concentration (pg / mL) of IL-12p70 in each supernatant sample was measured.

[0105] IV. Experimental Results and Analysis

[0106] 1. Flow cytometry results:

[0107] Table 1

[0108]

[0109] The above experiments show that the formulation of this invention has a similar effect to LPS, a strong activator of DCs, on CD80, CD86, and MHC-II in the population cells, and even shows a significantly better effect on MHC-II⁺ cells. Comparison of the experimental results of Comparative Examples 2, 5, and 6 shows that the absence of QS-21 alone, or the use of unmethylated β-glucan nanoparticles, results in a lower DC cell activation effect. However, the simultaneous absence of both significantly reduces the DC cell activation effect. Comparison of the experimental results of Comparative Examples 1, 4, and 6 shows that the simultaneous absence of both APS and methylated β-glucan nanoparticles does not significantly reduce the DC cell activation effect, and is essentially the same as the effect of lacking either one. Therefore, it can be concluded that QS-21 and carboxymethylated β-glucan nanoparticles have a synergistic effect in improving DC cell activation in the formulation of this invention.

[0110] The results of the ELISA assay for cytokine secretion are shown in Table 2:

[0111] Table 2

[0112]

[0113] The above experiments show that the IL-12p70 secretion level in Example 1 reached 1585.7 pg / mL, which is higher than that of the LPS positive control and significantly higher than any other experimental group. Comparison of the experimental results of Examples 2, 5, and 6 shows that the absence of QS-21 alone, or the use of unmethylated β-glucan nanoparticles, results in a lower DC cell activation effect. However, the simultaneous absence of both significantly reduces the DC cell activation effect. Conversely, comparison of the experimental results of Examples 1, 4, and 6 shows that the simultaneous absence of both APS and methylated β-glucan nanoparticles does not significantly reduce the DC cell activation effect, and is essentially the same as the effect of lacking either one. Therefore, it can be concluded that the carboxymethylated β-glucan nanoparticles and saponin (QS-21) have a synergistic effect in driving DC secretion, maximizing the secretion of key Th1 polarizing cytokines by DCs.

[0114] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A tumor vaccine containing a polysaccharide adjuvant, characterized in that, The composition comprises, by weight, 100 parts WT1-CM-βGNPs, 5 parts saponin QS-21, and 1 part TLR agonist. The preparation method of WT1-CM-βG NPs is as follows: 50 parts of the prepared CM-βG NPs are added to 10 parts EDC and 6 parts NHS to activate the carboxyl groups on the surface of CM-βG NPs; then, a WT1 polypeptide combination is added, the pH is adjusted to 6.5-7.5, and the mixture is purified after reaction to obtain WT1-CM-βG NPs. The preparation method of the carboxymethylated β-glucan nano-CM-βG NPs is as follows: 10 parts of yeast β-glucan are placed in a container, isopropanol is added as a dispersion medium, and the mixture is stirred and suspended; an alkaline solution is added dropwise for alkalization; 12 parts of sodium chloroacetate are dissolved in ultrapure water and added dropwise to the reaction system; after heating and reaction, the mixture is purified to obtain yeast β-glucan CM-βG; then, CM-βG is obtained by filtration after treatment with an ultrasonic cell disruptor and a high-pressure homogenizer. NPs; The WT1 polypeptide combination comprises, by weight parts: peptide 1: 2.5 parts, peptide 2: 2.5 parts, peptide 3: 5.0 parts; The peptide 1 is: RMFPNAPYL SEQ ID No: 01; The peptide 2 is: CMTWNQMNL SEQ ID No: 02; Peptide 3 is: PGCNKRYFKLSHLQMHSRK SEQ ID No:

03.

2. The tumor vaccine containing a polysaccharide adjuvant according to claim 1, characterized in that, The product also includes, by weight, 10 parts of Astragalus polysaccharide.

3. A tumor vaccine containing a polysaccharide adjuvant according to claim 1, characterized in that, The TLR agonists include one or a combination of several of the following: imidazoquinoline, CpG oligodeoxynucleotides, Poly(I:C), lipopolysaccharide, or monophospholipid A.

4. A tumor vaccine containing a polysaccharide adjuvant according to claim 3, characterized in that, The TLR agonist is Poly(I:C).

5. A tumor vaccine containing a polysaccharide adjuvant according to claim 1, characterized in that, The particle size of the WT1-CM-βGNPs is 100-200 nm.

6. A tumor vaccine containing a polysaccharide adjuvant according to claim 5, characterized in that, Particle size 150 ± 20 nm, PDI < 0.2.

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