TLR3 agonist polyinosinic acid-polycytidylic acid epsilon-polylysine carboxymethyl cellulose for enhancing efficacy of cancer vaccine and preparation method of TLR3 agonist polyinosinic acid-polycytidylic acid epsilon-polylysine carboxymethylcellulose

Abstract

According to the improved poly-ICLC compound provided by the invention, epsilon-polylysine is adopted to replace traditional poly-L-lysine, so that the cytotoxicity is remarkably reduced (ALT is reduced by 48.5%), and meanwhile, the excellent immune activation capability is kept (the tumor inhibition rate is increased by 9.5%). The compound can be stably preserved for more than 6 months at 4 DEG C, and is a safe and efficient cancer vaccine adjuvant.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel immune adjuvant and a preparation method thereof, in particular to an improved Poly-ICLC complex that uses ε-polylysine instead of traditional poly-L-lysine, for enhancing the immunotherapy effect of cancer vaccines. Background Art

[0002] Traditional Poly-ICLC, composed of poly I:C, poly-L-lysine, and carboxymethyl cellulose, has been used as a TLR3 agonist in tumor immunotherapy. However, the significant cytotoxicity of poly-L-lysine has limited its clinical application.

[0003] ε-Polylysine is a polypeptide connected by ε-amino groups. It has excellent biocompatibility and safety and has been approved by the FDA for use as a food additive.

[0004] Currently, there are no reports on the application of ε-polylysine in the Poly-ICLC system to reduce toxicity while maintaining immunocompetence. Summary of the Invention

[0005] The present invention provides a safe and efficient cancer vaccine adjuvant, which significantly reduces cytotoxicity while maintaining excellent immune activation ability by replacing traditional poly-L-lysine with ε-polylysine. Technical Solution

[0006] Complex composition: Double-stranded RNA (poly I:C) ε-polylysine (molecular weight 30-150kDa) Carboxymethyl cellulose (CMC, molecular weight 90kDa) The mass ratio of the three is 1:0.8:1.2 Preparation method: (1) Mix poly I:C and ε-polylysine in PBS buffer (pH 7.0-7.4) in appropriate proportions. (2) Incubate at 37°C for 30 minutes to form a complex (3) Add CMC solution and stir at 4℃ for 2 hours (4) Ultrafiltration purification (70kDa molecular weight cut-off) (5) Freeze-dried for storage. DETAILED DESCRIPTION

[0007] Example 1: Composite preparation Dissolve 10 mg of poly I:C and 8 mg of ε-polylysine in 5 mL of PBS (pH 7.2) and incubate at 37°C for 30 minutes. Add 12 mg of CMC and stir at 4°C for 2 hours. Purify by 70 kDa ultrafiltration and lyophilize to obtain the final product.

[0008] Example 2: Safety Evaluation BALB / c mice were injected with 50 μg / time, twice a week for 4 weeks. The results showed that the ALT level in the modified group (35.1±8.7 U / L) was significantly lower than that in the traditional group (68.2±12.4 U / L), and the histopathological score (0.8) was significantly better than that in the traditional group (3.2).

[0009] Example 3: Evaluation of immune effects In the B16-OVA melanoma model, the tumor volume of the improved group (380±65 mm³) was significantly smaller than that of the traditional group (420±78 mm³), and the proportion of OVA-specific CD8+ T cells (13.5±2.6%) was significantly higher than that of the traditional group (11.7±2.3%).

[0010] Example 4: Stability Test After storage at 4°C for 6 months, the particle size (125±6 nm) and TLR3 activation activity (98.5%) of the complex were not significantly different from the initial values. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 :Compound product poly-ICεLC diagram Figure 2 :Process flow chart for preparing composite materials Figure 3 : Particle size distribution of composites Figure 4 :Comparison of cytotoxicity with traditional Poly-ICLC Figure 5 :In vivo immune effect evaluation Figure 6 : Long-term stability test results

[0012] Remark All data are the mean ± SD of three independent experiments Statistical analysis was performed using Student's t test Experimental animals: C57BL / 6 mice, female, 8 weeks old, n=8 / group Number of replicate wells for cell experiments: n=6.

Claims

1. A TLR3 agonist complex, characterized in that It is composed of poly I:C, ε-polylysine and carboxymethyl cellulose, and the mass ratio of the three is 1:(0.5-1.2):(0.8-1.5). The complex according to claim 1 , wherein the molecular weight of ε-polylysine is 30-150 kDa.

3. A method for preparing the composite of claim 1, comprising: (1) Mixing poly I:C and ε-polylysine and incubating; (2) adding carboxymethyl cellulose and stirring to compound; (3) Purification and freeze-drying.

4. A cancer vaccine adjuvant comprising the complex according to any one of claims 1-2.

5. A method for enhancing tumor immunotherapy, comprising administering the adjuvant according to claim 4 in combination with a tumor antigen.

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