Double adjuvant-neoantigen tumor nanometer vaccine, and preparation method and application thereof

A nano-vaccine and antigen technology, which is applied in the field of preparation of tumor nano-vaccine drugs, can solve the problems of water solubility and pharmacokinetic limitation of joint use, and achieve the effect of inhibiting tumor cell growth, clear and simple synthesis steps, and activating immune response

Inactive Publication Date: 2020-04-28
SHANGHAI THERANOSTICS BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, differences in water solubility and pharmacokinetics limit the combined use of different types of vaccine drugs

Method used

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  • Double adjuvant-neoantigen tumor nanometer vaccine, and preparation method and application thereof
  • Double adjuvant-neoantigen tumor nanometer vaccine, and preparation method and application thereof
  • Double adjuvant-neoantigen tumor nanometer vaccine, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Preparation and related characterization of DNA primer-PEG-PLA nanoparticles

[0066] Take 50 μl of DNA primer template with a concentration of 120 μM (the 5’ end is modified with a disulfide bond), add 10 μl of dithiothreitol (DTT) at a concentration of 1 M, add 10 μL of 10× phosphate buffered saline solution and 30 μL of DEPC Water was used to prepare a 100 μL reaction system. Stir at 37°C for 1 hour. Prepare one NAP 5 purification column, wash it with ascorbate 3 times, after the above stirring, mix 100 μL of the stirred product and 400 μL phosphate-buffered saline into the NAP 5 purification column, wait until the 500 μL solution is exhausted, and then add Add 1 mL of phosphate-buffered saline to the purification column, and collect 1 mL of the filtered liquid to obtain DNA primers modified with sulfhydryl groups at the 5' ends. Subsequently, the concentration of polyethylene glycol-polylactic acid (MAL-PEG3000-PLA2000) with terminally modified maleamid...

Embodiment 2

[0067] Example 2: Preparation and Characterization of CpG Microparticles and Small Molecule R848 Loading

[0068]Take 20 μL of the final product of Example 1 (1.2 μM DNA primer concentration), add 1 μL (1 μM) of CpG or GpC single-stranded template, and add 4 μL 10×T4 ligase buffer solution and 15 μL aqueous solution to configure a 40 μL reaction system. The 40 μL reaction solution was heated at 95° C. for 5 minutes and gradually cooled to 25° C. within 1.5 hours to complete the complementary hybridization of the DNA primer fragment and the CpG (GpC) template fragment. The hybridized product was reacted with 1 μL of T4 ligase (20000 U / μL) at room temperature for 2 hours to seal the gap of the CpG (GpC) ring-mounted template. Then the circular DNA product was mixed with DNA polymerase (12μL, 2U / μL), dNTP (20μL, 2mM) and BSA (0.8μL, 100×), and left to react at 30°C for 2 days (Note: when synthesized To label CpG microparticles of fluorescent molecules, add 3 μL of Cy5-UTP to the...

Embodiment 3

[0070] Embodiment 3: Polypeptide-g-polyethylene glycol (PPT-g-PEG) synthesis

[0071] Synthesis of γ-propargyl-L-glutamic acid hydrochloride

[0072] Take a 500 mL round bottom flask, and disperse L-glutamic acid (6.0 g, 40.8 mmol) in 200 mL propargyl alcohol. Under the protection of nitrogen at room temperature, chlorotrimethylsilane (14.3 mL, 113 mmol) was added dropwise into the round bottom flask within 2 hours, and the stirring was continued for two days. After the reaction, the above reaction product was precipitated with diethyl ether (1.5 L) to obtain a black solid. The solid was first washed with methanol (50 mL), and then precipitated with diethyl ether (0.5 mL). This was repeated three times to purify the product. The product was filtered and dried under vacuum to obtain γ-propargyl-L-glutamic acid hydrochloride (4.71 g, yield: 52%) as a white solid. For its structural characterization see image 3 . Synthesis of γ-propargyl-L-glutamic acid N-carboxyanhydride (...

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Abstract

The invention provides a nanometer vaccine. The nanometer vaccine is a nanoparticle simultaneously loaded with tumor neoantigen and two adjuvants thereof. The invention also provides a composition used for preparing the nanometer vaccine. The composition comprises a micromolecular adjuvant R848, an amphiphilic diblock polymer containing a polyethylene glycol chain segment and with terminal modified with a maleamide bond, a nucleic acid adjuvant CpG, polyethylene glycol modified polypeptide and a tumor neoantigen. The nanometer vaccine disclosed by the invention has a significant immune activation effect and a good antigen-specific tumor inhibition effect in vivo and in vitro. The invention also provides a method for preparing the nanometer vaccine and an application of the nanometer vaccine in preparation of tumor vaccine drugs.

Description

technical field [0001] The invention belongs to the technical field of nano-medicines, in particular to a preparation method and application of tumor nano-vaccine drugs. Background technique [0002] Malignant tumors are one of the important causes of morbidity and mortality worldwide. Nearly one in six deaths worldwide is due to cancer, with approximately 70% of cancer deaths occurring in low- and middle-income countries. The immune system plays a vital role in maintaining human health. The function of the immune system in the prevention and treatment of major diseases such as HIV, atherosclerosis, autoimmune diseases and tumors has been extensively studied. Among them, the rise of the field of tumor immunotherapy has brought new hope to tumor treatment. Various immunotherapeutic drugs at the cellular or molecular level developed by interfering with each step of the tumor immune cycle have entered clinical trials, among which immune checkpoint inhibitors and CAR-T cell t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/39A61K47/60A61K47/64A61K47/69A61P35/00A61P37/04
CPCA61K39/00A61K39/39A61K47/60A61K47/64A61K47/6937A61K47/6935A61P35/00A61P37/04A61K2039/6093A61K2039/55511A61K2039/55516A61K2039/55561A61K2300/00
Inventor 陈小元倪倩倩
Owner SHANGHAI THERANOSTICS BIOTECH CO LTD
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