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Cationic liposome vaccine as well as preparation method and application thereof

A cationic liposome and cationic lipid technology, applied in the field of biomedicine, can solve the problems of low immunogenicity, disappearance, and high cost of tumors, and achieve inhibition of tumor growth and spread, activation and proliferation, and promotion of recognition and presentation Effect

Pending Publication Date: 2022-07-01
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] (1) Tumor cells are mainly targeted, and tumor-associated or specific antigens need to be screened; however, some dominant tumor antigens on the surface of tumor cells will disappear during immunotherapy, resulting in immunotherapy resistance
[0006] (2) Tumor samples obtained by biopsy are not necessarily representative; it is difficult to guarantee the reliability of epigenetics after gene transcription, translation and modification
[0007] (3) Standardized production is difficult and costly
[0009] (5) The therapeutic effect is limited by the tumor immunosuppressive microenvironment
[0010] The main challenge now is the tumor immunosuppressive microenvironment and the low immunogenicity of the tumor, making it difficult to generate sufficient intensity of tumor-specific CD8 + T immune response

Method used

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  • Cationic liposome vaccine as well as preparation method and application thereof
  • Cationic liposome vaccine as well as preparation method and application thereof
  • Cationic liposome vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0045] Preparation Example 1: Preparation of fusion protein 1 (rTCS-LEG(rTL))

[0046] Prokaryotic expression and purification of TCS-antigen peptide protein

[0047] a: The plasmid pET28a-TCS-legumain was transformed into E. coli BL21(DE3) competent cells.

[0048] b: Take 100 μL of transformed strains into 2 mL of LB medium containing 50 μg / mL Amp, cultivate at 37°C, 250 rpm for 3-4 h, and cultivate to logarithmic growth phase (absorbance at 600 nm is 0.6-0.8), adding the final concentration of 1 mM The IPTG was expressed overnight (16h) at 25°C, 150rpm.

[0049] c: Freeze centrifugation (9000rpm, 4°C, 3min) to collect the cells.

[0050] d: The bacteria were resuspended with HEPES buffer (containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5‰ Tween 20, pH 8.5).

[0051] e: Probe ultrasonic instrument to break the bacteria (over 3s, stop for 3s, power 400W, 40min, ice bath), until the bacteria liquid is clear and translucent.

[0052] f: centrifugal treatment of bacterial ...

preparation Embodiment 2

[0057] Preparation Example 2: cy5-labeled recombinant trichosanthin-antigen peptide vaccine

[0058] 1) cy5-NHS ester labeled rTL

[0059] Weigh a certain amount of cy5 and dissolve it in ultrapure water. According to the molar concentration ratio of rTL:cy5=1:3, slowly drop the cy5 solution into rTL, vortex while adding dropwise, and place it at 4°C to react overnight in the dark. , and then separated with a desalting column to remove unreacted cy5. The collected rTL-cy5 was ultrafiltered with an ultrafiltration tube with a molecular weight cut-off of 10 kDa, concentrated to 1.5 mL, and the amount of cy5 was determined by a fluorescence spectrophotometer, and the device was stored at -20 °C for use.

[0060] 2) rTL-cy5 standard curve

[0061] Fluorescence quantification of rTL-cy5 was performed by spectrofluorophotometry. Aspirate 50 μL of rTL-cy5 with a micropipette and dilute to 2 mL with water. After gradient dilution, the cy5 fluorescence intensity of samples with dif...

preparation Embodiment 3

[0062] Preparation Example 3: Preparation of Cationic Liposome Vaccine

[0063] 1) Preparation of cationic liposome membranes

[0064] Weigh 20 mg of soybean lecithin SPC-100, dissolve with chloroform, ultrasonicate for 10 min to make the final concentration 20 mg / mL; weigh 5 mg of cholesterol, dissolve in chloroform, ultrasonicate for 10 min, the final concentration is 5 mg / mL, weigh DOTAP 10 mg, dissolved in chloroform to a final concentration of 1 mg / mL. Take 1 mL of soybean phospholipid SPC-100 solution, add 1 mL of cholesterol solution, 0.4 mL of DOTAP solution, sonicate in a water bath for 20 seconds, mix well, add to a 250 mL round-bottomed flask, and spin dry the above solvent with a rotary evaporator at 37 °C , put it in a vacuum drying box to dry overnight to form a cationic liposome membrane.

[0065] 2) Add 1 mL of rTCS-LEG aqueous solution, add steel balls to hydrate for 10 min at room temperature, and continue to add 4 mL of ultrapure water to fully hydrate for...

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Abstract

The invention relates to a cationic liposome vaccine as well as a preparation method and application thereof. A fusion protein of an immunologic adjuvant and an antigen peptide, particularly a fusion protein of recombinant trichosanthin and legumain polypeptide, is entrapped in a cationic liposome to prepare the cationic liposome vaccine. Experiments prove that after local immunization is carried out on a mouse by using the liposome vaccine, on one hand, APCs can be effectively recruited, lymph node targeting can be realized through a size effect of nanoparticles, APCs in lymph nodes are sensitized, and recognition and presentation of antigens are promoted, so that activation and proliferation of antigen-specific T cells are promoted; and on the other hand, the TAM and the tumor immune microenvironment are regulated and controlled to promote the inhibitory microenvironment to realize'immune normalization ', so that the anti-tumor immunotherapy effect is achieved.

Description

technical field [0001] The present invention belongs to the field of biomedicine, and more particularly, the present invention relates to a trichosanthin-antigen peptide co-delivery cationic liposome vaccine and a preparation method and application thereof. Background technique [0002] Lung cancer is a common cancer model with high new morbidity and mortality in my country, and its five-year survival period is short. The occurrence and development of tumors are related to abnormal autoimmune functions. Tumor tissue is composed of tumor cells and their complex tumor microenvironment. Tumor cells adapt to the components of the microenvironment to form an immunosuppressive tumor microenvironment that is conducive to their occurrence and development. The immunotherapy strategy based on targeting and regulating the tumor microenvironment can "train" the autoimmune system, so that the body in a suppressed state can achieve "immunity normalization", so as to achieve the purpose of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/39A61K9/127A61P35/00
CPCA61K39/001154A61K39/39A61K9/127A61K47/28A61P35/00A61K2039/55516
Inventor 汤懿斯顾泽昀黄永焯
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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