Artificial antigen presenting cells and preparation method and application thereof

An artificial antigen and cell technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of difficult to form commercial products, complicated operation of DC cells, long culture period, etc., and achieve good biocompatibility, The effect of significant T cell expansion and specific surface area increase

Active Publication Date: 2019-11-19
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although DC cells have shown an important role in the activation of T cells, the operation of separating and inducing activated DC cells in vitro is complicated, the culture period is long, and the large-scale preparation of primary DC cells is difficult; in addition, the activation state of DC cells is unstable, resulting in stimulation of T cells. There are large differences in cell effects between batches; more importantly, DC cells will prevent excessive activation of T cells by expressing inhibitory signaling molecules. Due to the lack of control of this negative regulation, it will seriously affect the function of stimulating T cells and subsequent therapeutic effects (Steinman and Banchereau,2007), it is difficult to form commodity

Method used

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  • Artificial antigen presenting cells and preparation method and application thereof
  • Artificial antigen presenting cells and preparation method and application thereof
  • Artificial antigen presenting cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1. Preparation of PLLA rough microspheres

[0081] 1) Contains Fe 3 O 4 Preparation of PLLA rough microspheres

[0082] A. Centrifuge at 8,000 rpm for 5 min, collect smooth microspheres prepared with different molecular weights of PLLA (Mw: 5000, 10000, 50000), and fully remove the water.

[0083] B. Add 5 mL of tetrahydrofuran to the centrifuge tube, disperse evenly with a pipette, and suspend at 25°C for 30 minutes; centrifuge at 8,000 rpm for 5 minutes;

[0084] C. Repeat the above dissolution process 2 times; centrifuge at 8,000 rpm for 5 min, and wash with deionized water 3 times to characterize the appearance of the prepared microspheres.

[0085] Among them, the PLLA microspheres with a molecular weight of 5000 and 10000 are completely dissolved in tetrahydrofuran, and the PLLA microspheres with a molecular weight of 50,000 are partially dissolved and form a rough morphology with an average particle size of 10 μm. Therefore, the molecular weight range of PLLA is m...

Embodiment 2

[0086] Example 2. Preparation of artificial antigen presenting cells

[0087] 1) Contains Fe 3 O 4 Preparation of PLLA rough microspheres:

[0088] A. Centrifuge at 8,000 rpm for 5 min to collect Fe 3 O 4 The PLLA smooth microspheres fully remove moisture.

[0089] B. Add 5 mL of tetrahydrofuran to the centrifuge tube, disperse evenly with a pipette, and suspend at 25°C for 30 minutes; centrifuge at 8,000 rpm for 5 minutes;

[0090] C. Repeat the above dissolution process twice; centrifuge at 8,000 rpm for 5 min, wash with deionized water 3 times, and obtain Fe 3 O 4 PLLA rough microspheres (c-MS) with an average particle size of 10μm.

[0091] Select the smooth microspheres (s-MS) with a particle size of 2μm with ferroferric oxide distributed inside, and the others are the same as in this example, to prepare PLLA rough microspheres with holes on the surface, a deeper surface roughness, and an average particle size of 2μm .

[0092] 2) Physical adsorption method to modify avidin on the ...

Embodiment 3

[0101] Example 3. AAPCs prepared from rough PLLA microspheres are used for T cell activation and expansion

[0102] 1) Separate T cells in the spleen of C57 / B6 mice with CD3 negative kit magnetic beads;

[0103] 2) Mix aAPCs with primary mouse T cells in a ratio of 1:2, 1:10 and 1:20, and mix them with 10% FBS, 1% double antibodies, 1% non-essential amino acids and IL2 (300U / mL) co-cultured in RPMI1640 medium, 37℃, 5% CO 2 Cultivate for 5 days in an incubator, use CCK-8 kit to detect T cell proliferation, and CBA method to detect the level of IFN-γ secreted by T cell culture supernatant.

[0104] The examples prove that the activation effect is best when the ratio of aAPCs to primary mouse T cells is 1:2, and the final antibody concentration is 200ng / mL when aAPCs and T cells are co-cultured to stimulate T cell expansion and activation. Better than 40ng / mL and 1000ng / mL.

[0105] Rough microspherical appearance figure 2 , Which shows that the surface of the microspheres forms gaps ...

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Abstract

The invention discloses artificial antigen presenting cells and a preparation method and application thereof. According to the preparation method of the artificial antigen presenting cells, a polymermicrosphere surface topological structure morphological control method is established, an antigen presenting function of microspheres is achieved through signal molecule modification, and thus the microspheres can be used for stimulating immune cell amplification and activation. According to the artificial antigen presenting cells, adopted materials have biocompatibility and biodegradability, andthe preparation method of the microspheres with a rough surface structure is easy to operate, environmentally friendly and good in reproducibility.

Description

Technical field [0001] The invention belongs to the field of cell therapy and immunotherapy, and specifically relates to an artificial antigen presenting cell and a preparation method and application thereof. Background technique [0002] In recent years, the research of T cell immunotherapy has developed rapidly, especially the emergence of genetically engineered T cells has significantly improved the specificity and killing strength of immunotherapy, and achieved great success for the first time in the treatment of acute B lymphocytic leukemia (Brentjens et al., 2011). Up to now, genetically modified T cell immunotherapy has successively carried out a large number of clinical trials in a variety of malignant tumors, making T cell immunotherapy the most valuable and promising new anti-tumor therapy (Johnson and June, 2017). In order to obtain a sufficient amount of T cells, a large number of studies have been carried out using autologous DC cells to stimulate and expand T cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/69A61K39/395A61K47/02A61K47/59A61K47/66A61P35/00A61P37/02A61P37/06C12N5/0783
CPCA61K47/665A61K47/593A61K47/6927A61K47/02A61K39/3955A61P35/00A61P37/06A61P37/02C12N5/0636C12N2501/51C12N2501/515
Inventor 马洁张彤袁伟
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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