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Anti-tumor immune cell based on ligand-targeted cell conjugate (LTCC) technology as well as preparation method and application of anti-tumor immune cell

A technology of anti-tumor immunity and immune cells, applied in the field of biomedical engineering

Inactive Publication Date: 2021-04-20
AFFILIATED HOSPITAL OF JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In recent years, a new chemical biology technology combining metabolic glycoengineering and click chemical reaction has been developed. This technology can be used to make specific modifications to many cells. Many literatures report that the general process of this technology is to first convert azido sugar After cell uptake, the cells carry an azide group, and then the aptamer, chemical drug or fluorescent group coupled with an alkynyl group (such as DBCO or terminal alkynyl group) is used to connect the cell to a specific In the modification process of the above-mentioned alkynyl groups, they are all synthesized by pure chemistry, but because pure chemical synthesis requires a large amount of organic reagents as solvents, the alkynyl group is used to modify protein substances such as nanobodies, single-chain antibodies, and traditional antibodies. When the above methods are not applicable, more efficient and milder enzymatic methods are required for ligation

Method used

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  • Anti-tumor immune cell based on ligand-targeted cell conjugate (LTCC) technology as well as preparation method and application of anti-tumor immune cell
  • Anti-tumor immune cell based on ligand-targeted cell conjugate (LTCC) technology as well as preparation method and application of anti-tumor immune cell
  • Anti-tumor immune cell based on ligand-targeted cell conjugate (LTCC) technology as well as preparation method and application of anti-tumor immune cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of functionalized nanobodies

[0039] The preparation of functionalized nanobodies requires three processes, namely nanobody fermentation and purification, chemical synthesis of bio-orthogonal functional groups with triglycine peptides, and Sortase A enzymatic connection.

[0040] Nanobody fermentation purification:

[0041] Take 100 μL of glycerol-preserved Escherichia coli engineered bacteria that can produce Nanobody 7D12 (targeting EGFR) and inoculate them into 25 mL of LB culture containing 100 μg / mL kanamycin for overnight culture activation at 37°C and 200 rpm On the second day, transfer to TB medium containing 100μg / mL kanamycin at 37°C and 200rpm according to 2% inoculum size, and cultivate at 200rpm. The formula of TB is: peptone 12g / L, yeast powder 24g / L, glycerol 5g / L, K 2 HPO 4 ·3H 2 O 16.4g / L, KH 2 PO 4 2.31g / L, pending OD 600 When it is 0.6-0.8, add IPTG with a final concentration of 0.2mM to induce expression, the induction...

Embodiment 2

[0051] Example 2: Effect of azide sugar on the activity of NK cells

[0052] This example verifies the effect of added unnatural sugar on cell viability. The specific method is: after the unnatural sugar is taken up by the cells, culture for a certain period of time, and then use the CCK8 kit (Biyuntian) to measure the OD 450 , to analyze the difference in cell viability between the sugar-added group and the non-sugar-added group.

[0053] The specific method is as follows: put 1×10 5 NK cells were plated in a 96-well plate, the control group was added with 0.1% DMSO, and the experimental group was added with a final concentration of 50 μM Ac 4 ManNAz (the amount of DMSO is equivalent to 0.1%), and then cultivated in a 37°C cell incubator, and then added CCK8 reagent at 24h and 48h respectively, and detected OD by a microplate reader after incubation for 2h 450 , the result is as image 3 It shows that the addition of non-natural sugar has no toxicity to cells, but has a we...

Embodiment 3

[0054] Example 3: Nanobody modification of immune cells such as NK cells

[0055] This example is the acquisition of azide-labeled NK cells and the modification of 7D12. The principle process is as follows figure 1 shown. After the non-natural sugar is taken up by the cells, the bio-orthogonal reactive groups modified on the sugar are loaded onto the cell surface by using glycolysis, sialic acid metabolic pathway and other metabolic glycoengineering processes, and then the cells are collected and injected into the cell surface under similar physiological conditions. Functionalized nanobodies (1×10 4 NK cells were treated with 8-10 μg functionalized nanobody), and reacted at 37°C for 1-1.5h. Verify that cells have been successfully modified with Nanobodies by immunofluorescence.

[0056] The specific method is as follows:

[0057] The above chemically synthesized Ac 4 ManNAz was dissolved in DMSO to make a 50mM mother solution. After filtration and sterilization, it was ad...

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Abstract

The invention discloses a preparation method and application of an anti-tumor immune cell based on a ligand-targeted cell conjugate (LTCC)technology. The preparation method comprises the following steps: adding non-natural sugar modified with a biological orthogonal reaction group into a culture medium of immune cells, such as NK cells, so as to obtain immune cells modified with the biological orthogonal reaction group; then, modifying a targeting ligand, such as a nano antibody, of which one end is a biological orthogonal reaction pairing group capable of being matched with the biological orthogonal reaction group to generate a connection reaction to the surface of the immune cell through biological orthogonal reaction under physiological conditions, wherein the linking mode is a transpeptidase SrtA mediated chemical enzyme method, and ligand targeting has the characteristics of high-specificity recognition and high-expression receptor binding to the surface of the tumor cell. The ligand-targeted modified immune cell can specifically bind to cancer cells in a targeting manner, so that the modified immune cell generates an effect of secreting a large number of cytokines to enable the surfaces of the cancer cells to generate transmembrane pores or phagocytic cleavage of the cancer cells, and the immune cell has the effect of specifically killing the cancer cells.

Description

technical field [0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to an anti-tumor immune cell based on targeting ligand-cell coupling technology and its preparation method and application. Background technique [0002] Cancer is still the number one killer threatening human health and the second leading cause of human death. According to a report from the Global Medical Official Website: It is estimated that in 2020, there will be 18.9 million new cancer cases and 9.68 million deaths in the world, of which 3.854 million new cases and 2.496 million deaths are expected in my country, accounting for the total number of cancer cases and deaths. The number of people double tops the list in the world. [0003] Cancer immunotherapy is hailed as the third revolution in cancer treatment, and it is the most researched treatment method today. Adoptive cell therapy is a common means of immunotherapy, the most representative of whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61K35/15A61K47/68A61P35/00
CPCA61P35/00A61K47/6849A61K47/6889C12N5/0646C12N2537/10C12N2500/50A61K39/4613A61K2239/31A61K39/464404A61K2239/38A61K47/6901A61K47/6851
Inventor 黄朝晖吴志猛龚亮李艳春
Owner AFFILIATED HOSPITAL OF JIANGNAN UNIV
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