Membrane anchoring element and application thereof

An anchoring element and an anchoring technology, applied in the field of cells and their applications, and target molecules, can solve problems such as loss of recognition and killing of cancer cells, difficulty in achieving satisfactory therapeutic effects, and difficulty in achieving expected goals

Active Publication Date: 2018-06-12
SHANGHAI UNIV OF T C M +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, although CAR-NK has significant advantages, due to the limitation of NK cell transfection technology, the application of CAR-NK is currently limited to NK cell lines
[0006] NK cell line itself is a kind of cancer cell, therefore, before administering to patients, the cells must undergo lethal irradiation, the killing effect of such NK cells in the body Very low, it is difficult to achieve a satisfactory therapeutic effect
In addition, although the adoptive therapy of NK cells expanded in vitro has achieved good results in clinical trials for some hematological tumors, due to the influence of the cancer immunosuppressive environment, NK cells will be quickly trained after infusion into patients, m

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  • Membrane anchoring element and application thereof
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  • Membrane anchoring element and application thereof

Examples

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preparation example Construction

[0075] Preparation of membrane anchoring elements

[0076] The membrane anchoring element is mainly obtained by solid-phase synthesis of polypeptides, specifically, 1) using the Fmoc method to form the second part; 2) using the Fmoc method to couple the third part to the second part to form the second part-the second part Three parts; 3) Using the Fmoc method, the first part is coupled to the above-mentioned second part-third part to form a membrane anchoring element.

[0077] More specifically, the solid-phase synthesis method for the polypeptide of the membrane anchor element of an embodiment is as follows:

[0078] Transfer the Fmoc-modified resin containing amino acid 1 into a polypeptide synthesis reactor for swelling, and then remove the Fmoc protecting group. Add Fmoc-modified PEG, dissolve and react with nitrogen gas for coupling. Then add Fmoc-modified amino acid 2, dissolve and react with nitrogen gas for coupling. The third part of the fatty acid chain is then ...

Embodiment 1

[0105] Example 1 Preparation of Membrane Anchoring Elements

[0106] Peptide solid-phase synthesis:

[0107] 1. Use 0.4 mmol / g of Fmoc-Lys(Dde)-wang resin (purchased from Kerabaybio) as the starting resin.

[0108] Weigh 5 g of resin (2 mmol), transfer to a polypeptide synthesis reactor, and swell with 50 ml of DMF for 2 hours.

[0109] 2. Treat the resin with 50 ml of 20% piperidine in DMF for 30 minutes to remove the Fmoc protecting group, wash with methanol and DMF three times alternately, and vacuum dry.

[0110] 3. Weigh 2.31g Fmoc-PEG2-OH (3 times volume), HBTU: 2.24g (2.95 times volume), NMM: 1.34ml (6 times volume), add the above materials in sequence, add a small amount of DMF to dissolve, and blow nitrogen to react After 1 hour, take a sample for the ninhydrin test (Kaiser test). If the test result is negative, the coupling is complete. If the test result is positive, you need to repeat the feeding. After the coupling was completed, the resin was washed 4 times wi...

Embodiment 2

[0120] Example 2 Preparation of IL-21 Membrane Anchor Target Molecule

[0121] IL-21 is a cell membrane-bound factor that can induce the proliferation and differentiation of NK cells, enhance the cytotoxic function of NK cells, and promote the secretion of granzymes, perforin, and IFN-γ.

[0122] 1) IL-21 prokaryotic expression vector construction:

[0123] The amino acid sequence of IL-21 is shown in SEQ ID No.1, a cysteine ​​residue is introduced at the C-terminus, and its nucleotide sequence is shown in SEQ ID No.2. Primers were designed, and the nucleotide sequence of IL-21 was constructed on the prokaryotic expression vector PET21a to obtain the prokaryotic expression plasmid.

[0124] 2) Induced expression of IL-21

[0125] The above prokaryotic expression plasmids are transformed into prokaryotic host cells, such as Escherichia coli BL21, and the expression is induced by IPTG. The gene was expressed in E. coli as inclusion bodies.

[0126] 3) Inclusion body treatmen...

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Abstract

The invention discloses a membrane anchoring element at least composed of three parts: a chemical group connected with a target molecule, a hydrophilic compound connected with the chemical group and ahydrophobic compound combined with a cytomembrane; and the hydrophobic compound is connected to the hydrophilic compound. The invention further provides the target molecule and a cell which contain the membrane anchoring element and preparation methods of the target molecule and the cell. Due to the adoption of the membrane anchoring element, a novel cell engineering technology is provided, and new direction and operation way are provided for immunotherapy.

Description

technical field [0001] The invention relates to a membrane anchoring element capable of combining with cell membranes, as well as target molecules, cells and application thereof connected with the membrane anchoring element. Background technique [0002] Cancer is a major disease that seriously threatens human physical and mental health. Although with the smooth implementation of the Human Genome Project and the in-depth revelation of the molecular mechanism of cancer, cancer treatment methods have been continuously improved and updated, but the incidence and mortality of cancer are still high. In order to improve the efficiency of cancer treatment, prolong the survival period of cancer patients, and improve the quality of life of cancer patients, it is imperative to develop more effective cancer treatment methods. [0003] In recent years, cancer immunotherapy has developed rapidly and achieved remarkable results. In 2013, it was listed as the top ten scientific breakthrou...

Claims

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Application Information

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IPC IPC(8): C07D207/448C12N5/0783C12N5/0784C12N5/0786C12N5/09
CPCC12N5/0636C12N5/0638C12N5/0639C12N5/0645C12N5/0646C12N5/0693C07D207/448
Inventor 朱诗国张维龚陈媛刘慧杰
Owner SHANGHAI UNIV OF T C M
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