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Genetically engineered NK cell and preparation method and application thereof

A genetic engineering and NK cell technology, applied in the field of tumor immunotherapy, can solve the problems of not meeting the needs of critically ill patients, obtaining T cells is impractical, and obtaining T cells is cumbersome and other problems

Pending Publication Date: 2020-11-27
UNIVERSITY OF MACAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, for the above-mentioned immunotherapy, obtaining T cells from the patient is tedious work
In addition, the preparation of CART cells takes several weeks, which cannot meet the needs of critically ill patients
In particular, it is impractical to obtain T cells from patients with severe lymphopenia

Method used

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  • Genetically engineered NK cell and preparation method and application thereof
  • Genetically engineered NK cell and preparation method and application thereof
  • Genetically engineered NK cell and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] 1. Construction of CAR-engineered NK-92MI cells containing single nanobody Nab1

[0106] according to Figure 2A According to the design scheme shown in , by means of conventional techniques in the art (see, the "construction of the vector" part in the experimental method section), the vector is constructed, and the vector includes the transmembrane structure encoded by Nanobody Nab1 (NA-1), CD8 Nucleic acid of chimeric antigen receptor consisting of domain (TM), 4-1BB and CD3ζ.

[0107] For clarity, Figure 2A 5'LTR in means 5' long terminal repeat sequence; Ψ means viral packaging non-coding sequence; LS means coding sequence of leader sequence; NA-1 means coding sequence of Nanobody Nab1; CD8 TM means coding of CD8 transmembrane domain Sequences; 4-1BB and CD3ζ indicate the coding sequence of the intracellular signaling domain 4-1BB and CD3ζ, respectively; IRES indicates the coding sequence of the internal ribosome entry site; and ZsGreen1 indicates the coding sequ...

Embodiment 2

[0115] Example 2 was carried out using the same method as in Example 1, except that Figure 2B The schematic diagram of the design scheme of the vector used to construct the engineered NK cells comprising the bis-Nanobody of the present invention is shown in .

[0116] Figure 2B 2A in the scheme of design represents the coding sequence of Thoseaasigna virus 2A peptide; NA-2 represents the coding sequence of Nanobody Nab2.

[0117] Figure 3B A schematic diagram of the positive rate after sorting is shown in the plasmid containing the DNA sequence of the double nanobody CAR, transfected with lentivirus into NK-92MI cells. After sorting, the positive cell rate in the engineered CAR-NK-92MI cells was 68.1%. It was confirmed that engineered NK-92MI cells containing bis-Nanobodies Nab1 and Nab2 were obtained.

Embodiment 3

[0119] Example 3 was carried out using the same method as in Example 1, except that Figure 2C The design scheme for the construction of the vector used to construct the engineered NK-92MI cells comprising the single Nanobody Nab2 of the invention is shown in .

[0120] Next, the engineered NK-92MI cells obtained in Example 1, Example 2, and Example 3 above and the combination of Example 1 and Example 3 were tested through the evaluation example, that is, the engineered cell containing the single Nanobody Nab1 Cytotoxicity of NK-92MI cells and engineered NK-92MI cell combinations containing the single Nanobody Nab2.

[0121] Evaluation example

[0122] Calcein AM was used to perform cytotoxicity tests. First, LAN-1 cells were stained with calcein AM at 37 °C for 30 min, then the LAN-1 cells were washed with PBS, and the stained LAN-1 cells were prepared in this way. Since calcein AM can be converted to green fluorescent calcein inside the cells, after killing, dead LAN-1 ce...

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Abstract

The invention provides a genetically engineered NK cell as well as a preparation method and application thereof. The genetically engineered NK cell expresses an antigen chimeric receptor, the antigenchimeric receptor is composed of an extracellular domain, a transmembrane domain and an intracellular signal domain, and wherein the extracellular domain of the antigen chimeric receptor is an antibody or fragment thereof targeting an antigen IGF-1R, preferably a nano antibody Nab1. The genetically engineered NK cell provided by the invention can kill tumor cells expressing the IGF-1R.

Description

technical field [0001] The present invention relates to the field of immunotherapy of tumors, in particular to genetically engineered natural killer (NK) cells, their preparation method and application in tumor treatment. Background technique [0002] In the field of tumor treatment, chimeric antigen receptor T cell therapy has attracted more and more attention. [0003] Chimeric antigen receptor (CAR) is an artificially constructed transmembrane molecule encoded by a fusion gene, which can specifically target antigens on the surface of cancer cells such as T cells to eliminate target cancer cells. A CAR consists of an extracellular domain (eg, a single-chain antibody, scFv of an antibody), a transmembrane domain, and an intracellular domain. The scFv of the extracellular domain is responsible for the recognition of specific antigens. The intracellular domain is responsible for signal transduction. After the extracellular domain is specifically bound to the antigen, the i...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K39/00A61P35/00
CPCC07K16/2863C07K16/22C07K14/7051C12N5/0646C12N15/86A61K39/0011A61P35/00C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2740/15043
Inventor 赵琦张一驰周广宇曲书辉
Owner UNIVERSITY OF MACAU
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