Genetically engineered NK cell and preparation method and application thereof

[0104] Example 1

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

[0106] according to Figure 2A The design scheme shown in the design scheme, through the conventional technical means in the field (see, the "vector construction" part of the experimental method chapter), the vector is constructed, the vector includes the transmembrane structure encoded by Nanobody Nab1 (NA-1) and CD8 The nucleic acid of the chimeric antigen receptor composed of domain (TM), 4-1BB and CD3ζ.

[0107] For clarity, Figure 2A The 5'LTR represents the 5'long terminal repeat sequence; Ψ represents the virus packaging non-coding sequence; LS represents the coding sequence of the leader sequence; NA-1 represents the coding sequence of Nanobody Nab1; CD8 TM represents the coding of the CD8 transmembrane domain Sequence; 4-1BB and CD3ζ respectively represent the coding sequence of intracellular signal domain 4-1BB and CD3ζ; IRES represents the coding sequence of the internal ribosome entry site; and ZsGreen1 represents the coding sequence of a fluorescent protein.

[0108] Thus, the plasmid containing the CAR encoding the single Nanobody Nab1, pLVX-IRES-ZsGreen1 was obtained.

[0109] 2. 293T lentivirus packaging

[0110] Perform lentivirus packaging on 293T cells according to the steps in "293T Lentivirus Packaging" in the "Experimental Methods" section above. Obtain the virus including the pLVX-IRES-ZsGreen1 plasmid.

[0111] 3. Virus transfection of NK cells

[0112] Transfect NK-92MI cells with the virus obtained above according to the steps in "Virus Transfection of NK Cells" in the "Experimental Methods" section above. The transfected CAR-NK-92MI positive cells were obtained. And through the steps in "Testing the Transfection Rate and Sorting" in the "Experimental Methods" section above, test the positive rate of the transfected CAR-NK-92MI positive cells, and perform the sorting to obtain the engineered CAR- NK-92MI cells.

[0113] Figure 3A A schematic diagram showing the positive rate of NK-92MI cells transfected with lentivirus on the plasmid containing the DNA sequence of the single nanobody CAR. After sorting, the positive cell rate in the engineered CAR-NK-92MI cells was 73.4%. It was confirmed that CAR-engineered NK-92MI cells containing single Nanobody Nab1 were obtained.

[0114] Example 2

[0115] Example 2 was performed using the same method as in Example 1, except that it was used when preparing plasmids Figure 2B The design scheme of the vector used to construct the engineered NK cells containing the diabody of the present invention is shown in.

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

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

[0118] Example 3

[0119] Example 3 was performed using the same method as in Example 1, except that it was used when preparing plasmids Figure 2C The design scheme for constructing the vector used in the engineered NK-92MI cell containing the single Nanobody Nab2 of the present invention is shown in.

[0120] Next, the engineered NK-92MI cells obtained in Example 1, Example 2, and Example 3 and the combination of Example 1 and Example 3 were tested through evaluation examples, namely, the engineering of the single Nanobody Nab1 Cytotoxicity of the combination of NK-92MI cells and engineered NK-92MI cells containing the single Nanobody Nab2.

[0121] Evaluation example

[0122] Calcein AM is used for cytotoxicity testing. First, the LAN-1 cells were stained with Calcein AM at 37°C for 30 minutes, and 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 into green fluorescent calcein inside the cell, after killing, the dead LAN-1 cells will release green fluorescence into the supernatant for extraction and reading. A 96-well plate was used for this experiment. The engineered NK-92MI cells and LAN-1 cells obtained in Example 1 and Example 2 with ratios of 20:1, 10:1, 5:1, 2.5:1, and 1:1 were used, and the ratios were respectively The engineered NK-92MI cells and IMR-32 cells obtained in Example 1 and Example 2 of 20:1, 10:1, 5:1, 2.5:1, and 1:1. The experiment was repeated three times for each ratio, and the results were averaged. In addition, three control groups were used, including 100% killed LAN-1, spontaneous LAN-1, and LAN-1 medium.

[0123] In the present invention, the killing time varies from 3-5 hours. After reading the results, the cytotoxicity rate can be calculated by the following formula.

[0124]

[0125] For IMR-32 cells, the cytotoxicity rate was calculated using the above method similar to that for LAN-1 cells.

[0126] Figure 4A The results of the cytotoxicity test on LAN-1 cells of NK-92MI cells transfected with a plasmid containing the DNA sequence of a single Nanobody CAR and a plasmid containing the DNA sequence of a Double Nanobody CAR are shown.

[0127] Figure 4A In the figure, the curved single nanoCAR represents the results of the cytotoxicity test of NK-92MI cells engineered with single Nanobody Nab1 against LAN-1 cells; the curved double nanoCAR represents the double nanobody Nab1 and Nab2 engineered NK-92MI cells against LAN -1 Results of cell cytotoxicity test.

[0128] Figure 4B The results of the cytotoxicity test of NK-92MI cells transfected with the plasmid containing the DNA sequence of the single Nanobody CAR and the plasmid containing the DNA sequence of the Double Nanobody CAR respectively against IMR-32 cells are shown.

[0129] Figure 4B In, the curved single nano CAR represents the result of the cytotoxicity test of the single nano antibody Nab1 engineered NK-92MI cells against IMR-32 cells; the curved double nano CAR represents the double nano antibody Nab1 and Nab2 engineered NK-92MI cells against the IMR -Results of cytotoxicity test of 32 cells.

[0130] Figure 4C Shown are NK-92MI cells engineered with double Nanobody Nab1 and Nab2, NK-92MI cells engineered with single Nanobody Nab1, NK-92MI cells engineered with single Nanobody Nab2, and NK-92MI cells engineered with single Nanobody Nab1. The results of the cytotoxicity test of the mixture of 92MI cells and single Nanobody Nab2 engineered NK-92MI cells respectively against LAN-1 cells.

[0131] Figure 4C In the figure, the curved double nano CAR represents the results of the cytotoxicity test of NK-92MI cells engineered with double nano antibodies Nab1 and Nab2 against LAN-1 cells; the curved nano 1 CAR + nano 2 CAR represents the single nano antibody Nab1 engineered NK-92MI cells and The combination of single Nanobody Nab2 engineered NK-92MI cells is the result of a cytotoxicity test against LAN-1 cells, wherein the single Nanobody Nab1 engineered NK-92MI cells and the single Nanobody Nab2 engineered NK-92MI cells The ratio is 1:1; the curve nano 1CAR represents the result of the cytotoxicity test of the single Nanobody Nab1 engineered NK-92MI cells against LAN-1 cells; and the curve nano 2CAR represents the single Nanobody Nab2 engineered NK-92MI cells against The results of the cytotoxicity test of LAN-1 cells.

[0132] From Figure 4A It can be seen that the NK-92MI cells engineered by the double Nanobody Nab1 and Nab2, regardless of the cell ratio of 10:1 or 20:1, have obtained superior cytotoxicity than the NK-92MI cells engineered by the single Nanobody Nab1. test results. That is, the NK-92MI cells engineered with the double Nanobody Nab1 and Nab2 have stronger killing ability against LAN-1 cells.

[0133] From Figure 4B It can be seen that the NK-92MI cells engineered by the double Nanobody Nab1 and Nab2, regardless of the cell ratio of 10:1 or 20:1, have obtained better cytotoxicity than the NK-92MI cells engineered by the single Nanobody Nab1. test results. That is, the NK-92MI cells engineered with the double Nanobody Nab1 and Nab2 have stronger killing ability against IMR-32 cells.

[0134] From Figure 4C It can be seen that in the killing experiment against LAN-1 cells, NK-92MI cells engineered with double Nanobody Nab1 and Nab2 are most cytotoxic to LAN-1 cells, and are not only more cytotoxic than single Nanobody Nab1 engineered NK-92MI cells The NK-92MI cells engineered with single Nanobody Nab2 and NK-92MI cells are each more cytotoxic to LAN-1 cells, even than the combination of single Nanobody Nab1 engineered NK-92MI cells and single Nanobody Nab2 engineered NK-92MI cells The cytotoxicity against LAN-1 cells is also high. It was confirmed that the NK-92MI cells engineered by the double nanobody Nab1 and Nab2 achieved excellent synergistic effects against target cells.

[0135] Due to the simultaneous use of two antibodies on the surface of NK cells, the genetically engineered NK cells obtained have achieved the effect of killing tumor cells with high expression of IGF-1R antigen, and the effect is much higher than expected, proving that it has a synergistic effect . The engineered NK cells provided by the present invention can specifically and powerfully kill tumor cells expressing IGF-1R antigen. It can alleviate many side effects caused by the use of CAR-T cell therapy in patients, which makes it a promising "ready-made" product for immunotherapy.

[0136]

[0137]

[0138]