[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.
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