Pharmaceutical composition containing NK cells for treating cancer
A technology of NK cells and compositions, applied in the direction of drug combinations, medical preparations containing active ingredients, animal cells, etc., to achieve good application prospects, promote proliferation and kill activity
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Embodiment 1
[0031] Example 1 Preparation and Activity Verification of TNF-alpha Highly Active Mutants
[0032] According to the biological characteristics of tumor necrosis factor analyzed by bioinformatics, it was found that the factor contained a death domain, which formed a structure similar to helix-loop-helix and interacted with the intracellular death domain of TNFR1. Under the condition of ensuring the three-dimensional binding activity of TNF-α, 6 mutation sites are designed to improve the activity. The basic amino acid side chain often contains a basic chemical group that can be protonated to increase the polar effect and promote the binding. Speed and combining effects. Three of them include 83T / R, 89V / H, and 94A / K in the amino acid sequence of SEQ ID NO: 2 relative to the wild-type gene sequence shown in SEQ ID NO: 1 (A / K means wild type) Type A is replaced by K). Specifically, the upstream and downstream primers are designed according to the wild-type TNF-α gene sequence. ...
Embodiment 2
[0036] Example 2 Transfection of NK cells by IL-15 coupled with TNF-α
[0037] The IL-15 shown in SEQ ID NO: 3 is expressed as a fusion of the amino acid sequence of SEQ ID NO: 2 mutated with 89V / H using a T2A linker, and the expression form is as follows figure 2 shown. Specifically, the 89V / H mutant plasmid prepared in Example 1 was subjected to enzyme digestion, and the amplified IL-15 of SEQ ID NO: 4 was sequenced by overlapping PCR technology through the T2A linker to obtain the full-length sequence IL-15- T2A-89V / H TNF-α, the nucleotide sequence of which is shown in SEQ ID NO:5.
[0038] The fusion gene was double digested with the pCDH-CMV vector, then ligated with T4 DNA Ligase, 8 μL of the ligated reaction product was added to 50 μL of DH5α competent cells for plasmid transformation, and positive strains were identified by colony PCR. . After the plasmid of the positive strain was extracted, the 293T cells were activated and cultured, and the 293T cells were passa...
Embodiment 3
[0041] Example 3 Proliferation experiment of NK cells
[0042] NK92-IL-15-T2A-89V / H TNF-α, NK92-89V / H TNF-α, NK92-IL-15 cells and NK92 cell lines were adjusted to 1×10 5 / ml, cultured in 24-well plate at 37℃, counted once on the 3rd day and the 7th day, and changed half of the medium every two days, and cultured continuously for 7d. The result is as Figure 4 shown.
[0043] Figure 4 Indicates the growth effect of NK92-IL-15-T2A-89V / H TNF-α, NK92-89V / H TNF-α, NK92-IL-15 cells and NK92 cell line under culture conditions. It can be seen that the proliferation ability of NK92-IL-15-T2A-89V / H TNF-α is significantly higher than that of NK92-89V / H TNF-α, NK92-IL-15 cells and NK92 cell lines.
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