Chimeric antigen receptor targeting NK activated receptor

[0128] Example 1. Preparation of CAR T cells containing different scFvs and verification of their functions

[0129] 1.1 Preparation of CAR T cells

[0130] Sequences encoding the following proteins were synthesized and cloned into pLVX vector (Public Protein / PlasmidLibrary (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 17), anti-NKG2D scFv, CD8α hinge region (SEQ ID NO: 19), CD8α transmembrane region (SEQ ID NO: 3), 4-1BB intracellular region (SEQ ID NO: 9), CD3ζ intracellular signaling domain (SEQ ID NO: 11), and by sequencing Confirm correct insertion of the target sequence. Wherein, the amino acid sequence of the anti-NKG2D scFv contained in NKG2D-1CAR is shown in SEQ ID NO: 27; the amino acid sequence of the anti-NKG2D scFv contained in NKG2D-2CAR is shown in SEQ ID NO: 29; the anti-NKG2D scFv contained in NKG2D-3CAR The amino acid sequence of the anti-NKG2D scFv contained in NKG2D-4CAR is shown in SEQ ID NO: 31; the amino acid sequence of the anti-NKG...

[0137] Example 2: Preparation of CAR T cells and its killing effect on NK92 target cells and release of cytokines

[0138] 2.1 Preparation of CAR T cells

[0139] Sequences encoding the following proteins were synthesized and cloned into pLVX vector (Public Protein / PlasmidLibrary (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 17), anti-NKG2D scFv (SEQ ID NO: 27) , CD8α hinge region (SEQ ID NO: 19), CD8α transmembrane region (SEQ ID NO: 3), costimulatory domain, CD3ζ intracellular signaling domain (SEQ ID NO: 11), wherein the costimulatory domain includes CD28 intracellular region (SEQ ID NO: 7), or 4-1BB intracellular region (SEQ ID NO: 9) + DAP10 intracellular region (SEQ ID NO: 45), to obtain 2D28z-CAR and 2Dbb10z-CAR, and pass Sequencing confirms correct insertion of the target sequence. figure 1 The CAR structure constructed in this example is shown.

[0140] The preparation of CAR T cells was completed according to the method described in Example 1, ...

[0157] Example 3. Preparation of CAR-T cells in which targeted NK-activating receptors are knocked out

[0158] Knockout endogenous NKG2D in NT, 2Dbbz-CAR T cells, 2D28z-CAR T and 2Dbb10z-CAR T cells by CRISP / Cas9 system, and use PE mouse anti-human NKG2D (biolegend product number 302806) by flow cytometry The expression level of NKG2D in CAR T cells after knockout was detected (Table 1), and the proportion of CD8+ T cells in CAR-T cells was detected with APC-anti human CD8 (BDPharmingen, 555369) ( Figure 8 ). Non-knockout NT cells served as controls.

[0159] Table 1. NKG2D expression levels in CAR-T cells

[0160] gene name 2DbbzKO-CAR 2Dbb10zKO-CAR 2D28zKO-CAR NT NK2D 5.00％ 6.20％ 6.50％ 39.40％

[0161] The results in Table 1 show that NKG2D was effectively knocked out in each CAR-T cell.

[0162] From Figure 8 It can be seen that when the CAR targeting NKG2D is expressed, further knocking out endogenous NKG2D can significantly increase t...