Modified natural killer cells and natural killer cell lines targetting tumour cells
A natural killer and cell line technology, applied in the field of cancer treatment and blood cancer, can solve problems such as use restrictions
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[0087] According to the present invention, the following embodiments are provided:
[0088] CLAIMS 1. A natural killer (NK) cell or NK cell line modified to bind to tumor-associated mucin-1 (MUC-1) glycoforms.
[0089] 2. The NK cell or NK cell line according to embodiment 1, wherein, relative to the wild type MUC-1 glycoform, the NK cell or NK cell line is genetically modified to express a tumor-associated MUC-1 glycoform binding Membrane-bound moiety with high affinity.
[0090] 3. The NK cell or NK cell line according to embodiment 2, wherein the membrane-bound moiety is a chimeric antigen receptor (CAR).
[0091] 4. The NK cell or NK cell line according to embodiment 3, wherein the CAR comprises the HMFG2 sequence.
[0092] 5. The NK cell or NK cell line according to any one of the preceding embodiments, wherein, compared to the wild-type glycoform, the tumor-associated MUC-1 glycoform comprises a group selected from the group consisting of Tn, sialylated Tn (STn), A la...
Embodiment 1
[0123] Example 1 - Lentiviral plasmid encoding MUC-1-CAR
[0124] DNA nucleotides encoding the single-chain variable fragment (scFv) derived from the HMFG2 clone recognizing the tumor-associated antigen MUC-1 (SEQ ID NO: 1) were cloned into the pCDCAR1-GFP vector. This scFv sequence is followed by another immunoglobulin-based hinge region to overcome MUC-1 steric hindrance. The hinge region is followed by costimulatory activation domains of CD28, OX40, and CD3ζ to provide activation signals for NK cell cytotoxicity, thereby triggering cytolysis of MUC-1-positive tumor cells. After the chimeric antigen receptor (CAR) protein (SEQ ID NO: 2), the selectable marker enhanced green fluorescent protein (EGFP) is located downstream of the T2A sequence, separating CAR and EGFP. This sequence was cloned between the EcoRI and XbaI restriction sites of the pCDCAR1-GFP vector (see figure 1 and 2 ).
[0125]Thaw a 150 μl vial of E. coli competent on ice. Meanwhile, cool unlabeled tube...
Embodiment 2
[0127] Example 2 - Nucleofection of KHYG-1 cells with MUC-1 CAR plasmid
[0128] The day before nucleofection in T25 flasks, KHYG-1 cells were passaged 1:1 (5ml cells + 5ml medium), while the cells were in logarithmic growth phase. Use Lonza Nucleofection Kit T (Cat. No.: VCA-1002); one nucleofection sample contains 100 μl of nucleofection solution (standard cuvette) and 2 × 10 6 cells. Nucleofection solution contained freshly prepared 18 μl supplement and 82 μl Nulceofector solution (per sample) and incubated at 37°C for 10 minutes.
[0129] Solution T was warmed to room temperature. Prepare fresh 12ml aliquots of medium (CM) in 15ml tubes (without antibiotics) at 37°C, containing 2.4ml FBS, 9.6ml RPMI 1640 and supplemented with 6μl IL-2 (RPMI1640 + 20% FBS + 500IU / ml IL-2). Aliquot 4 ml of CM into T25 flasks and pre-incubate the plate for 20 min in a humidified 37 °C incubator. Make 10 ml cell cultures in 15 ml tubes and count cells to determine cell density. Will 2×10...
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Abstract
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