NK cell surface modified immune checkpoint inhibitor and application thereof
An immune checkpoint, NK cell technology, applied in the biological field, can solve the problems of high threshold, high cost, and inability to benefit cancer patients.
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Embodiment 1
[0032] The preparation route of the present embodiment is as follows:
[0033]
[0034] Including the following steps:
[0035] (1) Tos Synthesis of Sia methyl ester (2): Sialic acid (1) (15.0 g, 50 mmol), trifluoroacetic acid (1 mL) and anhydrous methanol (300 mL) were mixed, and the mixture was stirred at room temperature until clear. Evaporation of the solvent gave methyl sialate as a white solid without further purification. Combine solid with P 2 o 5 Dry in vacuo overnight, co-evaporate twice with anhydrous pyridine to remove traces of water, then dissolve in anhydrous pyridine (200 mL). The solution was cooled to 0°C and p-toluenesulfonyl chloride (10.0 g, 53 mmol) was added. The mixture was warmed to room temperature and stirred overnight. The solvent was removed by heating under reduced pressure, and the residue was purified by column chromatography (20:1 EtOAc:MeOH) to give Tos Sia methyl ester (2) (18.0 g, 38 mmol), yield 76%.
[0036] (2) N3 Sia methyl1 ...
Embodiment 2
[0039] 1 The immune checkpoint inhibitor modified on the surface of NK cells of the present invention modifies NK cells
[0040]Peripheral blood was drawn from healthy individuals, and cells were initially isolated by density gradient centrifugation, and then NK cells were further isolated by using an NK cell extraction kit (Invitrogen, Carlsbad, CA). Cell sorting was performed by flow cytometry, and the cells with phenotypes of NKp46+, CD3-, and CD56+ were the desired NK cells. The isolated cells were cultured in suspension in IMDM medium (containing 10% human serum, 1% non-essential amino acids, and 1% sodium pyruvate), and then replaced with an immune checkpoint inhibitor containing 100 μM of the NK cell surface modification of the present invention. IMDM medium continued to culture for 48h. The culture medium was replaced and washed three times, and the NK cells modified by the immune checkpoint inhibitor on the surface of NK cells of the present invention (named as NK+) ...
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