Genetically engineered erythrocytes carrying anti-pd-1 single chain antibody and preparation method therefor

A method for preparing genetically engineered erythrocytes carrying an anti-PD-1 single chain antibody, characterized by comprising the following steps:

• step S1:
• constructing a desired fragment sequence in a lentiviral expression vector to obtain a vector of the desired sequence, wherein the desired fragment is Anti-PD-1 scFv, and the Anti-PD-1 scFv represents an anti-PD-1 single-chain variable region fragment;
• step S2:
• lentivirally packaging the vector of the desired sequence in the step S1 and obtaining a high-titer lentiviral concentrate after centrifuging and concentrating;
• step S3:
• isolating Lin−CD34− cells from peripheral blood mononuclear cells and enriching the Lin−CD34− cells;
• step S4:
• changing the culture medium of the Lin−CD34− cells to a differentiation stage-1 culture medium, inducing the Lin−CD34− cells to differentiate into erythroid, and performing proliferation thereon;
• step S5:
• using the lentiviral concentrate in the step S2 to infect the Lin−CD34− cells in the step S4 to obtain anti-PD-1 scFv Lin−CD34− cells;
• step S6:
• changing the culture medium of the anti-PD-1 scFv Lin−CD34− cells to a differentiation stage-2 culture medium;
• wherein the anti-PD-1 scFv Lin−CD34− cells are subjected to erythrocyte denucleation to obtain mature anti-PD-1 scFv erythrocytes, i.e., the genetically engineered erythrocytes carrying an anti-PD-1 single chain antibody.