Plasmid combination and application thereof in preparing modified immune cells
Provided in the present disclosure is a method for using a four-plasmid system to prepare modified immune effector cells. The method comprises: forming a lentivirus by using four plasmids within 293T cells, extracting and obtaining the lentivirus, then transfecting immune effector cells by using the lentivirus, and expressing a chimeric antigen receptor. Also provided in the present disclosure is a use of the immune effector cell obtained by using the described method and of a composition containing the immune effector cell.
1. A method for treating a disease or disorder associated with CD19 expression, comprising administering, to a patient in need thereof,
a composition comprising a modified immune effector cell,
wherein the modified immune effector cell is prepared by a method comprising:
introducing a plasmid combination into a cell to prepare a lentiviral vector; wherein the plasmid combination comprises plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev present at a ratio of 3-6:1-1.5:1-3:1-1.5, wherein the plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO. 1; and
introducing the lentiviral vector into an immune effector cell to prepare the modified immune effector cell.
2. The method according to claim 1 , wherein the disease or disorder associated with CD19 expression comprises non-solid tumors.
3. The method according to claim 2 , wherein the non-solid tumor comprises leukemia and/or lymphoma.
4. The method according to claim 1 , wherein the disease or disorder associated with CD19 expression comprises acute lymphoblastic leukemia and/or B-cell lymphoma.
5. The method according to claim 4 , wherein the acute lymphoblastic leukemia comprises acute lymphoblastic leukemia in adults and/or acute lymphoblastic leukemia in children.
6. The method according to claim 5 , wherein the composition is administered at a dose of 0.25×10 8 to 0.5×10 8 CAR-positive T cells.
7. The method according to claim 4 , wherein the B-cell lymphoma comprises non-Hodgkin's lymphoma.
8. The method according to claim 7 , wherein the composition is administered at a dose of 1×10 8 to 2×10 8 CAR-positive T cells.
9. The method according to claim 5 , wherein the acute lymphoblastic leukemia is relapsed or refractory acute lymphoblastic leukemia.
10. The method according to claim 7 , wherein the non-Hodgkin's lymphoma is relapsed or refractory non-Hodgkin's lymphoma.
11. The method according to claim 1 , wherein the plasmid Seq1 comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the nucleic acid molecule comprises a nucleic acid sequence shown in SEQ ID NO. 2.
12. The method according to claim 1 , wherein the plasmids Seq1, PMD2.G, pMDLg-pRRE and pRSV-Rev are present at a ratio of 11.8:3.53:6.33:2.3, 13.8:3.48:5.31:2.54 or 14:4.67:4.67:4.67.
13. The method according to claim 1 , wherein the cell is 293T.
14. The method according to claim 1 , wherein the immune effector cell is selected from the group consisting of a T lymphocyte and a natural killer cell.