CN-121986171-A - Improved method in lentivirus manufacture for the production of CAR-T cell pharmaceutical products

Abstract

The present application relates to improvements in lentivirus manufacture for the production of CAR-T cell drug products, wherein the manufacturing method comprises a change in the time between transfection and harvesting of host cells, as well as a new vector ratio for transfection. Methods of preparing lentiviruses at various vector ratios and times between transfection and harvest of host cells, as well as transfection compositions comprising various vector ratios, are presented herein.

Inventors

• R. Batia
• N. Purnejad

Assignees

• 詹森生物科技公司

Dates

Publication Date

20260505

Application Date

20241004

Priority Date

20231005

Claims (20)

1. 1. A method of preparing a lentivirus, the method comprising: transfecting a host cell with a transfection composition, wherein the transfection composition comprises: A first vector comprising a polynucleotide encoding a CAR, A second carrier, a third carrier, and a fourth carrier, and Transfection medium; wherein the ratio of the first carrier to the second carrier to the third carrier to the fourth carrier is 2:1:1:1, 11:3:1:5 or 12:2:1:5, Culturing the transfected host cell to proliferate, and Harvesting the lentivirus, wherein the harvesting occurs about 24 hours after transfection.
2. 2. A method of preparing a lentivirus, the method comprising: transfecting a host cell with a transfection composition, wherein the transfection composition comprises: A first vector comprising a polynucleotide encoding a CAR, A second carrier, a third carrier, and a fourth carrier, and Transfection medium; Wherein the ratio of the first carrier to the second carrier to the third carrier to the fourth carrier is 11:3:1:5 or 12:2:1:5, Culturing the transfected host cell to proliferate, and Harvesting the lentivirus, wherein the harvesting occurs less than or equal to about 48 hours after transfection.
3. 3. The method of claim 1, wherein the ratio is 2:1:1:1.
4. 4. The method of claim 1 or 2, wherein the ratio is 11:3:1:5.
5. 5. The method of claim 1 or 2, wherein the ratio is 12:2:1:5.
6. 6. The method of any one of claims 3 to 5, wherein the harvesting occurs about 24 hours after transfection.
7. 7. The method of any one of claims 1 to 6, wherein the first, second, third, and fourth vectors comprise lentiviral vectors.
8. 8. The method of claim 1, wherein the ratio is 2:1:1:1 and the harvesting occurs about 24 hours after transfection.
9. 9. The method of claim 1, wherein the ratio is 11:3:1:5 and the harvesting occurs about 24 hours after transfection.
10. 10. The method of claim 1, wherein the ratio is 12:2:1:5 and the harvesting occurs about 24 hours after transfection.
11. 11. The method of any one of claims 1 to 10, wherein the second vector comprises a polynucleotide encoding a lentiviral envelope protein.
12. 12. The method of claim 11, wherein the lentiviral envelope protein is Vesicular Stomatitis Virus G (VSVG).
13. 13. The method of any one of claims 1 to 12, wherein the third vector comprises a polynucleotide encoding GAGs and POL.
14. 14. The method of any one of claims 1 to 13, wherein the fourth vector comprises a polynucleotide encoding REV.
15. 15. The method of any one of claims 1 to 14, wherein the culturing occurs in a bioreactor.
16. 16. The method of claim 15, wherein the bioreactor is a 2L, 10L or 50L bioreactor.
17. 17. The method of claims 1-16, wherein the host cell is a HEK 293 cell.
18. 18. The method of any one of claims 1 to 17, wherein the CAR immunospecifically targets B Cell Maturation Antigen (BCMA).
19. 19. A method of preparing a lentivirus, the method comprising: transfecting a host cell with a transfection composition, wherein the transfection composition comprises: A first vector comprising a polynucleotide encoding a CAR that immunospecifically targets BCMA, A second carrier, a third carrier, and a fourth carrier, and Transfection medium; wherein the ratio of the first carrier to the second carrier to the third carrier to the fourth carrier is 2:1:1:1, 11:3:1:5 or 12:2:1:5, Culturing the transfected host cell to proliferate, and Harvesting the lentivirus, wherein the harvesting occurs about 24 hours after transfection.
20. 20. A method of preparing a lentivirus, the method comprising: transfecting a host cell with a transfection composition, wherein the transfection composition comprises: A first vector comprising a polynucleotide encoding a CAR that immunospecifically targets BCMA, A second carrier, a third carrier, and a fourth carrier, and Transfection medium; Wherein the ratio of the first carrier to the second carrier to the third carrier to the fourth carrier is 11:3:1:5 or 12:2:1:5, Culturing the transfected host cell to proliferate, and Harvesting the lentivirus, wherein the harvesting occurs less than or equal to about 48 hours after transfection.

Description

Improved method in lentivirus manufacture for the production of CAR-T cell pharmaceutical products Cross Reference to Related Applications The present application claims the benefit of U.S. provisional application No. 63/588,148, filed on 5 of 10 th 2023, which is hereby incorporated by reference in its entirety. Sequence listing The present application encompasses a sequence table that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy was created at 28, 8, 2024, named "258199091502 (JBI 6821WOPCT 1) Sequence listing.xml" and was 31,678 bytes in size. Technical Field The present application relates to improvements in lentivirus manufacture for CAR-T cell preparation. Background Chimeric antigen receptor T cell (CAR-T) therapies utilize isolated T cells that have been genetically engineered to enhance their specificity for a particular tumor-associated antigen. These T cells are typically autologous T cells, wherein the T cells are isolated from the patient to be subjected to T cell therapy. These isolated T cells were then genetically engineered to express Chimeric Antigen Receptors (CARs) using lentiviruses. T cells expressing chimeric antigen receptors (CAR-T cells) can induce tumor immunoreactivity. Lentiviruses for T cell genetic engineering prepared using currently available vector packaging systems in the prior art have low concentrations of live virus and low infectious titers. In order to achieve the ideal T cell transduction effect, a higher dose of lentivirus needs to be added, but the disadvantages are higher cost, excessive residual substances and poor safety. Although four-carrier packaging systems are also used in the prior art in place of three-carrier packaging systems, no suitable ratio has been established at which carriers can be combined to provide higher infectious titer while ensuring good safety. Thus, there remains an urgent need to screen the appropriate ratios of the four vectors used to package lentiviruses to genetically engineer isolated T cells for CAR-T therapy. Disclosure of Invention Provided herein are methods of preparing a lentivirus for use in the manufacture of a Chimeric Antigen Receptor (CAR) T cell (CAR-T) drug product, the method comprising transfecting a host cell with a transfection composition, wherein the transfection composition comprises a first vector comprising a polynucleotide encoding a CAR, a second vector, a third vector and a fourth vector, and a transfection medium, wherein the ratio of the first vector to the second vector to the third vector to the fourth vector is 2:1:1, 11:3:1:5, or 12:2:1:5, culturing the transfected host cell to proliferate, and harvesting the lentivirus, wherein the harvesting occurs about 24 hours after transfection. Also provided herein are methods of preparing a lentivirus for use in the manufacture of a Chimeric Antigen Receptor (CAR) T cell (CAR-T) drug product, the method comprising transfecting a host cell with a transfection composition, wherein the transfection composition comprises a first vector comprising a polynucleotide encoding a CAR, a second vector, a third vector and a fourth vector, and a transfection medium, wherein the ratio of the first vector to the second vector to the third vector to the fourth vector is 11:3:1:5 or 12:2:1:5, culturing the transfected host cell to proliferate, and harvesting the lentivirus, wherein the harvesting occurs less than or equal to about 48 hours after transfection. In some embodiments, the ratio is 2:1:1:1. In some embodiments, the ratio is 11:3:1:5. In some embodiments, the ratio is 12:2:1:5. In some embodiments, harvesting occurs about 24 hours after transfection. In some embodiments, the first vector, the second vector, the third vector, and the fourth vector comprise lentiviral vectors. In some embodiments, the ratio is 2:1:1:1, and harvesting occurs about 24 hours after transfection. In some embodiments, the ratio is 11:3:1:5, and harvesting occurs about 24 hours after transfection. In some embodiments, the ratio is 12:2:1:5, and harvesting occurs about 24 hours after transfection. In one embodiment, the second vector comprises a polynucleotide encoding a lentiviral envelope protein. In some embodiments, the lentiviral envelope protein is Vesicular Stomatitis Virus G (VSVG). In some embodiments, the third vector comprises polynucleotides encoding GAGs and POL. In some embodiments, the fourth vector comprises a polynucleotide encoding REV. In some embodiments, the culturing is performed in a bioreactor. In some embodiments, the bioreactor is a 2L, 10L, or 50L bioreactor. In some embodiments, the host cell is a HEK 293 cell. In some embodiments, the host cell is a HEK 293F cell. In some embodiments, the CAR immunospecifically targets B Cell Maturation Antigen (BCMA). There is provided a method of preparing a lentivirus for use in the manufacture of a BCMA CAR-T drug product comprising transfecting a host ce