KR-20260064746-A - T cell manufacturing method

Abstract

For example, a method for producing engineered T cells is provided herein, which may include the step of genetically editing unstimulated T cells and expressing a chimeric antigen receptor before activation.

Inventors

• 시칠리아노, 니콜라스 에이.
• 루엘라, 마르코

Assignees

• 비토리아 바이오테라퓨틱스, 인코포레이티드.
• 더 트러스티스 오브 더 유니버시티 오브 펜실바니아

Dates

Publication Date

20260507

Application Date

20240531

Priority Date

20230531

Claims (20)

1. A method for producing T cells comprising the step of genetically editing non-stimulated T cells.
2. A method for producing T cells, comprising the step of genetically editing unstimulated T cells using an in vitro manufacturing process, wherein the T cells are cultured for up to 7 days.
3. In claim 1, the method further comprises the step of activating the gene-edited T cells.
4. The method of claim 1, wherein the step of activating the gene-edited T cells comprises the step of contacting the unstimulated gene-edited T cell population with anti-CD3 and anti-CD28 antibodies and/or the step of activating the gene-edited T cells comprises the step of activating the cells through the use of an αβ-T cell receptor (T-cell receptor, TCR) complex.
5. In claim 1, the method wherein the cell is genetically edited to modify the T cell locus.
6. In paragraph 5, the above-mentioned locus is the CD5 locus, method.
7. A method according to claim 1, wherein the step of genetically editing the T cell comprises contacting the T cell with a gene editing complex to genetically edit the T cell.
8. In claim 7, the method wherein the gene editing complex comprises a ribonucleoprotein complex.
9. In claim 7, the method wherein the gene editing complex comprises a nuclease and/or guide RNA (guide RNA, gRNA).
10. The method of claim 9, wherein the nuclease is a zinc finger nuclease system (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR-Cas nuclease, a base-editing nuclease, a prime-editing nuclease, a retron-based nuclease, or a programmable addition via site-specific targeting element nuclease (PASTE).
11. In claim 10, the method wherein the CRISPR-Cas nuclease is a Class 1 or Class 2 CRISPR-Cas nuclease.
12. In claim 11, the method comprises a Class 2 CRISPR-Cas system including a Type II Cas nuclease.
13. In paragraph 12, the above-mentioned Type II Cas nuclease is a Cas9 nuclease, method.
14. In paragraph 10, the above CRISPR-Cas nuclease is a high fidelity [HiFi] nuclease, method.
15. A method according to claim 7, wherein the contact comprises transfecting the cell with the gene editing complex or electroporating it.
16. In paragraph 15, the method comprises electroporating the cell using a fluid electroporation device or system.
17. The method of claim 1, wherein the gene editing comprises contacting the cell with a gene editing complex for about 30 minutes to about 72 hours before activating the gene editing cell, for about 6 hours to about 36 hours before activating the gene editing cell, for about 12 hours to about 36 hours before activating the gene editing cell, for about 18 hours to about 28 hours before activating the gene editing cell, for about 20 hours to about 26 hours before activating the gene editing cell, or for about 24 hours before activating the gene editing cell.
18. The method of claim 1 further comprises the step of contacting the activated gene-edited T cell with a vector comprising a heterogeneous nucleic acid molecule encoding a molecule of interest.
19. In paragraph 18, the method wherein the vector is a plasmid or a viral vector.
20. In claim 19, the method wherein the virus vector is a lentivirus-based virus vector.

Description

Method for manufacturing T cells Related applications This application claims the benefit of U.S. provisional application serial number 63/505,265 filed on May 31, 2023, the entire text of which is incorporated herein by reference. Reference to the electronically submitted sequence list The present application includes a sequence list that is submitted electronically in XML format, the full text of which is incorporated herein by reference. The XML copy created on May 23, 2024, has the filename 'VTB-007WO_SL' and is 18,711 bytes in size. Technology field The present embodiment relates to a method for producing genetically modified T cells that can be used to produce modified T cells, such as CAR-T cells, in a modified sequence of traditional steps and/or a shortened manufacturing period, and the product thereof can be used to treat, for example, cancer or other immunological conditions. Gene editing and gene integration technologies have expanded the ability to modify cells to contain heterologous nucleic acid molecules that can be used to express heterologous molecules, such as chimeric antigen receptors (CARs), nucleic acid molecules of interest, or proteins of interest. Although T-cell lymphomas and leukemias have poor prognoses and few available treatments, CAR T-cell (CART) therapy has demonstrated efficacy in certain malignancies. CAR-T cells have traditionally been manufactured using a process that takes more than seven days from cell harvesting to infusion, and the step of activating the T-cell population occurs prior to the gene editing step. A shortened manufacturing process is required, and by shortening and modifying the sequence of gene editing, fratricide in certain finished pharmaceutical products can be avoided, and more potent populations or groups of engineered T-cells can be developed for successful re-infusion into patients for the treatment of lymphomas and other cancers. The present embodiment meets these and other needs. In some embodiments, a method for producing T cells is provided, wherein the method comprises the step of genetically editing unstimulated T cells. In some embodiments, the method further comprises the step of activating the gene-edited T cells. In some embodiments, the cells are genetically edited to modify T cell loci in addition to introducing a heterologous sequence. In some embodiments, the loci are CD5, CD2, or CD7 loci. In some embodiments, a method for producing CAR-T cells is provided, wherein the method comprises the steps of: genetically editing isolated non-stimulated T cells; producing genetically edited T cells by electroporating the T cells together with a gene editing complex; activating the genetically edited T cells by CD3/CD28 microbead antigen-induced activation within 24 hours of electroporation; and transfecting the activated genetically modified T cells within 24 hours of activation using a lentivirus-based vector comprising a heterologous nucleic acid molecule encoding a chimeric antigen receptor. In some embodiments, the method further comprises the step of culturing the transfected T cells for a certain period, e.g., 1 to 5 days. In some embodiments, the method further comprises the step of freezing (cryopreserving) the transfected T cells. In some embodiments, a method for treating a cancer patient with CAR-T cells is provided, wherein the method comprises the steps of: genetically editing isolated non-stimulated T cells obtained from a subject; producing gene-edited T cells by genetically editing the T cells by electroporating the T cells together with a gene-editing complex; activating the gene-edited T cells to CD3/CD28 microbead antigen-induced activation within 24 hours of electroporation; transfecting the activated gene-modified T cells within 24 hours of activation using a lentivirus-based vector comprising a heterologous nucleic acid molecule encoding a chimeric antigen receptor; and administering the transfected T cells to a patient. In some embodiments, the subject is a cancer patient or the subject is a person who is not a patient (e.g., the cells are homologous to the patient). FIG. 1: Illustrates non-limiting embodiments of patient T cell harvesting, CART manufacturing process, and the possibility of re-injecting the finished CART drug into the patient. Figures 2a–2c: Show the evaluation results of CD5 KO CART5 cells produced through conventional and rapid manufacturing processes. Figure 2a shows the comparative results of CD5 expression. Figure 2b shows the comparative results of CAR5 expression. Figure 2c shows the comparative results of cell population doubling. Figure 3: Shows differences in memory phenotypes between mock cells, CD5 KO CART5 conventional cells and CD5 KO CART5 rapid cells. Fig. 4: Shows the difference in cytokine release between mock cells, CD5 KO CART5 conventional cells and CD5 KO CART5 rapid cells in an isolated state (upper panel) or after co-culture with Jurkat cells (lower panel). Figure 5: Shows the d