T cell manufacturing method

Abstract

For example, methods for producing engineered T cells are described herein, which may include genetically editing unstimulated T cells before activation and before the expression of chimeric antigen receptors.

Claims

1. A method for preparing T cells, comprising genetically editing unstimulated T cells.

2. A method for preparing T cells, comprising genetically editing unstimulated T cells using an ex vivo manufacturing process, wherein the T cells are cultured for up to seven days.

3. The method according to claim 1, further comprising activating the genetically edited T cells.

4. The method according to claim 1, wherein activating the genetically edited T cells comprises contacting the gene-edited unstimulated T cell population with anti-CD3 and anti-CD28 antibodies, and / or activating the genetically edited T cells comprises activating the cells by using the αβ-cell receptor (TCR) complex.

5. The method according to claim 1, wherein the cells are genetically edited to modify the T cell locus.

6. The method according to claim 5, wherein the gene locus is the CD5 gene locus.

7. The method according to claim 1, wherein the genetic editing of the T cells includes contacting the T cells with a gene editing complex to genetically edit the T cells.

8. The method according to claim 7, wherein the gene editing complex comprises a ribonucleoprotein complex.

9. The method according to claim 7, wherein the gene editing complex comprises a nuclease and / or guide RNA (gRNA).

10. The method according to claim 9, wherein the nuclease is a programmable addition via 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 site-specific targeted element nuclease (PASTE).

11. The method according to claim 10, wherein the CRISPR-Cas nuclease is a Class 1 or Class 2 CRISPR-Cas nuclease.

12. The method according to claim 11, wherein the Class 2 CRISPR-Cas system comprises a type II Cas nuclease.

13. The method according to claim 12, wherein the type II Cas nuclease is a Cas9 nuclease.

14. The method according to claim 10, wherein the CRISPR-Cas nuclease is a high-fidelity (HiFi) nuclease.

15. The method according to claim 7, wherein the contact includes transfecting the cells with the gene editing complex or performing electroporation.

16. The method according to claim 15, wherein the electroporation comprises electroporating the cells using a flow electroporation apparatus or system.

17. The method according to claim 1, wherein the gene editing comprises contacting the cells with the gene editing complex for approximately 30 minutes to approximately 72 hours before activating the gene editing cells, approximately 6 hours to approximately 36 hours before activating the gene editing cells, approximately 12 hours to approximately 36 hours before activating the gene editing cells, approximately 18 hours to approximately 28 hours before activating the gene editing cells, approximately 20 hours to approximately 26 hours before activating the gene editing cells, or approximately 24 hours before activating the gene editing cells.

18. The method according to claim 1, further comprising contacting the gene-edited activated T cells with a vector containing a heterogeneous nucleic acid molecule encoding a target molecule.

19. The method according to claim 18, wherein the vector is a plasmid or a viral vector.

20. The method according to claim 19, wherein the viral vector is a lentivirus-based viral vector.

21. The method according to claim 1, further comprising collecting the activated T cells.

22. The method according to claim 21, wherein the activated T cells are collected by the 5th day after activation, by the 6th day after activation, or by the 7th day after activation.

23. The method according to claim 21, wherein the activated T cells are collected between day 1 and day 7 after activation.

24. The method according to claim 21, wherein the activated T cells are collected on day 1, 2, 3, 4, 5, 6, or 7 after activation.

25. The method according to claim 18, wherein the heterologous molecule of the objective is siRNA, shRNA, non-coding RNA, peptide, polypeptide, protein, viral payload, viral genome, or a combination thereof.

26. The method according to claim 18, wherein the heterogeneous molecule for the purpose is a chimeric antigen receptor ("CAR").

27. The method according to claim 26, wherein the CAR includes an antigen-binding domain that binds to CD5.

28. The method according to claim 27, wherein the antigen-binding domain for CD5 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a heavy chain complementarity-determining region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 1, an HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and an HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and the VL comprising a light chain complementarity-determining region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 4, an LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and an LCDR3 containing the amino acid sequence of SEQ ID NO:

6.

29. The method according to claim 28, wherein VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 7, and VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

8.

30. The method according to claim 26, wherein the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

11.

31. The method according to claim 26, wherein the CAR comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

12.

32. The method according to any one of claims 18 to 31, wherein the T cells are transfected or transduced less than one day after activation of the T cells.

33. The method according to any one of claims 18 to 31, wherein the T cells are transfected or transduced more than one day after activation of the T cells.

34. The method according to any one of claims 1 to 33, wherein the manipulated T cells are patient-derived T cells.

35. The method according to claim 34, wherein the patient is a cancer patient.

36. The method according to claim 35, wherein the cancer is a hematological cancer (i.e., a hematological malignancy, including leukemia (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML)), lymphoma (Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) with subtypes such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL)), and multiple myeloma), or a solid tumor (including carcinoma (originating in epithelial cells), sarcoma (originating in connective tissue), and various other types, examples of which include brain tumors (glioma, meningioma), breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma (skin cancer), kidney cancer, bladder cancer, ovarian cancer, or endometrial cancer).

37. The method according to claim 35 or 36, wherein the cancer patient is a lymphoma patient.

38. The method according to any one of claims 1 to 37, further comprising isolating T cells from a patient before gene editing the cells.

39. The method according to any one of claims 1 to 38, further comprising cryopreserving the cells after any of the steps.

40. The method according to any one of claims 1 to 39, wherein the T cells are enriched before undergoing gene editing.

41. The method according to any one of claims 1 to 40, wherein the T cell is a CD5+ T cell.

42. The method according to claim 34, wherein the T cell population is activated by the use of CD3 / CD28 antigen activation, CD3 / CD28 / CD137 antigen activation, concanavalin A (conA), 12-myristate 13-acetate phorbol (PMA), or Treg proliferation.

43. The method according to any one of claims 1 to 42, wherein the gene-edited activated T cells are administered to a patient.

44. A method for producing CAR-T cells, The steps involve gene editing of isolated, unstimulated T cells, The steps include: electroperforating the T cells with a gene editing complex to genetically edit the T cells and produce gene-edited T cells; A step of activating the gene-edited T cells within 24 hours of electroporation by CD3 / CD28 microbead antigen-induced activation, Within 24 hours of activation, the gene-modified activated T cells are transduced with a lentivirus-based vector containing a heterologous nucleic acid molecule encoding a chimeric antigen receptor. The method comprising the above.

45. The method according to claim 44, further comprising culturing the transduced T cells for a certain period of time, such as 1 to 5 days.

46. The method according to any one of claims 44 to 45, wherein the gene editing involves editing the CD5 locus to disrupt the CD5 locus, to knock out the CD5 gene, and / or to reduce the expression of the CD5.

47. A method for producing CAR-T cells, i) Isolating unstimulated T cells from the subject, ii) Genetically editing the T cells by electroperforating the unstimulated T cells with a gene editing complex, thereby producing gene-edited unstimulated T cells, iii) By inducing CD3 / CD28 microbead antigen activation, the gene-edited unstimulated T cells are activated within 24 hours of electroporation to produce gene-edited activated T cells, iv) Transduction of the gene-edited activated T cells with a lentivirus-based vector containing a heterologous nucleic acid molecule encoding a chimeric antigen receptor within 24 hours of activation, v) Culturing the T cells for 1 to 7 days after transduction, vi) Collect the T cells 1 to 7 days after transduction, vii) Optionally, the collected cells may be frozen. The method comprising the above.

48. CAR-T cells produced according to the method described in any one of claims 1 to 47.

49. A method for treating cancer patients with CAR-T cells, Genetic editing of isolated, unstimulated T cells obtained from the target, The process involves electroporating the T cells with a gene editing complex to genetically edit the T cells and produce gene-edited T cells. The gene-edited T cells are activated within 24 hours of electroporation by CD3 / CD28 microbead antigen-induced activation, Within 24 hours of activation, the gene-modified activated T cells are transduced using a lentivirus-based vector containing a heterologous nucleic acid molecule encoding a chimeric antigen receptor. Administering the transduced T cells to the patient, The method comprising the above.

50. A method for treating cancer patients with CAR-T cells, i) Isolating unstimulated T cells from the subject, ii) Genetically editing the T cells by electroperforating the unstimulated T cells with a gene editing complex, thereby producing gene-edited unstimulated T cells, iii) By inducing CD3 / CD28 microbead antigen activation, the gene-edited unstimulated T cells are activated within 24 hours of electroporation to produce gene-edited activated T cells, iv) Transduction of the gene-edited activated T cells with a lentivirus-based vector containing a heterologous nucleic acid molecule encoding a chimeric antigen receptor within 24 hours of activation, v) Culturing the T cells for 1 to 7 days after transduction, vi) Collect the T cells 1 to 7 days after transduction, vii) Optionally, the collected cells may be frozen. viiii) Administering the collected T cells to the patient, The method comprising the above.

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