Methods and compositions for administration of allogeneic chimeric antigen receptor t cells targeting B7H3

Abstract

Belongs to the technical field of medical treatment, and particularly provides an administration method for allogeneic chimeric antigen receptor T cells, and also provides a composition and a product used in the method. The method comprises administering to a subject at least one dose of an allogeneic universal chimeric antigen receptor (CAR)-T cell (CAR-T cell) comprising anti-B7H3 for administration to a subject suffering from a recurrent high-level glioma.

Description

Methods and compositions for administration of allogeneic chimeric antigen receptor T cells targeting B7H3 Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a method for administering allogeneic chimeric antigen receptor T cells. Background Art

[0002] Glioma is the most common and aggressive malignant tumor of the central nervous system (CNS). It arises from CNS neuroepithelial cells and accounts for approximately 65% ​​of primary intracranial tumors. Its incidence rate approaches 10 per 100,000 patients and has been increasing worldwide in recent years. The peak age of onset is between 50 and 60 years old. Initial symptoms are generally insidious, primarily manifesting as nonspecific neurological symptoms such as headache and dizziness, or focal neurological symptoms such as altered mental status, muscle strength, and muscle tone. Low-grade gliomas (LGGs) can also present with epilepsy as the initial symptom.

[0003] In 2007, the World Health Organization classified gliomas into grades I-IV based on their malignancy. Grades I-II are classified as LGGs, which generally have a good prognosis; while grades III-IV are classified as high-grade gliomas (HGGs), which are highly invasive and have a very poor prognosis. Recurrence is a hallmark of gliomas. Despite various aggressive treatments, patients will eventually relapse, resulting in a very poor median overall survival (mOS).

[0004] Treatments for recurrent high-grade gliomas (rHGGs) usually include radiotherapy, chemotherapy, targeted therapy, electric field therapy, and immunotherapy.

[0005] Summary of the Invention

[0006] The present application intends to provide a CAR-T immunotherapy method for recurrent high-grade glioma and a method for administering CAR-T cells therein.

[0007] 1. A method for treating a subject with recurrent high-grade gliomas (rHGGs), the method comprising administering to the subject at least one dose comprising an allogeneic chimeric antigen receptor T cell (CAR-T cell) targeting B7H3, wherein the at least one dose is about 1.0×10 6 ~1.0×10 9 Cells / drugs.

[0008] 2. The method according to claim 1, wherein the high-grade glioma is a brain glioma.

[0009] 3. The method according to item 2, wherein the brain glioma is an astrocytoma (IDH mutant), an oligodendroglioma (IDH mutant with 1p / 19q combined deletion), a glioblastoma (IDH wild type), a diffuse astrocytoma (with MYB or MYBL1 alterations), an angiocentric glioma, a multiforme low-grade neuroepithelial tumor of the juvenile (PLNTY), a diffuse low-grade glioma (with MAPK signaling pathway alterations), a diffuse midline glioma (with H3 K27 alterations), a diffuse hemispheric glioma (with H3 G34 mutations), a diffuse childhood high-grade glioma (H3 and IDH wild type), an infantile hemispheric glioma, a pilocytic astrocytoma, a high-grade astrocytoma with hairy features, a pleomorphic xanthoastrocytoma, a subependymal giant cell astrocytoma, or a chordoid glioma.

[0010] 4. The method according to claim 1, wherein the at least one dose is selected from about 1.0×10 6 Cells / dose, approximately 2.0×10 6 cells / dose, approximately 3.0×10 6 cells / dose, approximately 4.0×10 6 Cells / dose, approximately 5.0×10 6 cells / dose, approximately 6.0×10 6 cells / dose, approximately 7.0×10 6 cells / dose, approximately 8.0×10 6 cells / dose, approximately 9.0×10 6 Cells / dose, approximately 1.0×10 7 Cells / dose, approximately 1.5×10 7 Cells / dose, approximately 2.0×10 7 Cells / dose, approximately 2.5×10 7 cells / dose, approximately 3.0×10 7 cells / dose, approximately 4.0×10 7 Cells / dose, approximately 5.0×10 7 cells / dose, approximately 6.0×10 7 cells / dose, approximately 7.0×10 7 cells / dose, approximately 8.0×10 7 cells / dose, approximately 9.0×10 7 Cells / dose, approximately 1.0×10 8 cells / dose, approximately 2.0×10 8 cells / dose, approximately 3.0×10 8 cells / dose, approximately 4.0×10 8 cells / dose, 5.0×10 8 cells / dose, approximately 6.0×10 8 cells / dose, approximately 7.0×108 cells / dose, approximately 8.0×10 8 cells / dose, approximately 9.0×10 8 Cells / dose 1.0×10 9 Cells / drugs.

[0011] 5. The method according to items 1-4, wherein when the subject has a weight of less than or equal to 50 kg, the at least one dose is selected from about 1.0×10 6 cells / dose, approximately 2.0×10 6 cells / dose, approximately 3.0×10 6 cells / dose, approximately 4.0×10 6 Cells / dose, approximately 5.0×10 6 cells / dose, approximately 6.0×10 6 cells / dose, approximately 7.0×10 6 cells / dose, approximately 8.0×10 6 cells / dose, approximately 9.0×10 6 Cells / dose, approximately 1.0×10 7 Cells / dose, approximately 1.5×10 7 Cells / dose, approximately 2.0×10 7 Cells / dose, approximately 2.5×10 7 cells / dose, approximately 3.0×10 7 cells / dose, approximately 4.0×10 7 Cells / dose, approximately 5.0×10 7 Cells / drugs.

[0012] 6. The method according to items 1-4, wherein when the subject has a weight greater than 50 kg, the at least one dose is selected from about 6.0×10 7 cells / dose, approximately 7.0×10 7 cells / dose, approximately 8.0×10 7 cells / dose, approximately 9.0×10 7 Cells / dose, approximately 1.0×10 8 cells / dose, approximately 2.0×10 8 cells / dose, approximately 3.0×10 8 cells / dose, approximately 4.0×10 8 cells / dose, 5.0×10 8 cells / dose, approximately 6.0×10 8 cells / dose, approximately 7.0×10 8 cells / dose, approximately 8.0×10 8 cells / dose, approximately 9.0×10 8 Cells / dose 1.0×10 9 Cells / drugs.

[0013] 7. The method of items 1-6, wherein CAR expression is detectable in the subject until at least 2 weeks after administration of the CAR-T cells.

[0014] 8. The method of items 1-7, wherein CAR expression is detectable in the subject until at least 4 weeks after administration of the CAR-T cells.

[0015] 9. The method of items 1-8, wherein the subject exhibits a CR state at least 4 weeks after administration of the CAR-T cells.

[0016] 10. The method of items 1-9, wherein the subject exhibits a CR state at least 8 weeks after administration of CAR-T cells.

[0017] 11. The method of items 1-10, wherein the subject exhibits a CR state of the CAR-T cells for at least 12 weeks after administration of the CAR-T cells.

[0018] 12. The method of items 1-11, wherein the subject exhibits a CR state of the CAR-T cells for at least 24 weeks after administration of the CAR-T cells.

[0019] 13. The method of items 1-12, wherein the subject exhibits a CR state for at least 48 weeks after administration of the CAR-T cells.

[0020] 14. The method according to items 1-13, wherein the CAR-T cells are administered locally.

[0021] 15. The method according to items 1-14, wherein the CAR-T cells are administered by intracerebroventricular administration or intrathecal administration.

[0022] 16. The method of items 1-15, wherein lymphocyte clearance is not required before administering the CAR-T cells.

[0023] 17. The method of items 1-16, wherein the subsequent doses are administered less than 4 weeks after the first dose.

[0024] 18. The method of items 1-16, wherein the subsequent dose is administered at least 4 weeks after the first dose.

[0025] 19. The method of claim 17-18, wherein the subsequent doses of CAR-T cells administered are substantially the same as the number of cells in the first dose.

[0026] 20. The method of claim 17-18, wherein subsequent doses of CAR-T cells are administered with a greater number of cells than the first dose.

[0027] 21. The method of claim 17-18, wherein subsequent doses of CAR-T cells administered contain fewer cells than the first dose.

[0028] 22. The method of items 1-18, wherein the subject exhibits a suboptimal response after administration of CAR-T cells.

[0029] 23. The method of clause 22, wherein the suboptimal response comprises:

[0030] Complete response (CR);

[0031] Partial response (PR); or

[0032] Stable disease (SD).

[0033] 24. The method according to any one of items 1-23, wherein the CAR-T cell expresses a CAR having an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 8.

[0034] 25. The method according to any one of items 1-23, wherein the CAR-T cell expresses a CAR having the amino acid sequence shown in SEQ ID NO:5.

[0035] 26. A method for producing an allogeneic chimeric antigen receptor T cell population targeting B7H3, the method comprising:

[0036] (a) Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors;

[0037] (b) activating T cells in the PBMCs;

[0038] (c) Transducing activated T cells with the complex or vector containing CAR

[0039] (d) destroying the expression of TRAC gene and HLA-A gene in T-cells to obtain T-cells with destroyed gene expression;

[0040] (e) expanding T-cells in which gene expression is disrupted; and

[0041] (f) T-cell population enriched for TRAC-negative, HLA-A-negative cells;

[0042] This generates a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells).

[0043] 27. The method according to item 26, wherein the vector of step (c) is a lentiviral vector.

[0044] 28. A method according to item 26, wherein the step (c) is to transduce the activated T-cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing the target CAR.

[0045] 29. The method according to any one of items 26 to 28, wherein the CAR is a CAR having an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 8.

[0046] 30. The method according to items 26 to 29, wherein the CAR is a CAR having the amino acid sequence shown in SEQ ID NO: 5.

[0047] 31. The method according to items 26-30, wherein the expression and / or activity of the TRAC gene and HLA-A gene of the CAR-T cell are downregulated.

[0048] 32. A kit or a pharmaceutical kit for treating a subject with recurrent high-grade gliomas (rHGGs), the kit or the pharmaceutical kit comprising: allogeneic chimeric antigen receptor T cells targeting B7H3.

[0049] 33. The kit or pharmaceutical kit of claim 32, wherein the kit or pharmaceutical kit comprises at least one dose of allogeneic chimeric antigen receptor T cells targeting B7H3, wherein the at least one dose is about 1.0×10 6 ~1.0×10 9 Cells / drugs.

[0050] 34. The kit or drug box according to item 32, wherein the CAR-T cell is the CAR-T cell involved in any one of items 1-25 or the CAR-T cell produced by the method of any one of items 26-31.

[0051] 35. A method of treatment, comprising:

[0052] 1) administering chimeric antigen receptor T cells targeting B7H3 to a subject having the disease for the first time; and

[0053] 2) administering a subsequent dose of CAR-T cells to the diseased subject at least four weeks after the administration of the CAR-T cells in 1).

[0054] 36. The method according to claim 35, wherein the subsequent dose is administered 2 times, or 3 times or more.

[0055] 37. The method of items 35-36, wherein the chimeric antigen receptor-T cells targeting B7H3 are allogeneic chimeric antigen receptor T cells targeting B7H3.

[0056] 38. The method of items 35-37, wherein the CAR-T cells are administered locally.

[0057] 39. The method according to items 35-37, wherein the CAR-T cells are administered by intracerebroventricular administration or intrathecal administration.

[0058] 40. The method according to items 35-39, wherein the first administration of CAR-T cells comprises a dose of about 1.0×10 6 ~1.0×10 9 cell.

[0059] 41. The method according to items 35-40, wherein the first dose of CAR-T cells administered comprises about 1.0×10 6 cells, about 2.0×10 6 cells, about 3.0×10 6 cells, about 4.0×10 6 cells, about 5.0×10 6 cells, about 6.0×10 6 cells, about 7.0×10 6 cells, about 8.0×10 6 cells, approximately 9.0×10 6 cells, about 1.0×10 7 cells, about 1.5×10 7 cells, about 2.0×10 7 cells, about 2.5×10 7 cells, about 3.0×10 7 cells, about 4.0×10 7 cells, about 5.0×10 7 cells, about 6.0×10 7 cells, approximately 7.0×10 7 cells, about 8.0×10 7 cells, approximately 9.0×10 7 cells, about 1.0×10 8 cells, about 2.0×10 8 cells, about 3.0×10 8 cells, about 4.0×10 8 cells, 5.0×10 8 cells, about 6.0×10 8 cells, approximately 7.0×10 8 cells, about 8.0×108 cells, approximately 9.0×10 8 cells and 1.0×10 9 cell.

[0060] 42. The method according to items 35-39, wherein the first administration of CAR-T cell dose comprises 1.5×10 4 ~2.0×10 7 cells / kg subject body weight.

[0061] 43. The method of claim 42, wherein the first administration of a CAR-T cell dose comprises approximately 1.5×10 4 , 2.0×10 4 , 2.5×10 4 , 3.0×10 4 , 3.5×10 4 , 4.0×10 4 , 4.5×10 4 , 5.0×10 4 , 5.5×10 4 , 6.0×10 4 , 6.5×10 4 , 7.0×10 4 , 7.5×10 4 , 8.0×10 4 , 8.5×10 4 , 9.0×10 4 , 9.5×10 4 , 1.0×10 5 , 1.5×10 5 , 2.0×10 5 , 2.5×10 5 , 3.0×10 5 , 3.5×10 5 , 4.0×10 5 , 4.5×10 5 , 5.0×10 5 , 5.5×10 5 , 6.0×10 5 , 6.5×10 5 , 7.0×10 5 , 7.5×10 5 , 8.0×10 5 , 8.5×10 5 , 9.0×10 5 , 9.5×10 5 , 1.0×10 6 , 1.5×10 6 , 2.0×10 6, 2.5×10 6 , 3.0×10 6 , 3.5×10 6 , 4.0×10 6 , 4.5×10 6 , 5.0×10 6 , 5.5×10 6 , 6.0×10 6 , 6.5×10 6 , 7.0×10 6 , 7.5×10 6 , 8.0×10 6 , 8.5×10 6 , 9.0×10 6 , 9.5×10 6 , 1.0×10 7 , 1.5×10 7 , 2.0×10 7 cells / kg subject body weight.

[0062] 44. The method of items 35-43, wherein the subsequent dose comprises about 1.0×10 6 ~1.0×10 9 cell.

[0063] 45. The method according to items 35-44, wherein the disease is a tumor or cancer.

[0064] 46. ​​The method of claim 45, wherein the tumor comprises a glioma.

[0065] 47. The method according to item 45, wherein the brain glioma is adult-type diffuse glioma, childhood-type diffuse low-grade glioma, childhood-type diffuse high-grade glioma, or focal astrocytic glioma.

[0066] 48. The method according to any one of items 46-47, wherein the brain glioma is an astrocytoma (IDH mutant), an oligodendroglioma (IDH mutant with 1p / 19q combined deletion), a glioblastoma (IDH wild type), a diffuse astrocytoma (with MYB or MYBL1 alterations), an angiocentric glioma, a juvenile multiform low-grade neuroepithelial tumor (PLNTY), a diffuse low-grade glioma (with MAPK signaling pathway alterations), a diffuse midline glioma (with H3 K27 alterations), a diffuse hemispheric glioma (with H3 G34 mutations), a diffuse childhood high-grade glioma (H3 and IDH wild type), an infantile hemispheric glioma, a pilocytic astrocytoma, a high-grade astrocytoma with hairy features, a pleomorphic xanthoastrocytoma, a subependymal giant cell astrocytoma, or a chordoid glioma.

[0067] 49. A method according to any one of items 35-48, wherein the method results in improvement in the disease to a greater extent and / or for a longer period of time compared to a method comprising an alternative dosing regimen in which a single dose of the cells in 1) and the cells in 2) is administered to the subject.

[0068] 50. The method of any one of items 35-49, wherein the disease persists after administration of the first dose and / or administration of the first dose is insufficient to eradicate the disease in the subject.

[0069] 51. The method of any one of items 35-50, wherein the subject exhibits one or more symptoms of the disease upon administration in 2).

[0070] 52. A method according to any one of items 35-51, wherein the cytokine release syndrome (CRS)-related outcome in the subject is not detectable or is decreased by about 20% to about 99% on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 after administration in 2), compared to a method comprising an alternative dosing regimen in which the cells in 2) are administered to the subject.

[0071] 53. A method according to any one of items 35-52, wherein the CAR-T cell is the CAR-T cell involved in items 1-25 or a CAR-T cell produced by the method of any one of items 26-31.

[0072] 54. A method of treatment, comprising administering a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells to a subject who has previously been administered a first dose of allogeneic CAR-T cells, wherein: the subsequent dose of cells is administered at a time point that is at least or more than about 4 weeks and less than about 24 weeks after the start of the first dose.

[0073] 55. A method of treatment, comprising administering a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells to a subject, wherein: prior to said administration, said subject has received a previous dose of CAR-T cells in an amount sufficient to demonstrate a clinical benefit in said subject; and at the time of said administration, said subject does not demonstrate a detectable adaptive host immune response specific for the CAR-T cells; and / or the time between said previous dose and said subsequent dose is greater than about 4 weeks and less than about 24 weeks.

[0074] 56. Use of a composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for the preparation of a medicament for treating a disease in a subject previously treated with CAR-T cells, wherein: the composition is for use between about 4 to about 24 weeks after the previous treatment; and / or the composition is formulated for administration of a subsequent dose in an amount sufficient to ameliorate the disease in a subject previously treated with CAR-T cells.

[0075] 57. A composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for treating a disease in a subject previously treated with CAR-T cells, wherein: the cells are for use between about 4 and 24 weeks after the previous treatment; and the cells are formulated for administration of a subsequent dose in an amount sufficient to ameliorate the disease in the subject previously treated with CAR-T cells.

[0076] 58. The use of claim 56 or the composition of claim 57, wherein the dose of CAR-T cells in the previous treatment improves one or more symptoms of the disease in the subject prior to administration of the subsequent dose.

[0077] 59. Use of allogeneic chimeric antigen receptor (CAR)-T cells in the preparation of a medicament for treating a disease, the use comprising:

[0078] 1) administering a first dose of CAR-T cells to a subject suffering from the disease, wherein the first dose comprises approximately 1×10 6 to about 1.0×10 9 Total cells, and

[0079] 2) administering a subsequent dose of CAR-T cells to the subject at a time point at least or more than about 4 weeks and less than about 24 weeks after the administration in 1).

[0080] 60. The use according to item 56 or 59 or the composition for use according to item 57, wherein the disease is a tumor or cancer.

[0081] 61. The method of any one of items 35 to 55, the use of any one of items 56 or 59, or the composition of item 57, wherein the CAR-T cells are allogeneic chimeric antigen receptor (CAR)-T cells targeting B7H3.

[0082] 62. An article of manufacture comprising: a plurality of sealable containers, each container comprising a unit dose of allogeneic chimeric antigen receptor (CAR)-T cells for administration to a subject, the unit dose comprising approximately 1×10 6 to about 1.0×10 9cells; packaging material; and a label or package insert comprising instructions for administering a plurality of said unit doses to said subject by performing a first administration and subsequent administrations, said first administration comprising delivering one of said unit doses to said subject, and said subsequent administrations comprising administering one or more of said unit doses to said subject.

[0083] 63. The article of manufacture of item 62, wherein the instructions specify that the subsequent administration be performed at a time between about 30 and 150 days after the first administration, optionally at about 30 days, about 60 days, about 90 days, or about 99 days.

[0084] 64. The article of claim 62 or 63, wherein the container is or comprises a flexible cell infusion bag or a cryovial.

[0085] 65. The method according to any one of items 35 to 55, the use according to any one of items 56 or 59, or the composition according to item 57, or the product according to any one of items 62 to 64, wherein the CAR-T cell is a CAR-T cell involved in items 1 to 25 or a CAR-T cell produced by the method of any one of items 26 to 31. In a specific embodiment of the present application, the CAR-T comprises: an extracellular antigen-binding domain targeting B7H3, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises at least two VHHs that bind to different epitopes of B7H3.

[0086] In a specific embodiment of the present application, the extracellular antigen-binding domain targeting B7H3 comprises a first heavy chain antibody heavy chain variable region (VHH-1) and a second heavy chain antibody heavy chain variable region (VHH-2) targeting B7H3, wherein the VHH-1 comprises the HCDR1 as shown in SEQ ID NO:9, the HCDR2 as shown in SEQ ID NO:11, and the HCDR3 as shown in SEQ ID NO:13; and / or the VHH-2 comprises the HCDR1 as shown in SEQ ID NO:10, the HCDR2 as shown in SEQ ID NO:12, and the HCDR3 as shown in SEQ ID NO:14.

[0087] In a specific embodiment of the present application, the VHH-1 and / or VHH-2 include human antibodies, humanized antibodies or chimeric antibodies.

[0088] In a specific embodiment of the present application, the VHH-1 and VHH-2 are directly connected or connected through a linker.

[0089] In a specific embodiment of the present application, from the N-terminus to the C-terminus, the extracellular antigen binding domain targeting B7H3 comprises:

[0090] i) VHH-1-linker-VHH-2; or

[0091] ii) VHH-2-linker-VHH-1.

[0092] In a specific embodiment of the present application, the linker comprises the amino acid sequence shown in SEQ ID NO:15.

[0093] In a specific embodiment of the present application, the VHH-1 comprises:

[0094] i) HFR1 comprising the amino acid sequence of SEQ ID NO: 16, HFR2 comprising the amino acid sequence of SEQ ID NO: 17, HFR3 comprising the amino acid sequence of SEQ ID NO: 18, and HFR4 comprising the amino acid sequence of SEQ ID NO: 19; or

[0095] ii) HFR1 comprising the amino acid sequence shown in SEQ ID NO:20, HFR2 comprising the amino acid sequence shown in SEQ ID NO:17, HFR3 comprising the amino acid sequence shown in SEQ ID NO:21, and HFR4 comprising the amino acid sequence shown in SEQ ID NO:22.

[0096] In a specific embodiment of the present application, the VHH-1 comprises the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24.

[0097] In a specific embodiment of the present application, the VHH-2 comprises:

[0098] i) HFR1 comprising the amino acid sequence of SEQ ID NO: 25, HFR2 comprising the amino acid sequence of SEQ ID NO: 26, HFR3 comprising the amino acid sequence of SEQ ID NO: 27, and HFR4 comprising the amino acid sequence of SEQ ID NO: 28; or

[0099] ii) HFR1 comprising the amino acid sequence shown in SEQ ID NO:29, HFR2 comprising the amino acid sequence shown in SEQ ID NO:26, HFR3 comprising the amino acid sequence shown in SEQ ID NO:21, and HFR4 comprising the amino acid sequence shown in SEQ ID NO:22.

[0100] In a specific embodiment of the present application, the VHH-2 comprises the amino acid sequence shown in SEQ ID NO: 30 or SEQ ID NO: 31.

[0101] In a specific embodiment of the present application, the extracellular antigen binding domain targeting B7H3 comprises the amino acid sequence shown in any one of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 34.

[0102] In a specific embodiment of the present application, the transmembrane domain comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8A, CD8B, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4 (CD244), FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, and SLAM.

[0103] In a specific embodiment of the present application, the transmembrane domain comprises a transmembrane domain derived from CD8A.

[0104] In a specific embodiment of the present application, the transmembrane domain comprises the amino acid sequence shown in any one of SEQ ID NO: 36 to SEQ ID NO: 84.

[0105] In a specific embodiment of the present application, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:36.

[0106] In a specific embodiment of the present application, the intracellular co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the following group: CD28, CD137, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

[0107] In a specific embodiment of the present application, the intracellular costimulatory signaling domain is derived from the costimulatory signaling domain of 4-1BB.

[0108] In a specific embodiment of the present application, the intracellular co-stimulatory signaling domain comprises the amino acid sequence shown in any one of SEQ ID NO: 85 to SEQ ID NO: 117.

[0109] In a specific embodiment of the present application, the intracellular co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 86.

[0110] In a specific embodiment of the present application, the intracellular signal transduction domain comprises an intracellular signal transduction domain derived from one or more proteins selected from the following group: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12 and a domain comprising at least one ITAM.

[0111] In a specific embodiment of the present application, the intracellular signal transduction domain comprises a signaling domain derived from CD3ζ.

[0112] In a specific embodiment of the present application, the intracellular signal transduction domain comprises the amino acid sequence shown in any one of SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 103, SEQ ID NO: 118 to SEQ ID NO: 128.

[0113] In a specific embodiment of the present application, the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:118.

[0114] In a specific embodiment of the present application, a hinge region is included between the extracellular antigen binding domain and the transmembrane domain, and the hinge region comprises a hinge region derived from one or more proteins selected from the following group: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30 and LIGHT.

[0115] In a specific embodiment of the present application, the hinge region comprises a hinge region derived from CD8A.

[0116] In a specific embodiment of the present application, the hinge region comprises the amino acid sequence shown in any one of SEQ ID NO: 129 to SEQ ID NO: 150.

[0117] In a specific embodiment of the present application, the hinge region comprises the amino acid sequence shown in SEQ ID NO:137.

[0118] In a specific embodiment of the present application, the non-targeting portion of the CAR-T comprises the hinge region of CD8A, the transmembrane domain of the CD8A molecule, the intracellular co-stimulatory signaling domain of 4-1BB, and the intracellular signaling domain of CD3ζ.

[0119] In a specific embodiment of the present application, the non-targeting portion of the CAR-T comprises the amino acid sequence shown in SEQ ID NO: 151.

[0120] In a specific embodiment of the present application, the CAR-T further comprises a signal peptide fragment, the C-terminus of the signal peptide fragment is connected to the N-terminus of the extracellular antigen binding domain, and from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide fragment, an extracellular antigen binding domain, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular signal transduction domain.

[0121] In a specific embodiment of the present application, the signal peptide fragment includes a CD8A signal peptide fragment.

[0122] In a specific embodiment of the present application, the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:152.

[0123] In a specific embodiment of the present application, the CAR-T comprises the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 38.

[0124] In a specific embodiment of the present application, the expression and / or activity of one or more genes related to immune rejection in the CAR-T cells are downregulated.

[0125] In a specific embodiment of the present application, the immune rejection-related gene is selected from any one, two, three, four, five or six of the TRAC gene, TRBC gene, HLA-A gene, HLA-B gene, B2M gene and CIITA gene.

[0126] In a specific embodiment of the present application, the expression and / or activity of the TRAC gene and HLA-A gene of the CAR-T cell are downregulated.

[0127] In a specific embodiment of the present application, any one of the two TRAC alleles in the CAR-T cells is knocked out and any one of the two HLA-A alleles is knocked out.

[0128] In a specific embodiment of the present application, the TRAC gene exon of the CAR-T cell is knocked out and the HLA-A gene exon is knocked out.

[0129] In one specific embodiment of the present application, antisense RNA is administered to CAR-T cells.

[0130] In a specific embodiment of the present application, the antisense RNA comprises the nucleotide sequence shown in any one of SEQ ID NO: 153 to SEQ ID NO: 156.

[0131] In a specific embodiment of the present application, sgRNA targeting the exon portion of the TRAC gene is administered to CAR-T cells.

[0132] In a specific embodiment of the present application, the sgRNA targeting the exon portion of the TRAC gene includes the nucleotide sequence shown in any one of SEQ ID NO: 157 to SEQ ID NO: 171.

[0133] In a specific embodiment of the present application, sgRNA targeting the exon portion of the HLA-A gene is administered to CAR-T cells.

[0134] In a specific embodiment of the present application, the sgRNA targeting the exon portion of the HLA-A gene comprises a nucleotide sequence shown in any one of SEQ ID NO: 172 to SEQ ID NO: 212.

[0135] In a specific embodiment of the present application, sgRNA targeting the exon portion of the HLA-B gene is administered to CAR-T cells.

[0136] In a specific embodiment of the present application, the sgRNA targeting the exon portion of the HLA-B gene comprises the nucleotide sequence shown in any one of SEQ ID NO: 213 to SEQ ID NO: 215.

[0137] In one embodiment of the present application, a method for producing an allogeneic chimeric antigen receptor (CAR)-T cell (CAR-T cell) population against a target of interest comprises:

[0138] (a) Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors;

[0139] (b) activating T cells in the PBMCs;

[0140] (c) transducing the activated T-cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing a target CAR;

[0141] (d) disrupting the expression of TRAC genes and HLA-A genes in activated T-cells transduced with lentiviral vectors to obtain T-cells in which gene expression is disrupted;

[0142] (e) expanding T-cells in which gene expression is disrupted; and

[0143] (f) T-cell population enriched for TRAC-negative, HLA-A-negative cells;

[0144] This generates a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells).

[0145] In a specific embodiment of the present application, the lentiviral vector further expresses a safety switch.

[0146] In a specific implementation manner of the present application, the safety switch is RQR8.

[0147] In a specific embodiment of the present application, the CAR-T cells are administered intrathecally.

[0148] In a specific embodiment of the present application, the allogeneic chimeric antigen receptor-T cells are universal CAR-T cells, which can also be called allogeneic universal CAR-T, etc. Any of the above names or terms are regarded as allogeneic CAR-T cells (UCAR-T).

[0149] In a specific embodiment of the present application, the expression of the TRAC gene and the HLA-A gene is disrupted so that the expression level and / or activity of the related genes are downregulated, including downregulating the expression and / or activity of the nucleic acid molecules encoding the genes; and / or downregulating the expression and / or activity of the protein products encoded by the genes.

[0150] In a specific embodiment of the present application, achieving downregulation of gene expression level and / or activity comprises administering to T cells one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA and CRISPR / Cas9 system.

[0151] Beneficial effects of the present application: The present application discloses a CAR-T immunotherapy method for recurrent high-grade glioma, and provides a method for administering the immunotherapy method to patients by local administration of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) targeting B7H3. Depending on the patient's condition, the CAR will show a CR state at least 4 weeks after the administration of the CAR-T cells; the toxic effects of the subjects under B7H3 UCAR-T cell treatment are all grade 1 or 2, with good safety; the treatment significantly prolongs the overall survival of the subjects and has a higher objective response rate; compared with the results of historical treatments, it has a longer survival period and a higher objective response rate; the B7H3UCAR-T cells are well retained in the subjects and the effect is sustained. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] The accompanying drawings are provided to facilitate a better understanding of the present application and do not constitute an undue limitation on the present application.

[0153] Figure 1 shows the structure of the dual-epitope B7H3 CAR gene lentiviral expression vector of the present application.

[0154] Figure 2 shows the baseline characteristics of the patients in Example 4. A) Analysis stratified by sex; B) Analysis stratified by tumor type; C) Analysis stratified by age.

[0155] FIG3 shows the cumulative incidence of adverse events (AEs) reported following treatment in Example 4.

[0156] FIG4 shows the secretion levels of IL-6 in peripheral blood and cerebrospinal fluid (CSF) after injection.

[0157] FIG5 shows the tumor size of a subject at different time periods.

[0158] Figure 6 shows the best percent change from baseline in tumor size based on the investigator's target lesion assessment.

[0159] FIG7 is a bar graph of 12-month overall survival for the treatments in Example 4 and historical data.

[0160] FIG8 is a bar graph showing the statistical data of the average overall survival rate of the treatment in Example 4.

[0161] Figure 9 shows the CAR copy number in the cerebrospinal fluid (CSF) detected after each administration in Example 4.

[0162] FIG10 shows the results of CAR copy number detection in the cerebrospinal fluid (CSF) of the response group and the non-response group in Example 4.

[0163] Figure 11 shows the pharmacokinetic characteristics of single-dose administration in different dose groups in Example 5 - peak CAR copy number.

[0164] Figure 12 shows the pharmacokinetic characteristics of single-dose administration in different dose groups in Example 5 - CAR copy number.

[0165] Figure 13 shows the pharmacokinetic characteristics of multiple doses in different dose groups in Example 5 - CAR copy number.

[0166] FIG14 is the pharmacodynamic characteristics of multiple administrations in different dosage groups in Example 5—the detection results of IL6 levels in patients.

[0167] FIG. 15 is the pharmacodynamic characteristics of multiple administrations in different dosage groups in Example 5—the detection results of TNF-α levels in patients.

[0168] FIG. 16 is the pharmacodynamic characteristics of multiple administrations in different dosage groups in Example 5—the detection results of IFN-α levels in patients.

[0169] Figure 17 is a flow chart of patient recruitment and grouping in the study of Example 5

[0170] Figure 18 shows four statistical analysis sets in the study of Example 5.

[0171] Figure 19 is the KM survival curve and median survival time (mOS) based on 3 PPS in Example 5

[0172] Figure 20 shows the 12-month OS rate (12m-OS) based on 3 PPS in Example 5

[0173] Figure 21 is a forest plot based on univariate subgroup analysis in Example 5 - PPS1

[0174] Figure 22 shows the efficacy data in Example 5: A) waterfall chart: based on PPS2.B) spider chart: based on PPS2.C) swimming chart: based on PPS1. DETAILED DESCRIPTION

[0175] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0176] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

[0177] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0178] Generally speaking, the terms used in this specification have the following meanings.

[0179] The term "intrathecal drug delivery" refers to the process of injecting drugs directly into the cerebrospinal fluid, bypassing the blood-brain barrier and entering the central nervous system. It is a common method of drug delivery for studying the mechanisms of action of drugs at the spinal level and is widely used in research areas such as injury, pain, central nervous system diseases, and cancer treatment. Through intrathecal catheterization, drugs can be selectively injected into the spinal cord as needed, such as through continuous infusion with a sustained-release pump or multiple injections with a syringe. Common intrathecal drug delivery methods include intrathecal catheterization via the cisterna magna and intrathecal catheterization of the lumbar spinal cord.

[0180] The term "glioma" refers to tumors originating from brain glial cells. It is the most common primary intracranial tumor. The 2021 edition of the WHO Central Nervous System Tumor Classification divides gliomas into grades 1 to 4, with grades 1 and 2 being low-grade gliomas and grades 3 and 4 being high-grade gliomas. High-grade gliomas are mainly glioblastomas or diffuse midline gliomas. Glioblastomas are characterized by irregular peripheral enhancement and massive central necrosis, with edema visible outside the enhancement. Diffuse midline gliomas often occur in midline structures such as the thalamus and brainstem. MRI shows long T1 and long T2 signals, and enhanced scans may have varying degrees of enhancement.

[0181] The term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, which are generally two in the simplest embodiment, and when in immune effector cells, provide cell specificity to target cells (usually cancer cells) and generate intracellular signals. In some embodiments, CAR includes at least one extracellular antigen binding domain (such as VHH, scFv or part thereof), a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some embodiments, the group of polypeptides is in the same polypeptide chain (for example, comprising a chimeric fusion protein). In some embodiments, the group of polypeptides is discontinuous with each other, for example, in different polypeptide chains. In some aspects, the group of polypeptides includes a dimerization switch, which can couple polypeptides to each other in the presence of a dimerization molecule, for example, the antigen binding domain can be coupled to the intracellular signaling domain. On the one hand, the stimulatory molecule of CAR is a zeta chain associated with a T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-ζ). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined below. On the one hand, the costimulatory molecule can be selected from 4-1BB (i.e., CD137), CD27, ICOS and / or CD28. On the one hand, CAR comprises a chimeric fusion protein that may include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein that may include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. On the one hand, CAR includes a chimeric fusion protein, which may include an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain includes a functional signal transduction domain derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR includes a chimeric fusion protein, which may include an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain includes at least two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. In one aspect, CAR includes an optional leader sequence on the amino terminus (N-ter) of the CAR fusion protein.In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., VHH) during cellular processing and localizes the CAR to the cell membrane.

[0182] The term "antibody" is generally used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species.

[0183] A full-length antibody typically refers to an antibody composed of two "full-length antibody heavy chains" and two "full-length antibody light chains". A "full-length antibody heavy chain" is generally a polypeptide that, in the N-terminal to C-terminal direction, consists of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and in the case of antibodies of the IgE subclass, optionally also includes an antibody heavy chain constant domain 4 (CH4). In some embodiments, a "full-length antibody heavy chain" is a polypeptide composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is generally a polypeptide composed of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). The two full-length antibody chains are linked together by an inter-polypeptide disulfide bond between the CL domain and the CH1 domain and an inter-polypeptide disulfide bond between the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE).

[0184] The term "antigen-binding fragment" (also referred to herein as "targeting portion" or "antigen-binding portion") generally refers to a portion of an antibody molecule that comprises the amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is referred to as an "epitope" as described above. An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily comprise both. An Fd fragment, for example, has two VH regions and generally retains some antigen-binding function of an intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) a Fab fragment, a monovalent fragment having a VL, VH, constant light chain (CL) and CH1 domains; (2) a F(ab')2 fragment, a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is incorporated herein by reference in its entirety), which has a VH domain; (6) isolated complementarity determining regions (CDRs); and (7) single-chain Fv (scFv), for example, derived from an scFV library.Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods via a synthetic linker that enables them to be prepared as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)); and (8) VHH, "VHH" refers to the variable antigen-binding domain of heavy chain antibodies from the Camelidae family (camel, dromedary, llama, alpaca, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and reviewed in Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs may also be referred to as nanobodies (Nb) and / or single-domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same manner as for intact antibodies.

[0185] The term "single domain antibody" or "VHH" generally refers to a class of antibodies that lack an antibody light chain and have only a heavy chain variable region. In some cases, a single domain antibody can be derived from a Bactrian camel, a dromedary camel, an alpaca, a llama, a nurse shark, a white shark, or a ray (for example, see Kang Xiaozhen et al., Journal of Biotechnology, 2018, 34(12): 1974-1984). For example, a single domain antibody can be derived from an alpaca. A single domain antibody can be composed of a heavy chain variable region (VH). The term "heavy chain variable region" generally refers to the amino terminal domain of the heavy chain of an antigen binding fragment. The heavy chain variable region can be further divided into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The heavy chain variable region contains a binding domain that interacts with an antigen.

[0186] The term "complementarity determining region" (CDR) generally refers to the complementarity determining region within the variable region of an antigen binding fragment. In this application, there are three CDRs in the heavy chain variable region, which are named HCDR1, HCDR2, and HCDR3 for each variable region. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antigen-binding fragment, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that despite great diversity at the amino acid sequence level, certain subsections within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subsections are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs. CDRs, such as the Chothia CDRs, have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5): 732-45 (1996)). In addition, other CDR boundary definitions may not strictly follow one of the above systems but will still overlap with the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. In this application, the IMGT numbering system is used.

[0187] The term "functional variant" generally refers to an amino acid sequence that has substantially the same function (e.g., can possess the properties of a chimeric antigen receptor) and has at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity thereto. In certain embodiments, the amino acid sequence variant is an amino acid sequence that has substantially the same function (e.g., can possess the properties of a chimeric antigen receptor) and comprises one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid substitutions, deletions, or additions thereto.

[0188] The term "specific binding" refers to the interaction between a binding molecule (e.g., an antibody) and its binding partner (e.g., an antigen), and generally refers to the presence of a specific structure (e.g., an antigenic determinant or epitope) on the binding partner. In other words, even when the binding partner is present in a mixture of other molecules or organisms, the antibody will still preferentially bind to or recognize the binding partner. Binding can be mediated by covalent or non-covalent interactions or a combination of the two. In other words, the term "specific binding" generally refers to immunospecific binding to an antigenic determinant or epitope and not immunospecific binding to other antigenic determinants or epitopes. The binding molecule of an immunospecific binding antigen can bind to other peptides or polypeptides with lower affinity, as measured by (e.g., radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BIACORE, or other analyses known in the art. The binding molecule of an immunospecific binding antigen, or a fragment thereof, can cross-react with related antigens with the same epitope. In some cases, the binding molecule of an immunospecific binding antigen, or a fragment thereof, does not cross-react with other antigens.

[0189] The term "KD" is used interchangeably with "KD" and generally refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, expressed in M ​​(mol / L). KD can be calculated by taking the concentrations of substance A and the dissociated substances A and B: KD = c(A) * c(B) / c(AB). This formula indicates that a larger KD value indicates greater dissociation, meaning a weaker affinity between substances A and B; conversely, a smaller KD value indicates less dissociation, meaning a stronger affinity between substances A and B.

[0190] The term "isolated" generally refers to a molecule (e.g., an antibody, nucleic acid, etc.) that is at least partially separated from other molecules with which it is normally associated in its native state. An "isolated polypeptide" is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, cellular debris, and growth medium. An "isolated nucleic acid" is at least partially separated from the nucleic acids that normally flank the polynucleotide in its native state.

[0191] The term "isolated polypeptide" generally refers to any polypeptide containing more than one different polypeptide chain. In some embodiments, the isolated polypeptide comprises two polypeptide chains, each comprising a chimeric antigen receptor and an enhancer. In some embodiments, the polypeptide chain comprising the chimeric antigen receptor and the polypeptide chain comprising the enhancer can be linked together by one or more intermolecular bonds (including but not limited to disulfide bonds) to form a macromolecular complex of two or more polypeptides. In some embodiments, the isolated polypeptide may comprise a single polypeptide of two different polypeptide chains (e.g., a chimeric antigen receptor and an enhancer) connected by a polypeptide linker. In this case, the polypeptide may physically represent a single chain, but two or more parts of the single chain may function as if they were two independent polypeptide chains. In other embodiments, the linker is a cleavable peptide linker (e.g., a 2A peptide), and the chimeric antigen receptor and enhancer on the same peptide chain can be cleaved into two polypeptide chains.

[0192] The term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides, or ribonucleotides of any length, or their analogs, separated from their natural environment or artificially synthesized.

[0193] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct entry of internal ribosomes into the start codon of a cistron (protein coding region), such as ATG, resulting in cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In specific embodiments, the vectors contemplated by the present invention comprise one or more polynucleotides of interest encoding one or more polypeptides. In specific embodiments, in order to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

[0194] The term "self-cleaving peptide" or "sequence encoding a self-cleaving peptide" generally refers to a linker sequence used to include a site in a vector that promotes ribosome skipping and thereby produces two polypeptides from a single promoter. Such self-cleaving peptides include but are not limited to T2A and P2A peptides or sequences encoding self-cleaving peptides.

[0195] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression by transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0196] The term "viral vector" is broadly used to refer to nucleic acid molecules (e.g., transfer plasmids) or viral particles that mediate nucleic acid transfer, including virally derived nucleic acid elements that typically facilitate transfer or integration of nucleic acid molecules into the cellular genome. Viral particles typically include various viral components and sometimes host cell components in addition to nucleic acid. A viral vector can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or the nucleic acid itself.

[0197] The term "lentivirus" generally refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedivirus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In a particular embodiment, a lentivirus is used to deliver a polynucleotide comprising a CAR to a cell.

[0198] The term "host cell" or "cell" generally refers to an individual cell, cell line or cell culture that can or already contains a vector comprising the isolated nucleic acid molecule described herein, or that is capable of expressing the isolated antigen-binding fragment described herein. The host cell may include the progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but they may be capable of expressing the isolated antigen-binding fragment described herein. The host cell can be obtained by in vitro transfection of cells using the vectors described herein. The host cell can be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell). For example, the host cell can be an Escherichia coli cell. For example, the host cell can be a yeast cell. For example, the host cell can be a mammalian cell. For example, the mammalian cell can be a CHO-K1 cell.

[0199] The term "T cell" or "T lymphocyte" can be any T cell, such as a cultured T cell, for example a primary T cell, or a T cell from a cultured T cell line, or a T cell obtained from a mammal (preferably a primate, species including monkey, dog or human). If obtained from a mammal, the T cell can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus or other tissues or fluids. The T cell can also be enriched or transfected. The T cell can be obtained by maturing hematopoietic stem cells into T cells in vitro or in vivo. In exemplary aspects, the T cell is a human T cell. In exemplary aspects, the T cell is a T cell isolated from a human. T cells can be any type of T cell, including NKT cells, and can be of any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells; CDA+ helper T cells; such as Th1 and Th2 cells, CD8+ T cells (such as cytotoxic T cells); peripheral blood mononuclear cells (PBMC); peripheral blood leukocytes (PBL); tumor infiltrating cells (TIL); memory T cells; untreated T cells and the like. Preferably, the T cells are CD8+ T cells or CD4+ T cells. In some alternatives, the T cells are allogeneic (different donors from the same species) to the recipient of the cells or the cells to be received (such as the cells are in the form of a therapeutic composition); in some alternatives, the T cells are autologous (the donor and the recipient are the same); in some alternatives, the T cells are syngeneic (the donor and the recipient are different, but are identical twins).

[0200] The term "natural killer cell" ("NK cell") generally refers to a type of cytotoxic lymphocyte of the immune system. NK cells provide a rapid response to virus-infected cells and respond to transformed cells. Generally speaking, immune cells detect peptides presented by the major histocompatibility complex (MHC) molecules on the surface of infected cells from pathogens, triggering cytokine release, causing lysis or apoptosis. However, NK cells are unique because they have the ability to recognize stressed cells, regardless of whether the peptides from pathogens are present on the MHC molecules. Because they do not need to be pre-activated to kill the target's initial understanding, they are named "natural killer cells." NK cells are large granular lymphocytes (LGL) and are known to differentiate and mature in the bone marrow (where they then enter the circulation). In some embodiments, NK cells are mammalian NK cells. Examples of "mammals" or "mammals" include primates (e.g., humans), canines, felines, rodents, pigs, ruminants, etc. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats, and mice. In one embodiment, the mammalian NK cells are human NK cells.

[0201] The term "immune effector cell" generally refers to an immune cell that participates in an immune response and performs effector functions. For example, performing effector functions may include clearing foreign antigens or promoting immune effector responses. Immune effector cells may include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0202] The immune effector cells of the present application can be autologous / self (autologous / autogeneic) ("one's own") or non-autologous ("non-one's own", such as allogeneic, isogenic or allogeneic). In the present application, the term "autologous" generally refers to cells from the same subject. "Allogenic" generally refers to cells that are of the same species but genetically different from the compared cells. "Isogenic" generally refers to cells of a different subject that are genetically identical to the compared cells. "Allogenic" generally refers to cells of a species different from the compared cells. In some embodiments, the cells of the present application are autologous or allogenic.

[0203] The term "modification" generally refers to changing the state or structure of a cell and / or a change in the state or structure of a cell. The change is generally compared to the state or structure of a corresponding cell without the modification, and the change may include a change in the expression level or function of an endogenous gene, such as downregulating, upregulating, or not expressing the expression level of an endogenous gene in the cell through genetic engineering means, such as homologous recombination, CRISPR / Cas9 system gene editing, etc.; the change may also include a change in the expression, structure, or function of a cell protein, such as a change in the expression level or function of a corresponding protein achieved by a change in the expression level or function of the endogenous gene, such as a change in protein expression, structure, or function achieved by regulating protein translation or post-translational modification; the change may also include the introduction of exogenous genes, the expression of exogenous proteins, etc.

[0204] The term "TRAC" generally refers to the T cell receptor alpha chain constant region (T cell receptor alpha constant). T cell receptor (TCR) generally refers to a specific receptor located on the surface of T cells that can recognize antigens bound to major histocompatibility complex (MHC) molecules. TCR is usually composed of two different protein chains (i.e., a heterodimer). In humans, the TCR in most T cells is composed of an α chain and a β chain (encoded by TRA and TRB, respectively). This type of T cell is called an αβT cell. In a small number of T cells, the TCR is composed of a γ chain and a δ chain (encoded by TRG and TRD, respectively). This type of T cell is called a γδT cell. Normally, αβT cells account for about 95% of the total T cell population, and γδT cells account for about 5% of the total T cell population. This ratio changes during individual development and in disease states (such as leukemia), and also varies between species. Each TCR chain contains a variable region and a constant region. In humans, the gene encoding the α chain (TRA, for example, as shown in HGNC:12027) is located on chromosome 14 and consists of multiple gene segments, including the variable segment (V), the joining segment (J), and the constant region (C). The TRAC gene generally refers to the gene sequence encoding the constant region (C) of the T cell receptor α chain (for example, as shown in HGNC:12029), which is located on chromosome 14 (14q11.2; 14: 22,547,505-22,552,131). Typically, one of the variable segment (V) genes encoding the N-segment antigen recognition domain rearranges with one of the joining segments (J) to produce a functional V region exon. This exon is transcribed and, through splicing, joins with the constant region (C), forming the coding sequence for the T cell receptor α chain.

[0205] The term "major histocompatibility complex antigen" ("MHC", also referred to as "human leukocyte antigen" ("HLA") in the case of humans) generally refers to proteins expressed on the surface of cells that confer a unique antigenic identity to the cell. MHC / HLA antigens are target molecules that are recognized by T cells and NK cells as either originating from the same hematopoietic stem cell source as the immune effector cells ("self") or as originating from another hematopoietic reconstituting cell source ("non-self"). Two major classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, C in humans) enable each cell to be recognized as "self", while HLA class II antigens (DR, DP, and DQ in humans) participate in the reaction between lymphocytes and antigen-presenting cells. Both have been implicated in the rejection of transplanted organs. An important aspect of the HLA gene system is its polymorphism. Different alleles exist for each gene, MHC class I (A, B, and C) and MHC class II (DP, DQ, and DR). HLA alleles are represented by numbers and subscripts. For example, two unrelated individuals might carry the class I HLA-B genes B5 and Bw41, respectively. The allele products differ in one or more amino acids in the α and / or β domains. A wide range of specific antibodies or nucleic acid reagents are used to type an individual's HLA haplotype using white blood cells expressing class I and class II molecules. The genes commonly used for HLA typing are the six MHC class I and class II proteins: HLA-A; HLA-B; and HLA-DR, each with two alleles. The HLA genes are clustered in a "superlocus" located at chromosomal location 6p21. This "superlocus" encodes the six classical HLA genes and at least 132 protein-coding genes that play important roles in the regulation of the immune system and several other fundamental molecular and cellular processes. The entire locus roughly measures 3.6 Mb and contains at least 224 loci. One effect of this clustering is a "haplotype," a set of alleles present on a single chromosome, inherited from one parent, that tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, and some alleles tend to associate together. Identifying a patient’s haplotype can help predict the probability of finding a matching donor and aid in developing search strategies, because some alleles and haplotypes are more common than others, and their frequencies vary among different racial and ethnic groups.

[0206] The term "HLA-A" generally refers to a class of human leukocyte antigen (HLA-A) polypeptide chains, encoded by the HLA-A gene located on human chromosome 6p21.3 (e.g., information shown as HGNC:4931). HLA-A is one of the three major types of polypeptides that make up the class I MHC molecules on the surface of human cells, the others being HLA-B and HLA-C. The HLA-A class MHC I molecule is a heterodimer composed of the α chain encoded by the HLA-A gene and the β chain (β2-microglobulin) encoded by the B2M gene. The α chain encoded by the HLA-A gene can include an α1 domain, an α2 domain, an α3 domain, a transmembrane region, and a cytoplasmic region. The α1 and α2 domains can bind to peptides, allowing the peptides to be presented to immune cells by MHC I molecules (e.g., HLA-A class). In humans, similar to most mammals, the α chain of the MHC I molecule is polymorphic, with significant variation in its primary structure. As of December 2013, there were 2,432 known HLA-A alleles, encoding 1,740 active proteins and 117 inactive proteins. For the purposes of this application, HLA-A alleles may include sequence information for different HLA-A alleles named by the WHO HLA Factor Nomenclature Committee, as listed in the IMGT / HLA Database, version 3.38.0 (https: / / www.ebi.ac.uk / ipd / imgt / hla / ).

[0207] The term "HLA-B" generally refers to the gene family that is part of the human leukocyte antigen (HLA) complex. HLA is the human version of the major histocompatibility complex (MHC), a gene family present in many species. Genes within this complex are divided into three basic groups: class I, class II, and class III. In humans, the HLA-B gene and two related genes, HLA-A and HLA-C, are the major genes of MHC class I. The HLA-B gene is located on the short (p) arm of chromosome 6, at cytoband 21.3, from base pairs 31,353,871 to 31,357,211. HLA-B is one of the three major HLAs that should be matched between donor and recipient. These are HLA-A and HLA-B (all MHC class I), and HLA-DR (MHC class II). If both tissues have the same genes encoding these three HLAs, the likelihood and severity of rejection is minimized. Hundreds of versions (alleles) of HLA-B are known, each with a specific number (for example, HLA-B27). Closely related alleles are grouped together; for example, at least 28 very similar alleles are subtypes of HLA-B27. These subtypes are designated HLA-B*2701 to HLA-B*2728.

[0208] The term "HLA-mismatched" refers to a donor-recipient pair in which at least one HLA antigen (particularly for HLA-A, HLA-B and HLA-DR) is mismatched between the donor and the recipient, such as a donor who provides a hematopoietic stem cell transplant to a recipient in need of hematopoietic stem cell transplantation therapy. In some embodiments, one haplotype is matched and the other is mismatched. HLA-mismatched donor-recipient pairs may have an increased risk of graft rejection relative to HLA-matched donor-recipient pairs because, in the case of HLA-mismatched donor-recipient pairs, endogenous T cells and NK cells are more likely to recognize the incoming graft as foreign, and such T cells and NK cells are therefore more likely to generate an immune response against the graft.

[0209] The term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides that have similar binding properties to a reference nucleic acid (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include variants thereof modified in a conservative manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0210] The term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence.

[0211] The term "gene mutation" generally refers to a change in the base pair composition or arrangement order of a gene structure, such as a point mutation caused by a single base change, or a deletion, duplication, or insertion of multiple bases.

[0212] The term "gene silencing" generally refers to the use of regulatory mechanisms to prevent the expression of certain genes. There are two main types: one is gene silencing (TGS) at the transcriptional level due to factors such as DNA methylation, heterochromatinization, and position effects; the other is post-transcriptional gene silencing (PTGS), which affects gene expression by specifically intervening in the target RNA at the level after gene transcription. Usually, when a gene is silenced, the expression of the corresponding gene is downregulated / reduced. When a gene is knocked out, it is usually manifested as non-expression. For example, in a cell, when all alleles of a specific gene are knocked out, the expression of the gene disappears. Gene silencing is generally considered to be a gene knockdown mechanism, and methods commonly used to silence genes include RNAi.

[0213] The term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0214] The term "exogenous" refers to any substance introduced from outside of or generated outside of an organism, cell, tissue, or system.

[0215] The term "antisense RNA" generally refers to a single-stranded RNA that is complementary to the transcribed product, mRNA (messenger RNA). Antisense RNA can inhibit gene expression by binding to mRNA. For example, antisense RNA binding to the target mRNA increases the susceptibility of the double-stranded RNA molecule to RNase III, leading to its degradation. For example, antisense RNA can bind to the upstream noncoding region of an mRNA, thereby directly inhibiting translation of the target mRNA.

[0216] The term "siRNA" is usually an abbreviation for Small interfering RNA or short interfering RNA. siRNA is a type of double-stranded non-coding RNA molecule with a length of approximately 18-28 base pairs. It can interfere with the expression of specific genes by causing the degradation of mRNA through complementary binding to mRNA. In some embodiments, siRNA can be a product obtained by treating long double-stranded RNA or shRNA with the Dicer enzyme. In some embodiments, siRNA enters the cell and forms an RNA-induced silencing complex (RISC) with other proteins. The sense strand is degraded, and the antisense strand can bind to the complementary targeting sequence, thereby achieving gene silencing.

[0217] The term "shRNA" is usually an abbreviation for short hairpin RNA. shRNA usually consists of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure. It can also usually include 5-6 T bases as a transcription terminator for RNA polymerase III. In certain embodiments, shRNA can enter cells via a viral vector or plasmid, and be transcribed under the action of polymerase II or polymerase III. The transcription product is exported from the cell nucleus (usually via Exportin 5) and then processed by Dicer and transported to RISC. The sense strand is degraded, and the antisense strand can bind to the complementary targeting sequence, thereby achieving gene silencing.

[0218] The term "CRISPR / Cas system" generally refers to a group of molecules comprising RNA-guided nucleases or other effector molecules and gRNA molecules, which can guide and enable the modification of nucleic acids at target sequences by RNA-guided nucleases or other effector molecules, such as causing target sequence degradation. In certain embodiments, the CRISPR system comprises gRNA and Cas proteins, for example, Cas9 proteins. Systems comprising Cas9 or its functional mutants are referred to as "Cas9 systems" or "CRISPR / Cas9 systems" in this application. In certain embodiments, gRNA molecules and Cas molecules can be compounded to form a ribonucleoprotein (RNP) complex.

[0219] The terms "gRNA molecule" or "guide RNA", "guide RNA", "guide RNA", "guide RNA molecule", "gRNA" are used interchangeably and generally refer to a nucleic acid molecule that can promote a specific guide RNA-guided nuclease or other effector molecule (generally in complex with a gRNA molecule) to a target sequence. In certain embodiments, the guidance is achieved by hybridizing a portion of the gRNA to the DNA (e.g., through a gRNA guide domain) and by binding a portion of the gRNA molecule to the RNA-guided nuclease or other effector molecule (e.g., at least through gRNA tracr). In certain embodiments, the gRNA molecule consists of a single continuous polynucleotide molecule, referred to herein as a "single guide RNA" or "sgRNA", etc. In other embodiments, the gRNA molecule consists of multiple (e.g., two) polynucleotide molecules that can themselves associate (generally by hybridization), referred to herein as a "dual guide RNA" or "dgRNA", etc.

[0220] The terms "tumor" and "cancer" are used interchangeably and generally refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "cancer" or "tumor" include pre-malignant and malignant cancers and tumors, and also encompass solid tumors and non-solid tumors.

[0221] The term "pharmaceutically acceptable" generally refers to those compounds, materials, compositions and / or dosage forms which are, commensurate with a reasonable benefit / risk ratio, suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications.

[0222] The term "pharmaceutically acceptable carrier" generally refers to any of those conventionally used carriers and is limited only by physical-chemical considerations (such as solubility and lack of reactivity with active binding agents) and by route of administration. Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and is a carrier that does not have adverse side effects or toxicity under the conditions of use. In some embodiments, when administered to an animal or human, the carrier does not produce adverse, allergic, or other inappropriate reactions. In some aspects, the pharmaceutical composition does not contain pyrogens and other impurities that may be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc.; their use is well known in the art.

[0223] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0224] The term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability due to the disease.

[0225] A "therapeutically effective amount" or "effective amount" of B7H3 targeted CAR-T cells is also an amount or dose in which the beneficial effects of treatment outweigh any toxic or deleterious effects, such as CRS, of the B7H3 targeted CAR-T cells. The term "therapeutically effective amount" encompasses an amount that effectively "treats" a subject (e.g., a patient). In one embodiment, the therapeutically effective dose is the minimum effective dose (MED) of B7H3 targeted CAR-T cells for treating multiple myeloma in a subject. In one embodiment, the therapeutically effective dose is the maximum tolerated dose (MTD) of B7H3 targeted CAR-T cells that does not result in unresolved CRS in the subject.

[0226] The term "no lymphocyte clearing" or "no lymphocyte depletion" refers to not depleting the subject's lymphocytes. This includes not administering a lymphocyte depletion agent, whole-body radiation therapy, a combination thereof, or other means of causing lymphocyte depletion; and also includes administering a lymphocyte depletion agent, whole-body radiation therapy, a combination thereof, or other means of causing lymphocyte depletion, where the lymphocyte depletion rate in the subject is less than 60%.

[0227] "Include" usually means to include, encompass, contain or encompass. In some cases, it also means "to be" or "to be composed of."

[0228] "About" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0229] Complete response (CR) means complete disappearance of all target lesions except nodular disease. All target nodules must shrink to normal size (short axis <10 mm). All target lesions must be evaluable.

[0230] Partial response (PR) means the sum of the diameters of all measurable target lesions is ≥30% lower than baseline. The short diameter is used for the sum of target nodules, while the longest diameter is used for the sum of all other target lesions. All target lesions must be evaluated.

[0231] Disease progression (PD) is defined as the minimum sum of the diameters of all target lesions measured during the entire experimental study, with a relative increase of at least 20% (if the baseline measurement value is the minimum, the baseline value is used as the reference); in addition, the absolute value of the sum of the diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered disease progression).

[0232] Stable disease (SD) refers to the target lesion that has neither decreased to the level of PR nor increased to the level of PD, but is somewhere in between. The minimum value of the sum of the diameters can be used as a reference for research.

[0233] The objective response rate (ORR) is the proportion of patients whose tumor volume shrinks to a predetermined value and can be maintained for the minimum time required. It is the sum of the complete response (CR) and partial response (PR) rates, ORR = CR + PR.

[0234] Disease control rate (DCR) is the percentage of cases achieving remission (PR+CR) and stable lesions (SD) after treatment among the evaluable cases, DCR = CR+PR+SD.

[0235] Progression-free survival (PFS) is the time from the start of treatment to disease progression or death from any cause. Generally, a longer PFS indicates a longer, better quality of life for the patient. Clinically, median PFS is often used to express this. For example, if 50% of patients have a progression-free survival of 5 months, the median PFS is 5 months.

[0236] Overall survival (OS) is the time from the start of treatment to the death of the patient due to any cause, that is, the time the patient survives treatment.

[0237] Pharmacokinetics (PK) is a discipline that quantitatively studies the processes of drugs in vivo (absorption, distribution, metabolism, and excretion), applying mathematical principles and methods to explain the dynamic laws governing drug interactions within the body. The basis for determining drug dosage and dosing intervals is whether the drug can achieve a safe and effective concentration at its site of action. Drug concentration at the site of action changes dynamically as a result of the drug's in vivo processes.

[0238] The KPS score is the Karnofsky functional status scale (KPS, percentage method). The higher the score, the better the health and the more tolerable the side effects of treatment, which makes it more likely that patients will receive complete treatment. A Karnofsky score of 80 or above is generally considered independent, meaning patients can take care of themselves. A score of 50 to 70 is considered semi-independent, meaning patients can only take care of themselves partially. A score below 50 is considered dependent, meaning patients need help from others. Patients with a score of 80 or above have better postoperative conditions and longer survival rates.

[0239] The ECOG performance status score is an indicator of a patient's general health and treatment tolerance based on their physical activity. The ECOG performance status score has six levels: 0, 1, 2, 3, 4, and 5. Patients with activity levels 3 and 4 are generally considered unsuitable for chemotherapy.

[0240] Cerebrospinal fluid (CSF) is produced in the choroid plexus within the lateral ventricles. It is mostly an ultrafiltrate of plasma, but it also contains components actively secreted by the choroid plexus. Mechanical and permeability barriers exist between the blood and CSF, and between the CSF and the brain, known as the blood-CSF barrier and the CSF-brain barrier, respectively. CSF is produced by specialized blood vessels within the brain and reabsorbed by specialized venous tributaries. It provides shock absorption and support for the central nervous system. A normal adult has 130 ml of clear CSF; CSF is often collected for testing to diagnose neurological disorders. It is one of the inherent contents of the cranial cavity. It is primarily produced by the choroid plexus within the lateral, third, and fourth ventricles. The secretory pressure of CSF is primarily determined by the difference between mean arterial pressure and intracranial pressure (ICP). CSF absorption occurs primarily through the arachnoid granulations, where CSF flows unidirectionally into the venous sinuses at a constant rate. The rate of absorption depends on the pressure difference between ICP and venous pressure. Secretion and absorption are in relative equilibrium, with ICP being crucial for maintaining this balance.

[0241] Peripheral blood is the blood outside the bone marrow. In clinical practice, some methods are often used to release hematopoietic stem cells in the bone marrow into the blood, and then extract and separate hematopoietic stem cells from the blood. We call the stem cells obtained in this way peripheral blood stem cells.

[0242] Cytokine release syndrome (CRS), also known as cytokine storm, is a severe systemic inflammatory response syndrome caused by the activation of immune cells and the release of large amounts of cytokines. Clinically, it is characterized by fever and multiple organ dysfunction. It is a supraphysiological response to immunotherapy, resulting from the activation or participation of endogenous or infused T cells and / or other immune effector cells. Symptoms can be progressive and must include fever at onset, but may also include hypotension, capillary leak, hypoxia, and end-organ dysfunction.

[0243] Immune effector cell-associated neurotoxicity syndrome (ICANS) is a unique neurological syndrome that occurs after CAR-T cell infusion. Symptoms primarily include hallucinations, confusion, and delirium. While onset is rapid, the duration is short. Very few patients experience aphasia, and in some severe cases, severe CNS reactions can cause loss of consciousness for several days. The median onset is four days after infusion. ICANS is more common in patients treated with CD19- and BCMA C-T cells. ICANS can occur concurrently with CRS or shortly after CRS resolves, with some patients experiencing delayed onset up to one month after infusion. ICANS is typically self-limited, with symptoms typically lasting 5-17 days. The onset, duration, and severity of ICANS may depend on the CAR product and the patient's disease status. The ICANS scoring system utilizes the Immune Effector Cell-Associated Encephalopathy (ICE) score, which categorizes patients based on the most severe symptom across five domains: level of consciousness, severe motor weakness, seizures, and signs of increased intracranial pressure or cerebral edema. The mainstay of treatment for ICANS is supportive care and corticosteroids. Tocilizumab does not resolve ICANS and may worsen it. Therefore, when both occur simultaneously, the management of ICANS may take precedence over the management of low-grade CRS.

[0244] A "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0245] Example 1 Design of anti-B7H3 chimeric antigen receptor and modification of lentiviral vector

[0246] First, an anti-B7H3 chimeric antigen receptor (CAR) needs to be designed. As shown in Figure 1, the anti-B7H3 CAR structure includes: two B7H3 antigen-binding regions (derived from a combination of different anti-B7H3 single-domain antibodies), a CD8A extracellular hinge region, a CD8A transmembrane region, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ activation signaling domain.

[0247] The sequence of B7H3 CAR is shown in SEQ ID NO:5.

[0248] Secondly, it is necessary to construct the B7H3 CAR lentiviral expression vector based on the B7H3 sequence information and CAR vector structure.

[0249] The commercial lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP was selected as the backbone for the vector's component modifications. First, the vector's ampicillin resistance gene, β-lactamase, was replaced with an aminoglycoside phosphotransferase derived from Tn5, conferring kanamycin resistance. Second, the CMV promoter and its adjacent downstream multiple cloning site, which pose a potential threat in in vivo applications, were deleted. Finally, the copGFP gene, expressed from the original vector under the EF1 promoter, was deleted, retaining the SalI restriction site. A SmaI restriction site was added to the 5' end of SalI for vector construction, creating the final destination vector. The added SmaI restriction site is a single restriction site in the final destination vector; it is absent from other sequences in the vector. After optimization, the chimeric antigen receptor lentiviral expression vector was constructed. After sequence confirmation by Sanger sequencing, lentiviral packaging was performed.

[0250] Example 2 Preparation of TRAC, HLA-A double knockout B7H3-specific UCAR-T cells

[0251] This application provides a B7H3 UCAR-T cell with higher safety, which is mainly manifested in two strategies:

[0252] The primary donor screening strategy is to select donors based on HLA-A and / or HLA-B homozygosity in the population. Cells from these donors can cover a large patient population, minimizing rejection reactions caused by HLA-A and / or HLA-B subtype discordance. For HLA-A selection, the more frequent A*02 homozygotes, A*11 homozygotes, A*02 / A11 heterozygotes, and A*24 homozygotes are selected. HLA-B mainly selects B*40 homozygotes, B*15 homozygotes, B*46 homozygotes, B*13 homozygotes, B*51 homozygotes, B*58 homozygotes, B*07 homozygotes, B*35 homozygotes, B*44 homozygotes, B*52 homozygotes, B*57 homozygotes, B*54 homozygotes, and B*55 homozygotes, which are more frequent in the population.

[0253] The second knockout strategy targets HLA-A and / or HLA-B molecules, which are highly correlated with TCR and rejection, while retaining other HLA-I molecules. This not only reduces allogeneic cell rejection but also prevents complete HLA knockout and clearance by NK cells, significantly extending the in vivo half-life of allogeneic CAR-T cells. Simultaneously, knocking out the TCR can reduce GVHD caused by allogeneic cell therapy. The TCR gene encoding the TCRα chain, TRAC, is selected. HLA-A targets A*02 homozygotes, A*11 homozygotes, and A*02 / A11 heterozygotes, which are relatively common in the population. HLA-B targets B*40 homozygotes and B*46 homozygotes, which are relatively common in the population.

[0254] Peripheral blood was collected from healthy individuals and HLA typing was performed based on the first donor screening strategy. The type that met our needs was selected. PBMC separation: The blood in the blood collection tube or blood bag was transferred to a 50ml centrifuge tube and centrifuged. The upper plasma layer was discarded. An equal volume of PBS was added to the lower blood cell layer and mixed. The mixture was then slowly added to the centrifuge tube containing lymphocyte separation medium (DPBS: blood: lymphocyte separation medium = 1:1:1). After centrifugation, the buffy coat layer was collected and washed with PBS by centrifugation. The cells were diluted and counted, and 1x10 6 Place cells in EP tubes for flow cytometry; PBMC cryopreservation: Determine whether to cryopreserve according to actual conditions. CD3+ T cell sorting: Resuspend cells with a certain amount of buffer, mix well, and add 20ul CD3 magnetic beads / 10 7 Add CD3 magnetic beads to the cells, mix well and incubate in a refrigerator at 4 degrees. Add buffer to wash the cells and centrifuge. Resuspend the cells for magnetic bead separation. First, place the column on the magnetic pole, place the corresponding centrifuge tube below, and soak the column with buffer (LS); add cells to the column without generating bubbles, wash the column with buffer three times, remove the column and place it in a clean 15ml centrifuge tube to collect CD3+ T cells; aspirate buffer and add it to the column, elute the CD3-labeled cells with the piston, and take some cells for cell counting. CD3CD28 magnetic bead activation: according to the cell count results, according to 1×10 6Resuspend the cells at a density of 1:1 / ml, prepare the activation reagent, add it to the complete culture medium (containing cytokines IL2, IL7, IL15), fully resuspend the cells, and incubate at 37°C and 5% CO2. Virus transfection: 24 hours after magnetic bead activation, add the virus according to the MOI of the B7H3 virus carrying the B7H3 CAR sequence expressed in Example 1, centrifuge at room temperature, and culture at 37°C and 5% CO2 for 24 hours after the centrifugation. Gene knockout: After sampling and counting, collect the cells and centrifuge them, resuspend the cell pellet with PBS, and prepare the electrotransfer buffer: Nucleofector Solution: Supplement is configured according to 82:18; use 1×10 7 The cells were distributed with RNP complex (Cas9:sgRNA=2:1) ​​and electroporated. After 48 hours of electroporation, the cells were harvested and the editing efficiency was detected by Sanger sequencing. At the same time, the cells were harvested and the knockout efficiency was detected by FACS.

[0255] The sgRNA sequence TRAC sgRNA is shown in SEQ ID NO: 157;

[0256] The A02 sgRNA sequence is shown in SEQ ID NO: 174;

[0257] The A11 sgRNA sequence is shown in SEQ ID NO: 204.

[0258] Example 3

[0259] This example illustrates the disease progression in subjects with recurrent high-grade glioma treated with allogeneic CAR-T cells.

[0260] The purpose of this example is to evaluate the safety and tolerability of the B7H3 UCAR-T cell (i.e., MT027) injection in the treatment of patients with advanced glioma. At the same time, the pharmacokinetic characteristics of the B7H3 UCAR-T cell injection and its metabolites in single and multiple doses at steady state were explored; and the preliminary efficacy of the B7H3 UCAR-T cell injection in the treatment of patients with advanced glioma was evaluated. The B7H3 UCAR-T of this application comprises an extracellular antigen binding domain targeting B7H3 and a 4-1BB+CD3ζ intracellular domain, while the expression and / or activity of one or more of its cells and immune rejection-related genes are downregulated, such as TCR and HLA-A genes expressed by the cells are knocked out.

[0261] Some patient selection criteria include:

[0262] 1. Applicants must be between 18 and 70 years old, male or female, with an expected survival time of at least 3 months;

[0263] 2. Patients with advanced, locally advanced, or recurrent or metastatic tumors confirmed by histology or cytology;

[0264] 3. Patients who have failed previous standard treatment; or patients who have given up treatment for various reasons after first-line treatment was ineffective;

[0265] 4. Patients with pathologically diagnosed high-grade glioma (referring to grades 3 and 4 in the WHO (2021) Classification of Central Nervous System Tumors), who have progressed during standard treatment or relapsed after standard treatment;

[0266] 5. Enhanced MRI shows space-occupying lesions in the brain, with at least one measurable lesion (according to the RANO 2010 standard);

[0267] 6. The patient's tumor tissue is B7H3-positive and the subject voluntarily provides the most recent FFPE tissue or pathological sections (at least 8 consecutive white slides) for B7H3 expression detection (IHC);

[0268] 7. Expected survival time ≥ 3 months;

[0269] 8. KPS (Karnofsky) score ≥ 40 points

[0270] 9. Routine blood test: White blood cell count (WBC) ≥ 3×10 9 / L, lymphocyte percentage (LY%) ≥ 15%, hemoglobin Hbo (Hb) ≥ 90g / L, platelet (PLT) ≥ 60×10 9 / L;

[0271] 10. Liver and kidney function: alanine aminotransferase and aspartate aminotransferase (ALT, AST) < 3 times the normal value, total bilirubin (TBiL) < 1.5 times the normal value, serum creatinine (SCr) < 1.5 times the normal value;

[0272] 11. The subjects voluntarily participate in this study, sign the informed consent form, have good compliance, and cooperate with follow-up;

[0273] Some patient exclusion criteria include:

[0274] 1. Patients with any of the following conditions are not eligible for inclusion in this study:

[0275] 2. Patients with brainstem recurrence, spinal cord dissemination, or extracranial metastasis;

[0276] 3. The maximum diameter of a single tumor lesion is greater than 5 cm, or the maximum diameter of multiple lesions combined is greater than 6 cm;

[0277] 4. Patients who have received radiotherapy within 3 months before cell therapy or surgery, chemotherapy (except lymphocyte depletion therapy), immunotherapy, molecular targeted therapy or any other anti-tumor treatment within 4 weeks before cell therapy;

[0278] 5. Participated in other drug clinical trials within 4 weeks before screening;

[0279] 6. Previous CAR-T cell therapy;

[0280] 7. The patient has a history of severe allergy to any component of the investigational drug or biological product;

[0281] 8. Patients with other primary malignant tumors (except cured cervical carcinoma in situ and basal cell carcinoma of the skin);

[0282] 9. Suffering from active systemic infection, coagulation disorder and other major diseases;

[0283] 10. Suffering from severe heart, lung, liver, or kidney dysfunction; heart function: Class III or above according to the New York Heart Association (NYHA) criteria; liver function: Child-Puget classification C or above; kidney function: Chronic kidney disease (CKD) stage 4 or above; renal insufficiency stage III or above; lung function: severe respiratory failure symptoms involving other organs;

[0284] 11. People with severe autoimmune diseases;

[0285] 12. Subjects who have previously received allogeneic tissue / solid organ transplantation;

[0286] 13. Subjects who have received live vaccines within 2 weeks before the first cell therapy or plan to receive live vaccines during the study;

[0287] 14. Active hepatitis B virus infection; or hepatitis C virus infection (defined as HCV antibody positive, if HCV-RNA is below the detection limit, enrollment is allowed); or human immunodeficiency virus infection (defined as HIV antibody positive); or Treponema pallidum antibody positive;

[0288] 15. Patients with epilepsy or chronic intracranial hypertension that is difficult to control with medication (e.g., daily use of mannitol >500ml, dexamethasone >15mg, or methylprednisolone >80mg, or equivalent doses of other hormones);

[0289] 16. The subject suffers from severe neurocognitive disorder as determined by the researcher;

[0290] 17. Pregnant and lactating women;

[0291] 18. Patients who the researcher believes are unsuitable to participate in this clinical study due to any clinical or laboratory test abnormalities or other reasons.

[0292] Example 4: In this study, 7 patients were first evaluated, including 4 males and 3 females; there were 5 glioblastoma patients and 2 patients with mutant diffuse midline glioma, with a median age of 40.4 years. The baseline characteristics of the patients are shown in Figure 2.

[0293] In this example, TRAC prepared in Example 2 and HLA-A double knockout B7H3-specific UCAR-T cells (i.e., MT027 in this study) were used to administer B7H3-targeting allogeneic chimeric antigen receptor-T cells (MT027) to the above 7 subjects by intrathecal administration at a dose of 2.5×10 7 Total cells / dose, injected once every 4 weeks, while the health status of the subjects was monitored.

[0294] Safety: As shown in Figure 3, the most common adverse reactions in all 7 subjects were fever and headache. The most common laboratory abnormalities were increased cerebrospinal fluid and blood IL-6 levels. However, no typical or severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) were observed, and no cases of graft-versus-host disease (GVHD) were reported. All adverse events were grade 1-2. In addition, all adverse events were resolved or improved during or after treatment without intervention. As shown in Figure 4, the level of IL-6 in the blood was significantly reduced.

[0295] Efficacy: As of April 30, 2023, as shown in Figures 6 and 7, of all 7 subjects, 3 subjects demonstrated partial response (PR) and 4 subjects had stable disease (SD). The objective response rate (ORR) was 42.9% (3 / 7), and the disease control rate (DCR) was 100% (7 / 7). The 12-month survival rate after relapse was 85.7%, significantly higher than the historical data of 14%. Due to the deaths of 3 patients, the median overall survival (OS) was not reached. As shown in Figure 8, the median OS was 14.5 months, significantly higher than the historical data of 6-8 months. These data demonstrate that intrathecal administration of allogeneic CAR-T cells targeting B7H3 (MT027) to subjects has a significant effect on prolonging overall survival.

[0296] As shown in Figure 5, the tumor size of one subject was significantly reduced after treatment. The tumor image is shown in Figure 5. The tumor size of this subject before treatment was 1422.78 mm. 2 , 40 weeks after administration of allogeneic chimeric antigen receptor-T cells targeting B7H3 (MT-027), the tumor size became 1038.27 mm 2 After 54 weeks, the tumor size was greatly reduced to 267.35 mm2 The data suggest that intrathecal administration of allogeneic CAR-T cells targeting B7H3 (MT-027) to subjects has a significant effect on controlling tumor size.

[0297] The subjects were administered allogeneic CAR-T cells (MT-027) targeting B7H3 by intrathecal administration to detect the CAR copy number in vivo. The results are shown in Figure 9. It was found that the CAR copy number amplified significantly on the first day after injection. At the same time, the allogeneic CAR-T cells targeting B7H3 persisted in the body for more than 30 days. Subgroup analysis was performed based on the existing sample size. As shown in Figure 10, by comparing the response rates of different subgroups, the data showed that the efficacy seemed to be positively correlated with the peak CAR copy number (Cmax) after the initial administration.

[0298] Example 5:

[0299] In this study, all patients who received B7H3 UCAR-T (MT027) as of March 7, 2024 were included in the study to evaluate the safety, tolerability, and efficacy of multiple-dose treatment with the allogeneic B7H3 CAR-T of the present invention.

[0300] This study adopted a single-arm, open-label, single-center clinical trial design. The study population included patients aged 18-70 years with B7H3 antigen-positive, histologically or cytologically confirmed high-grade gliomas that relapsed or progressed after standard treatment. Participants received topical administration, intraventricular administration via the Ommaya capsule, and intrathecal administration via lumbar puncture into the lumbar cistern. The dose was divided into four levels: 1.0, 1.5, 2, and 2.5 × 10 7 cells / time, with a dosing frequency of once every four weeks. During the study, adverse events were observed and recorded, with a focus on product-specific adverse reactions such as graft-versus-host disease (GvHD), cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS). Cerebrospinal fluid (CSF) and blood samples were collected to analyze PK and PD indicators such as CAR copy number and cytokines in the samples. Efficacy was evaluated once every cycle, and efficacy indicators such as overall survival (OS), 12-month overall survival rate (12m-OS), objective response rate (ORR), and disease control rate (DCR) were calculated.

[0301] Baseline characteristics:

[0302] As of March 7, 2024, a total of 50 subjects received at least one dose of the allogeneic B7H3CAR-T (MT027) of the present invention, of which 32 received at least 3 doses and 16 received at least 5 doses (see Figure 17 for details of patient flow). Based on this, four statistical analysis data sets were defined: (1) Full Analysis Set (FAS): defined as all subjects who were enrolled and received at least one dose of study treatment, n = 50; (2) Per-Protocol Set 1 (PPS1): defined as all subjects who received at least 3 doses of study treatment, n = 32; (3) Per-Protocol Set 2 (PPS2): including all subjects who received at least 3 doses of study treatment and had at least 1 efficacy follow-up record, and including 1 subject who only received 2 doses of treatment but had a complete efficacy follow-up record, n = 15; (4) Per-Protocol Set 3 (PPS3): defined as subjects who received at least 5 doses of study treatment, n = 16 (Figure 18).

[0303] Safety analysis was based on FAS, efficacy analysis was based on three PPSs, of which PPS1 was the primary efficacy analysis set, and BOR analysis (Best of response) was based only on PPS2.

[0304] Among all 50 subjects, the median age was 48 years (range, 18-66 years), 28 (56%) were male and 22 (44%) were female, 48 (96%) had high B7H3 expression, and 48 (96%) had failed prior temozolomide (TMZ) therapy. The baseline characteristics of PPS1 were similar to those of FAS and are shown in Tables 1 and 2.

[0305] Table 1 Baseline characteristics of FAS (n=50)

[0306] Table 2 Baseline characteristics of PPS1 (n=32)

[0307] Safety results:

[0308] Among all 50 subjects, the most common adverse reactions were fever and headache. No typical or severe CRS or ICANS occurred, and no GvHD was observed. Grade ≥3 AEs occurred in 4 patients (8%) and SAEs in 3 patients (6%). All adverse events resolved or improved with treatment or no intervention. No drug-related deaths occurred. See the tables below, and see Tables 3 and 4 for details.

[0309] Table 3 Safety results of the full analysis set (FAS)

[0310] CRS: cytokine release syndrome; ICANS: immune effector cell-associated neurotoxicity; GVHD: graft-versus-host disease; AE: adverse event; SAE: serious adverse event.

[0311] Table 4 Common AEs in FAS

[0312] CTCAE, Common Terminology Criteria for Adverse Events, Common Terminology Criteria for Adverse Events.

[0313] Pharmacokinetic results:

[0314] Single-dose pharmacokinetics: The CAR copy number in all dose groups reached 10,000-100,000 copies / μg, significantly higher than similar products (Phase I result of MB101[IL13Rα2CAR-T] at City of Hope), and showed persistence of >60 days after a single dose (Figures 11-12).

[0315] Multiple-dose pharmacokinetics: After multiple doses, the number of MT027 CAR-T cells remained at a high level (median: 8850 copies / μg (range: 200-385900)) and could be maintained for a long time (Figure 13).

[0316] Pharmacodynamic results:

[0317] Repeated-dose pharmacodynamics: After repeated administration, IL6, TNF-α, and IFN-γ levels in the cerebrospinal fluid increased significantly and remained at high levels for a long time (Figures 14-16). Cytokine levels were significantly higher than those of similar products (X. Tang et al., Administration of B7H3 targeted chimeric antigen receptor-T cells induce regression of glioblastoma. Signal Transduction and Targeted Therapy 6, (2021)).

[0318] Efficacy results:

[0319] Based on the three PPSs, KM survival curves were derived using SPSS 25.0. As shown in Figures 19A-C , the median survival (mOS) were: PPS1, 13.54 m (95% CI: 7.45; 19.63); PPS2, 20.73 m (95% CI: 16.12; 25.34); and PPS3, 16.76 m (95% CI: 8.78; 24.74). As shown in Figure 20 , the mOS were: PPS1, 53.30% (95% CI: 37.5%-75.7%); PPS2, 84.60% (95% CI: 67.1%-100%); and PPS3, 73.30% (95% CI: 54.0%-99.5%). These were all significantly higher than historical data by 14%. Based on PPS1, univariate analysis was performed with gender, age, B7H3 expression, KPS, and WHO grade as subgroup stratification factors. Univariate analysis showed that B7H3 expression was significantly different between high expression and low-medium expression (9.068 months (95% CI: 1.274; 16.861) vs. 20.731 months (95% CI: 9.983; 31.479), p = 0.052) (Figure 21).

[0320] The objective response rate (ORR) of the 15 PPS2 patients was 33%, the disease control rate was 80%, and the 12-month overall survival (OS) rate was 83.3%, significantly higher than the historical data of 14% (Figure 22).

[0321] In patients with recurrent high-grade gliomas, the allogeneic B7H3 CAR-T (MT027) injection of the present invention has good safety and tolerability when administered intrathecally via lumbar puncture, and the toxicity of grade 3 or above is 1 / 5 of that of similar products; the pharmacokinetics / pharmacodynamics of single and multiple administrations are stable, the persistence in the cell body is long, and the number of cells in the body and the induced cytokine response are 1-2 orders of magnitude higher than those of similar products. In terms of efficacy, it is significantly better than historical data and data of similar products, with a significantly prolonged overall survival and a higher objective response rate. The B7H3 UCAR-T (MT027) of the present invention has good therapeutic potential for recurrent high-grade gliomas.

[0322] During the treatment of the above subjects, it was found that:

[0323] 1) All toxic effects in subjects treated with B7H3 UCAR-T cells were grade 1 or 2, indicating a good safety profile.

[0324] 2) Significantly prolong the overall survival of the subjects and have a higher objective response rate; compared with historical treatment results, they have a longer survival period and a higher objective response rate.

[0325] 3) B7H3 UCAR-T cells remained well in the subjects and the effects were sustained.

[0326] The foregoing merely illustrates the principles of the present application. It should be understood that the scope of the present application is not intended to be limited to the exemplary aspects described herein, but rather includes all currently known and future developed equivalents. In addition, it should be noted that, without departing from the technical principles of the present application, several improvements and modifications may be made, and such improvements and modifications shall also be considered within the scope of the present application.

Claims

1. A method for treating a subject with recurrent high-grade gliomas (rHGGs), the method comprising administering to the subject at least one dose comprising an allogeneic chimeric antigen receptor T cell (CAR-T cell) targeting B7H3, wherein the at least one dose is about 1.0×10 6 ~1.0×10 9 Cells / drugs.

2. The method according to claim 1, wherein: The high-grade glioma is a brain glioma.

3. The method according to claim 2, wherein: The brain glioma is astrocytoma (IDH mutant), oligodendroglioma (IDH mutation with 1p / 19q combined deletion), glioblastoma (IDH wild type), diffuse astrocytoma (with MYB or MYBL1 changes), angiocentric glioma, juvenile multiform low-grade neuroepithelial tumor (PLNTY), diffuse low-grade glioma (with MAPK signaling pathway changes), diffuse midline glioma (with H3 K27 changes), diffuse hemispheric glioma (with H3 G34 mutation), diffuse childhood high-grade glioma (H3 and IDH wild type), infantile hemispheric glioma, pilocytic astrocytoma, high-grade astrocytoma with hair-like features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, and chordoid glioma.

4. The method according to claim 1, wherein: The at least one dose is selected from about 1.0×10 6 Cells / dose, about 2.0×10 6 Cells / dose, about 3.0×10 6 Cells / dose, about 4.0×10 6 Cells / dose, about 5.0×10 6 Cells / dose, about 6.0×10 6 Cells / dose, about 7.0×10 6 Cells / dose, about 8.0×10 6 Cells / dose, about 9.0×10 6 Cells / dose, about 1.0×10 7 Cells / dose, about 1.5×10 7 Cells / dose, about 2.0×10 7 Cells / dose, about 2.5×10 7 Cells / dose, about 3.0×10 7 Cells / dose, about 4.0×10 7 Cells / dose, about 5.0×10 7 Cells / dose, about 6.0×10 7 Cells / dose, about 7.0×10 7 Cells / dose, about 8.0×10 7 Cells / dose, about 9.0×10 7 Cells / dose, about 1.0×10 8 Cells / dose, about 2.0×10 8 Cells / dose, about 3.0×10 8 Cells / dose, about 4.0×10 8 Cells / dose, 5.0×10 8 Cells / dose, about 6.0×10 8 Cells / dose, about 7.0×10 8 Cells / dose, about 8.0×10 8 Cells / dose, about 9.0×10 8 Cells / dose 1.0×10 9 Cells / drugs.

5. The method according to claims 1-4, wherein: When the subject has a weight of less than or equal to 50 kg, the at least one dose is selected from about 1.0×10 6 Cells / dose, about 2.0×10 6 Cells / dose, about 3.0×10 6 Cells / dose, about 4.0×10 6 Cells / dose, about 5.0×10 6 Cells / dose, about 6.0×10 6 Cells / dose, about 7.0×10 6 Cells / dose, about 8.0×10 6 Cells / dose, about 9.0×10 6 Cells / dose, about 1.0×10 7 Cells / dose, about 1.5×10 7 Cells / dose, about 2.0×10 7 Cells / dose, about 2.5×10 7 Cells / dose, about 3.0×10 7 Cells / dose, about 4.0×10 7 Cells / dose, about 5.0×10 7 Cells / drugs.

6. The method according to claims 1-4, wherein: When the subject has a weight greater than 50 kg, the at least one dose is selected from about 6.0×10 7 Cells / dose, about 7.0×10 7 Cells / dose, about 8.0×10 7 Cells / dose, about 9.0×10 7 Cells / dose, about 1.0×10 8 Cells / dose, about 2.0×10 8 Cells / dose, about 3.0×10 8 Cells / dose, about 4.0×10 8 Cells / dose, 5.0×10 8 Cells / dose, about 6.0×10 8 Cells / dose, about 7.0×10 8 Cells / dose, about 8.0×10 8 Cells / dose, about 9.0×10 8 Cells / dose 1.0×10 9 Cells / drugs.

7. The method according to claims 1-6, wherein: CAR expression is detectable in the subject until at least 2 weeks after administration of the CAR-T cells.

8. The method according to claims 1-7, wherein: CAR expression is detectable in the subject until at least 4 weeks after administration of the CAR-T cells.

9. The method of claims 1-8, wherein the subject exhibits a CR state at least 4 weeks after administration of the CAR-T cells.

10. The method according to claims 1-9, wherein: The subject exhibits a CR status for at least 8 weeks after CAR-T cell administration.

11. The method of claims 1-10, wherein the subject's CAR-T cells exhibit a CR state for at least 12 weeks after CAR-T cell administration.

12. The method of claims 1-11, wherein the subject's CAR-T cells exhibit a CR state for at least 24 weeks after CAR-T cell administration.

13. The method of claims 1-12, wherein the subject exhibits a CR state for at least 48 weeks after administration of the CAR-T cells.

14. The method according to claims 1-13, wherein: The CAR-T cells are administered locally.

15. The method according to claims 1-14, wherein: CAR-T cells are administered intraventricularly or intrathecally.

16. The method according to claims 1-15, wherein: No lymphodepletion is required before administration of CAR-T cells.

17. The method according to claims 1-16, wherein: Administer subsequent doses when the interval between first doses is less than 4 weeks.

18. The method according to claims 1-16, wherein: Subsequent doses were administered at least 4 weeks after the first dose.

19. The method according to claims 17-18, wherein: Subsequent doses of CAR-T cells were administered with essentially the same number of cells as the first dose.

20. The method according to claims 17-18, wherein: Subsequent doses of CAR-T cells are administered with a greater number of cells than the first dose.

21. The method according to claims 17-18, wherein: Subsequent doses of CAR-T cells are administered with fewer cells than the first dose.

22. The method according to claims 1-18, wherein: The subject showed a suboptimal response after administration of CAR-T cells.

23. The method according to claim 22, wherein: The suboptimal response includes: Complete response (CR); Partial response (PR); or, Stable disease (SD).

24. The method according to any one of claims 1 to 23, wherein: The CAR-T cell expresses a CAR having an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO:

8.

25. The method according to any one of claims 1 to 23, wherein: The CAR-T cell expresses the CAR with the amino acid sequence shown in SEQ ID NO:

5.

26. A method for producing an allogeneic chimeric antigen receptor T cell population against a B7H3 target, the method comprising: (a) Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors; (b) activating T-cells in the PBMCs; (c) Transducing activated T cells with a complex or vector containing CAR (d) destroying the TRAC gene and HLA-A gene expression of T-cells to obtain T-cells with destroyed gene expression; (e) expanding T cells in which gene expression is disrupted; and (f) T-cell population enriched for TRAC-negative, HLA-A-negative cells; This generates an allogeneic chimeric antigen receptor (CAR)-T cell (CAR-T cell) population.

27. The method according to claim 26, wherein: The vector in step (c) is a lentiviral vector.

28. The method according to claim 26, wherein: The step (c) is to transduce the activated T-cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing the target CAR.

29. The method according to any one of claims 26 to 28, wherein: The CAR is a CAR having an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO:

8.

30. The method according to claims 26 to 29, wherein: The CAR is a CAR having the amino acid sequence shown in SEQ ID NO:

5.

31. The method according to claims 26-30, wherein: The expression and / or activity of the TRAC gene and HLA-A gene of the CAR-T cell are downregulated.

32. A kit or a pharmaceutical kit for treating a subject with recurrent high-grade gliomas (rHGGs), the kit or the pharmaceutical kit comprising: allogeneic chimeric antigen receptor T cells targeting B7H3.

33. The test kit or kit according to claim 32, wherein The kit or pharmaceutical kit comprises at least one dose of allogeneic chimeric antigen receptor T cells targeting B7H3, wherein the at least one dose is about 1.0×10 6 ~1.0×10 9 Cells / drugs.

34. The test kit or kit according to claim 32, wherein The CAR-T cell is a CAR-T cell according to any one of claims 1 to 25 or a CAR-T cell produced by the method according to any one of claims 26 to 31.

35. A method of treatment, wherein: include: 1) administering chimeric antigen receptor T cells targeting B7H3 to a subject having the disease for the first time; as well as 2) administering a subsequent dose of CAR-T cells to the diseased subject at least four weeks after the administration of the CAR-T cells in 1).

36. The method of claim 35, wherein: The subsequent doses are administered 2 times, or 3 times or more.

37. The method of claims 35-36, wherein the chimeric antigen receptor-T cell targeting B7H3 is an allogeneic chimeric antigen receptor T cell targeting B7H3.

38. The method according to claims 35-37, wherein: The CAR-T cells are administered locally.

39. The method according to claims 35-37, wherein: CAR-T cells are administered intraventricularly or intrathecally.

40. The method according to claims 35-39, wherein: The first administration of CAR-T cells contains a dose of about 1.0×10 6 ~1.0×10 9 cell.

41. The method according to claims 35-40, wherein: The first dose of CAR-T cells contained approximately 1.0×10 6 cells, about 2.0×10 6 cells, about 3.0×10 6 cells, about 4.0×10 6 cells, about 5.0×10 6 cells, about 6.0×10 6 cells, about 7.0×10 6 cells, about 8.0×10 6 cells, about 9.0×10 6 cells, about 1.0×10 7 cells, about 1.5×10 7 cells, about 2.0×10 7 cells, about 2.5×10 7 cells, about 3.0×10 7 cells, about 4.0×10 7 cells, about 5.0×10 7 cells, about 6.0×10 7 cells, about 7.0×10 7 cells, about 8.0×10 7 cells, about 9.0×10 7 cells, about 1.0×10 8 cells, about 2.0×10 8 cells, about 3.0×10 8 cells, about 4.0×10 8 cells, 5.0×10 8 cells, about 6.0×10 8 cells, about 7.0×10 8 cells, about 8.0×10 8 cells, about 9.0×10 8 Cells and 1.0×10 9 cell.

42. The method of claims 35-39, wherein: The first CAR-T cell dose contains 1.5×10 4 ~2.0×10 7 cells / kg subject body weight.

43. The method of claim 42, wherein: The first administration of CAR-T cell doses includes about 1.5×10 4 , 2.0×10 4 , 2.5×10 4 , 3.0×10 4 , 3.5×10 4 4.0×10 4 , 4.5×10 4 5.0×10 4 , 5.5×10 4 , 6.0×10 4 , 6.5×10 4 , 7.0×10 4 , 7.5×10 4 , 8.0×10 4 , 8.5×10 4 9.0×10 4 , 9.5×10 4 , 1.0×10 5 , 1.5×10 5 , 2.0×10 5 , 2.5×10 5 , 3.0×10 5 , 3.5×10 5 4.0×10 5 , 4.5×10 5 5.0×10 5 , 5.5×10 5 , 6.0×10 5 , 6.5×10 5 , 7.0×10 5 , 7.5×10 5 , 8.0×10 5 , 8.5×10 5 9.0×10 5 , 9.5×10 5 , 1.0×10 6 , 1.5×10 6 , 2.0×10 6 , 2.5×10 6 , 3.0×10 6 , 3.5×10 6 4.0×10 6 , 4.5×10 6 5.0×10 6 , 5.5×10 6 , 6.0×10 6 , 6.5×10 6 , 7.0×10 6 , 7.5×10 6 , 8.0×10 6 , 8.5×10 6 9.0×10 6 , 9.5×10 6 , 1.0×10 7 , 1.5×10 7 , 2.0×10 7 cells / kg subject body weight.

44. The method according to claims 35-43, wherein: The subsequent dose contains approximately 1.0×10 6 ~1.0×10 9 cell.

45. The method of claims 35-44, wherein: The disease is a tumor or cancer.

46. ​​The method of claim 45, wherein: The tumor includes a brain glioma.

47. The method of claim 45, wherein: The brain glioma is adult diffuse glioma, childhood diffuse low-grade glioma, childhood diffuse high-grade glioma, or focal astrocytic glioma.

48. The method according to any one of claims 46-47, wherein: The brain glioma is astrocytoma (IDH mutant), oligodendroglioma (IDH mutation with 1p / 19q combined deletion), glioblastoma (IDH wild type), diffuse astrocytoma (with MYB or MYBL1 changes), angiocentric glioma, juvenile multiform low-grade neuroepithelial tumor (PLNTY), diffuse low-grade glioma (with MAPK signaling pathway changes), diffuse midline glioma (with H3 K27 changes), diffuse hemispheric glioma (with H3 G34 mutation), diffuse childhood high-grade glioma (H3 and IDH wild type), infantile hemispheric glioma, pilocytic astrocytoma, high-grade astrocytoma with hair-like features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, and chordoid glioma.

49. The method according to any one of claims 35 to 48, wherein: The method results in improvement of the disease to a greater extent and / or for a longer period of time than a method comprising an alternative dosing regimen in which a single dose of the cells in 1) and the cells in 2) is administered to the subject.

50. The method according to any one of claims 35 to 49, wherein: The disease persists after administration of the first dose and / or administration of the first dose is insufficient to eradicate the disease in the subject.

51. The method according to any one of claims 35 to 50, wherein: The subject exhibits one or more symptoms of the disease upon administration in 2).

52. The method according to any one of claims 35 to 51, wherein: Cytokine release syndrome (CRS)-related outcomes in the subject are not detectable or are decreased by about 20% to about 99% on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 after administration in 2), compared to a method comprising an alternative dosing regimen in which the cells in 2) are administered to the subject.

53. The method according to any one of claims 35 to 52, wherein: The CAR-T cell is a CAR-T cell involved in claims 1-25 or a CAR-T cell produced by the method of any one of claims 26-31.

54. A method of treatment, wherein: The invention comprises administering a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells to a subject who has previously been administered a first dose of allogeneic CAR-T cells, wherein: the subsequent dose of cells is administered at a time point at least or more than about 4 weeks and less than about 24 weeks after the start of the first dose.

55. A method of treatment, wherein: Comprising administering a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells to a subject, wherein: prior to said administration, said subject has received a previous dose of CAR-T cells in an amount sufficient to show a clinical benefit in said subject; and at the time of said administration, said subject does not exhibit a detectable adaptive host immune response specific to CAR-T cells; and / or the time between said previous dose and said subsequent dose is greater than about 4 weeks and less than about 24 weeks.

56. Use of a composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for the preparation of a medicament for treating a disease in a subject previously treated with CAR-T cells, wherein: The composition is for use between about 4 to about 24 weeks after the prior treatment; and / or the composition is formulated for administration of a subsequent dose in an amount sufficient to ameliorate disease in a subject previously treated with CAR-T cells.

57. A composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for treating a disease in a subject previously treated with CAR-T cells, wherein: The cells are for use between about 4 and 24 weeks after a prior treatment; and the cells are formulated for administration of a subsequent dose in an amount sufficient to ameliorate disease in a subject previously treated with CAR-T cells.

58. The composition for use according to claim 56 or the composition for use according to claim 57, wherein the dose of CAR-T cells in the previous treatment improves one or more symptoms of the disease in the subject before the subsequent dose is used.

59. Use of allogeneic chimeric antigen receptor (CAR)-T cells in the preparation of a drug for treating a disease, the use comprising: 1) administering a first dose of CAR-T cells to a subject suffering from the disease, wherein the first dose comprises about 1×10 6 About 1.0×10 9 Total cells, and 2) administering a subsequent dose of CAR-T cells to the subject at a time point at least or more than about 4 weeks and less than about 24 weeks after the administration in 1).

60. The use according to claim 56 or 59 or the composition for use according to claim 57, wherein The disease is a tumor or cancer.

61. The method according to any one of claims 35 to 55, the use of any one of claims 56 or 59, or the composition according to claim 57, wherein the CAR-T cell is an allogeneic chimeric antigen receptor (CAR)-T cell targeting B7H3.

62. An article of manufacture comprising: a plurality of sealable containers, each container comprising a unit dose of allogeneic chimeric antigen receptor (CAR)-T cells for administration to a subject, the unit dose comprising about 1×10 6 About 1.0×10 9 cells; packaging material; and a label or package insert comprising instructions for administering a plurality of said unit doses to said subject by performing a first administration and a subsequent administration, said first administration comprising delivering one of said unit doses to said subject, and said subsequent administration comprising administering one or more of said unit doses to said subject.

63. The article of manufacture of claim 62, wherein the instructions specify that the subsequent administration be performed at a time between about 30 and 150 days after the first administration, optionally at about 30 days, about 60 days, about 90 days, or about 99 days.

64. The article of manufacture of claim 62 or 63, wherein the container is or comprises a flexible cell infusion bag or a cryopreservation vial.

65. The method according to any one of claims 35-55, the use of any one of claims 56 or 59, or the composition according to claim 57, or the preparation according to any one of claims 62-64, wherein the CAR-T cell is a CAR-T cell involved in claims 1-25 or a CAR-T cell produced by the method of any one of claims 26-31.

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