Methods and compositions for dosing of allogeneic chimeric antigen receptor t cells

Abstract

To provide methods for administering chimeric antigen receptor (CAR)-T cells for treatment of human patients susceptible to or diagnosed with a disease, such as cancer.SOLUTION: A method of treating a subject who has refractory and / or relapsed non-Hodgkin's lymphoma comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3zeta CAR, where the at least one dose is about 20×106 cells / dose to about 360×106 cells / dose.SELECTED DRAWING: Figure 4A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 750,215, filed October 24, 2018, U.S. Provisional Patent Application No. 62 / 716,898, filed August 9, 2018, and U.S. Provisional Patent Application No. 62 / 579,426, filed October 31, 2017, each of which is incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates to a therapeutic regimen for the treatment of disorders, including proliferative disorders and immune disorders. The therapeutic regimen comprises the administration of one or more doses of immune cells, including allogeneic chimeric antigen receptor T cells. The therapeutic regimen can be used to prevent and / or treat disorders, including cancer.

[0003] Description of electronically submitted text files The contents of the electronically submitted text file are incorporated herein by reference in their entirety: Computer-readable copy of the Sequence Listing (Filename: ALGN-012-03WO Sequence Listing.txt, Recorded on: October 30, 2018, File size: 39,283 bytes). [Background technology]

[0004] Various methods are available for adoptive cell therapy using engineered cells expressing recombinant receptors, such as chimeric antigen receptor (CAR)-T cells. However, the dose of allogeneic CAR-T cells required to effectively treat disease and achieve remission in human patients is unknown. Provided herein are methods, compositions, and articles of manufacture for administering and re-administering allogeneic CAR-T cells to patients. The methods of the present disclosure reduce the risk of toxicity and / or increase efficacy, for example, by increasing the subject's exposure to the administered cells (e.g., by improving the proliferation and / or persistence of the administered cells). Summary of the Invention

[0005] The present disclosure relates to a therapeutic regimen for treating disorders such as cancer. More specifically, the present disclosure relates to a therapeutic regimen for treating disorders such as cancer by administering allogeneic CAR-T cells. Features of the method include the timing of the dose and number of cells administered, and provide various advantages, such as improving efficacy and / or reducing toxicity, by increasing the subject's exposure to administered cells.

[0006] For example, methods are provided for administering CAR-T cells to a subject to treat the subject's disease and / or disorder.The methods generally include administering one or more doses of such cells, and / or administering subsequent doses to a subject who has previously been treated with a previous (e.g., first) dose of such cells.Also provided are cells, compositions, and articles of manufacture for use in such methods.

[0007] In some embodiments, the method includes treating an adult subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR (e.g., UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells)), wherein the at least one dose comprises about 6x10 5 cells / dose, approximately 6x10 6 cells / dose, approximately 6-8x10 7 cells / dose, and approximately 1.8-2.4x10 8 cells / dose.

[0008] In some embodiments, the method includes treating a pediatric subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR (e.g., UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells), or UCART19 (CD19CAR / R2+_TCRαβ-_T cells), or UCART19 (CD19CAR / TCRαβ-_T cells), wherein the at least one dose comprises about 2-8 x 10 7 cells / dose.

[0009] In some embodiments, a method includes treating a subject with refractory and / or relapsed non-Hodgkin's lymphoma (e.g., large B-cell lymphoma or follicular lymphoma), the method includes administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR (CD19CAR / RQR8+_TCRαβ-_T cells), wherein the at least one dose is about 20x10 6 cells / dose ~ approx. 360x10 6 cells / dose, e.g., about 20x10 6 cells / dose, approximately 40x10 6 cells / dose, approximately 80x10 6 cells / dose, approximately 120x10 6 cells / dose, 240x10 6 cells / dose, or approximately 360x10 6 cells / dose.

[0010] In some embodiments, the method includes (a) administering to a subject having a disease a first dose of allogeneic CAR-T cells, and (b) administering to the subject a subsequent dose of allogeneic CAR-T cells. In other embodiments, the method is carried out by administering the subsequent dose, as in (b), to a subject who has previously been administered the first dose, as in (a). In some embodiments, the CAR-T cells are tumor antigen-specific CAR-T cells. In some embodiments, the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells)). In some embodiments, the disease is a tumor. In some embodiments, it is a cancer, malignancy, neoplasm, or other proliferative disease or disorder, such as a leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL (e.g., relapsed / refractory ALL)), non-Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma. In some embodiments, the disease is selected from the group consisting of DLBCL, multiple myeloma, follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer (e.g., small cell and non-small cell lung cancer), liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, head and neck squamous cell carcinoma (HNSCC), and / or mesothelioma. In some embodiments, the disease is leukemia or lymphoma. In some embodiments, the disease is acute lymphoblastic leukemia. In some embodiments, the disease is relapsed or refractory acute lymphoblastic leukemia. In some embodiments, the disease is non-Hodgkin's lymphoma (NHL). In some embodiments, the disease is relapsed or refractory large B-cell lymphoma. In some embodiments, the disease is relapsed or refractory follicular lymphoma.

[0011] In some embodiments, the disease is acute lymphoblastic leukemia (ALL). In some embodiments, the disease is childhood acute lymphoblastic leukemia (ALL). In some embodiments, the disease is an aggressive lymphoid malignancy such as B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the disease is refractory B-ALL, e.g., adult refractory B-ALL. In some embodiments, the disease is relapsed B-ALL, e.g., adult relapsed B-ALL. In some embodiments, the disease is extramedullary disease, e.g., associated with one or more of the cancers described herein. In some embodiments, the subject exhibits detectable molecular disease and / or minimal residual disease upon administration of the subsequent dose. In some embodiments, the subject exhibits one or more symptoms of the disease upon administration of the subsequent dose. In some embodiments, the disease is cancer and the subject exhibits a relapse at the start of administration of the subsequent dose. In some embodiments, the disease is leukemia or lymphoma and the subject exhibits greater than 5% blasts in the bone marrow upon administration of the subsequent dose.

[0012] In some embodiments, the disease is cancer and the subject exhibits morphologic disease. In some embodiments, the disease is leukemia or lymphoma and the subject exhibits extramedullary disease (i.e., the presence of blasts outside the bone marrow) at the time of administration of the subsequent dose. In some embodiments, the disease is leukemia or lymphoma and the subject exhibits more than 5% blasts in the bone marrow at the time of administration of the subsequent dose. In other embodiments, the disease is cancer and the subject does not exhibit morphologic disease at the start of administration of the subsequent dose. In some embodiments, the disease is leukemia or lymphoma and the subject exhibits less than 5% blasts in the bone marrow at the time of administration of the subsequent dose.

[0013] In some embodiments, the method includes (a) administering to a subject having a disease a first dose of allogeneic CAR-T cells. In some embodiments, the first dose is about 1 x 10 4 cells, approximately 5 x 10 4 cells, approximately 1 x 10 5 cells, approximately 5 x 10 5 cells, approximately 1 x 10 6 cells, approximately 5 x 10 6 cells, approximately 6 x 106 cells, approximately 1 x 10 7 cells, approximately 6 x 10 7 cells, approximately 1 x 10 8 , about 1.8×10 8 cells, or approximately 4.8 x 10 8 In some embodiments, the first dose contains about 20x10 cells. 6 Cell ~ approx. 360x10 6 Cells, e.g., about 20x10 6 cells, approximately 40x10 6 cells, approximately 80x10 6 cells, approx. 120x10 6 cells, 240x10 6 cells, or approximately 360x10 6 In some embodiments, the method further comprises (b) administering to the subject a subsequent dose of CAR-T cells at least about 5 weeks, or more than about 5 weeks, after initiation of administration in (a) and less than about 24 weeks after initiation of administration in (a).

[0014] In some embodiments, subjects with relapsed / refractory ALL receive 6×10 6 A first and subsequent doses of allogeneic CAR-T cells containing cells are administered, and the subsequent dose of CAR-T cells in (b) is administered about 99 days after the start of administration in (a).

[0015] In some embodiments, the method further includes administering additional subsequent doses, such that the first and multiple subsequent doses are administered, e.g., according to a specified dosing and timing schedule for the first and subsequent doses. In some embodiments, the first of the one or more subsequent doses is administered at least 5 weeks or more after initiation of administration of the subsequent doses. In some embodiments, administering the first, second, and subsequent doses includes administering at least three of the doses within about 5 weeks. In some embodiments, the first dose is administered about 16 weeks after initiation of administration, and the additional subsequent dose is administered 17 weeks after initiation of administration of the first dose. In some embodiments, the additional subsequent dose is administered 17 weeks and / or 34 weeks after initiation of administration of the first dose.

[0016] In some embodiments, the time for administering the subsequent dose is a further time during which the subject has not exhibited an immune response, e.g., has not exhibited a detectable adaptive host immune response specific for CAR-T after said first (or previous) dose.

[0017] In some embodiments, the time between administration of the first dose (initial dose), e.g., start of administration of the first or previous dose, and start of administration of the subsequent dose (e.g., start of administration of the subsequent dose) is more than about 4 weeks, e.g., more than about 5, 6, 7, 8, or 9 weeks, e.g., more than about 20 weeks, e.g., between about 9 weeks and about 35 weeks, between about 14 weeks and about 28 weeks, between 15 weeks and 27 weeks, or between 16 weeks and about 18 weeks, and / or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 weeks. In some embodiments, the administration (e.g., beginning, etc.) of a subsequent dose is more than about 5 weeks but less than about 24 weeks after the administration (e.g., beginning, etc.) of the first or previous dose. In some embodiments, the administration of a subsequent dose is initiated 17 weeks after the initiation of the first dose. In some embodiments, the interval between the administration of a first dose and a subsequent dose (e.g., beginning, etc.) or between the administration of a previous dose and the next subsequent dose is more than about 5 weeks but less than about 24 weeks, e.g., between 10 and 24 weeks, e.g., about 17 weeks. In some embodiments, the time between the administration of a first dose and the administration (e.g., beginning, etc.) of a subsequent dose is about 17 weeks. In some embodiments, the administration (e.g., beginning, etc.) of a subsequent dose is more than about 7 days but less than about 365 days after the administration (e.g., beginning, etc.) of the first or previous dose. In some embodiments, the administration (e.g., the start of) of a subsequent dose is more than about 30 days and less than about 110 days after the administration (e.g., the start of) of the first or previous dose.

[0018] In some embodiments, the subject exhibits an absence of CAR-T cell persistence upon administration of subsequent doses. In some embodiments, the subject exhibits a suboptimal response upon administration of subsequent doses. In some embodiments, the suboptimal response can include any one or more of the following: (i) complete response (CR), complete response with incomplete recovery of blood counts (CRi), with absence of detectable minimal residual disease (leukemia patients) and cytogenetic response, (ii) complete bone marrow response, (iii) partial response, or (iv) stable response.

[0019] In some embodiments, administration of the first dose leads to an improvement in one or more symptoms of the disease in the subject after administration of the first dose, hi some embodiments, at the time of administration of the subsequent dose, the subject has relapsed and / or experienced an increase in one or more symptoms of the disease after an initial improvement (e.g., remission) experienced after the first dose.

[0020] In some embodiments, the subsequent dose of cells contains a quantity of CAR-T cells sufficient to ameliorate the subject's disease. In some embodiments, administration of the subsequent dose leads to further improvement of the subject's disease. In some embodiments, administration of the subsequent dose causes improvement of the disease in the subject compared to immediately before the start of administration of the subsequent dose. In some embodiments, administration of the subsequent dose causes MRD negativity. In some embodiments, the method ameliorates disease to a greater extent and / or for a longer period of time compared to another dosing regimen method, wherein the subject is administered a first dose of cells and a subsequent dose of cells in a single administration. The improvement may include a decrease in the total number of disease cells, e.g., tumor cells, in the subject, in the subject's organ, in the subject's tissue, or in the subject's bodily fluid. The improvement may include a decrease in molecular detection by flow cytometry or quantitative PCR, a decrease in tumor mass or volume, and / or a decrease in the number and / or extent of metastases. In some embodiments, the improvement includes improved subject survival, e.g., an increase in survival time or event-free interval, progression-free, or recurrence-free.

[0021] In some embodiments, the disease persists after administration of the first dose and / or administration of the first dose is not sufficient to eradicate the disease in the subject, hi some embodiments, administration of said subsequent dose causes an improvement in the disease in the subject compared to immediately before the start of administration of the subsequent dose.

[0022] In some embodiments, administration of the first dose does not induce severe cytokine release syndrome (CRS) in the subject. The severity of cytokine release syndrome (CRS) may be assessed according to the modified grading system described in Lee DW, et al., Blood 2014;124(2):188-195, which is incorporated herein by reference in its entirety. In some embodiments, administration of the first dose does not induce CRS in the subject. In some embodiments, based on clinical data, administration of the first dose does not induce severe CRS in the majority of subjects. In some embodiments, administration of the first dose does not induce CRS including the combination of (1) persistent fever (a fever of at least about 38 degrees Celsius for at least 3 days) and (2) a serum level of C-reactive protein (CRP) of at least 20 mg / dL or about 20 mg / dL, and / or does not induce CRS including hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation.

[0023] In some embodiments, administration of the first dose does not induce Grade 3 or higher neurotoxicity in the subject. In some embodiments, based on clinical data, administration of the first dose does not induce Grade 3 or higher neurotoxicity in a majority of subjects. In some embodiments, clinical risk of neurotoxicity and / or symptoms associated with Grade 3 or higher neurotoxicity include confusion, delirium, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (optionally confirmed by electroencephalogram [EEG]), high levels of beta amyloid (Aβ), high levels of glutamate, and high levels of oxygen radicals.

[0024] In some embodiments, the subject has been treated with a tumor-targeting therapeutic agent prior to administration of the first dose. In some aspects, the subject is refractory or non-responsive to the therapeutic agent at the time of administration of the first dose and / or subsequent doses. In some embodiments, after administration of the first dose and before administration of the subsequent doses or before administration of the first dose, the method further comprises assessing the serum level of a factor indicative of CRS, disease symptoms, and / or indicators of a host anti-recombinant receptor (e.g., anti-CAR) immune response in the subject, such as humoral or cell-mediated immune responses. In some such embodiments, the detected disease symptoms are or include molecular detection by flow cytometry or quantitative PCR, the total number of diseased cells in the subject, the subject's organ, the subject's tissue, or the subject's bodily fluids, the mass or volume of a solid tumor, or the number or extent of metastases.

[0025] In some embodiments, the method includes assessing disease symptoms before administration of the subsequent dose, and determining the subsequent dose of cells to be administered to the subject based on the results of the assessment. In some embodiments, if the assessment determines that the subject has morphological disease, the subject is administered a subsequent dose containing a number of allogeneic CAR-T cells that is less than, greater than, or approximately the same as the number of allogeneic CAR-T cells in the first dose. In some embodiments, if the assessment determines that the subject has minimal residual disease, the subject is administered a subsequent dose containing an increased number of allogeneic CAR-T cells compared to the first dose. In some embodiments, the subsequent dose contains approximately the same number of allogeneic CAR-T cells as in the first dose. In some embodiments, the number of allogeneic CAR-T cells per kilogram administered in the subsequent dose is less than, the same as, or approximately the same as the number of allogeneic CAR-T cells per kilogram administered in the first dose. In other embodiments, the number of allogeneic CAR-T cells administered in the subsequent dose is greater than the number of allogeneic CAR-T cells administered in the first dose. In some embodiments, the subsequent dose comprises an increased number of cells compared to the first dose, e.g., at least 2-fold, 5-fold, or 10-fold greater than the number in the first dose. In some embodiments, the number of CAR-T cells per kilogram administered in the subsequent dose is at least 2-fold, or about 2-fold, or 3-fold, greater than the number of receptor-expressing (e.g., CAR-expressing) cells per kilogram administered in the first dose.

[0026] In some embodiments, following administration of the first dose and / or following administration of subsequent doses, the population of allogeneic CAR-T cells from the first dose expands in the subject. In some embodiments, the expansion is evidenced by an increase in serum C-reactive protein (CRP) levels after administration of the first dose and / or subsequent doses compared to immediately before administration. In some embodiments, the expansion is evidenced by PK, for example, but not limited to, as assessed by flow cytometry. In some embodiments, the expansion is evidenced by an increase in the level of CAR-encoding nucleic acid in serum after administration of the first dose and / or subsequent doses compared to immediately before administration, as measured by qPCR. In some embodiments, the increase is at least 1-, 2-, or 3-fold.

[0027] In some embodiments, the first dose of cells and the second or subsequent dose of cells are from the same donor, hi some embodiments, the first dose of cells and the second or subsequent dose of cells are from different donors.

[0028] In some embodiments, the first and / or subsequent doses are not split doses. For example, in some embodiments, the first dose of cells is administered in a single pharmaceutical composition comprising the first dose of cells, and / or the subsequent doses of cells are administered in a single pharmaceutical composition comprising the subsequent doses of cells. In other embodiments, the first and / or subsequent doses are split doses, e.g., where the first dose of cells is administered in multiple compositions collectively comprising the first dose of cells over a period of no more than 3 days, and / or the subsequent doses are split doses, where the subsequent doses of cells are administered in multiple compositions collectively comprising the subsequent doses of cells over a period of no more than 3 days.

[0029] In some embodiments, the method comprises administering a subsequent dose of allogeneic CAR-T cells to a subject who has previously received a first dose of allogeneic CAR-T cells. In some embodiments, the subsequent dose of cells is administered at least about 5 weeks, or more than about 5 weeks, and less than about 24 weeks after the initiation of the first dose. In some embodiments, the number of allogeneic CAR-T cells administered in the subsequent dose is the same as in the first dose. In some embodiments, the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).

[0030] In some embodiments, the number of cells administered in the first dose is about 0.5 x 10 6 cells ~5×10 8 Between cells, approximately 0.75 × 10 6 cells ~8×10 7 between cells, or approximately 5 x 10 6 cells ~7×10 6 In some embodiments, the number of cells administered in the first dose of CD19-specific CAR-T cells is between about 0.5 x 10 6 cells ~1×10 9 Between cells, approximately 1 x 10 5 cells ~3×10 8 between cells, or approximately 6 x 10 5 cells ~2.4×10 8 In some embodiments, the number of cells administered in the first dose of UCART19 cells is between about 6 x 10 and about 10 x 10 cells, including each of the following: 5 cells, approximately 6 x 10 6 cells, approximately 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8 cells, or approximately 2.4 x 10 8 In some embodiments, the number of cells administered in the first dose of UCART19 cells is about 6 x 10 6 It is a cell.

[0031] In some embodiments, the number of cells administered in subsequent doses of CD19-specific CAR-T cells is about 0.5 x 10 6 cells ~1×109 Between cells, approximately 1 x 10 5 cells ~3×10 8 between cells, or approximately 6 x 10 5 cells ~2.4×10 8 In some embodiments, the number of cells administered in subsequent doses of UCART19 cells is between about 6 x 10 and about 6 x 10 cells, including each of the following: 5 cells, approximately 6×10 6 cells, approximately 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8 cells, or approximately 2.4 x 10 8 In some embodiments, the number of cells administered in the subsequent dose of UCART19 cells is about 6 x 10 6 In some embodiments, the number of cells administered in the first and subsequent doses of UCART19 cells is about 6 x 10 6 It is a cell.

[0032] In some embodiments, the method further comprises administering a lymphodepleting regimen prior to the first dose and / or prior to administration of a subsequent dose. In some embodiments, the lymphodepleting regimen comprises cyclophosphamide, fludarabine, and / or a combination thereof. In some embodiments, the lymphodepleting regimen comprises one or more of cyclophosphamide, fludarabine, a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10), and / or a combination thereof. In some embodiments, administering the lymphodepleting regimen comprises administering cyclophosphamide and fludarabine prior to administration of the first dose, and administering a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) after administration of the first dose. In some embodiments, administering the lymphodepleting regimen comprises administering the lymphodepleting regimen prior to administration of the first dose, and optionally, not before administration of a subsequent dose. In some embodiments, the lymphodepleting regimen is administered between 2 and 10 days prior to administration of the first dose. In some embodiments, the lymphodepleting regimen is administered 2-10 days before the administration of the second dose. In some embodiments, the lymphodepleting regimen is administered 2-10 days before the administration of the first dose and 2-14 days before the administration of the second dose. In some embodiments, the lymphodepleting regimen is administered over the course of 1, 2, 3, 4, or 5 days.

[0033] In some embodiments, the lymphodepleting regimen is about 90-120 mg / m 2 of fludarabine, and approximately 1500 mg / m 2 In some embodiments, the lymphodepleting regimen comprises administering about 150 mg / m of cyclophosphamide. 2 of fludarabine, and approximately 120 mg / m 2 of cyclophosphamide.

[0034] In some embodiments, the lymphodepleting regimen is about 30-150 mg / m 2 Fludarabine at a dose of approximately 300-4000 mg / m 2In some embodiments, the method comprises administering cyclophosphamide at a dose of about 0.3 to 1 mg / kg, or a CD52 antibody (e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) at a dose of about 0.3 to 1 mg / kg. In some embodiments, fludarabine is administered at a dose of about 30 to 150 mg / m 2 and cyclophosphamide is administered at a dose of approximately 300-4000 mg / m 2 and the CD52 antibody (e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) is administered at a dose of about 10-13 mg. In some embodiments, fludarabine is administered at a dose of about 30 mg / m / day and cyclophosphamide is administered at a dose of about 300 mg / m 2 In some embodiments, fludarabine is administered at a dose of about 30 mg / m 2 or a CD52 antibody (e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) is administered at a dose of about 10 to about 13 mg / m 2 . 2 / day, and cyclophosphamide is administered at a dose of approximately 300 mg / m 2 The CD52 antibody (eg, a CD52 antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) is administered at a dose of about 10 to about 13 mg / day.

[0035] In some embodiments, the lymphodepleting regimen comprises administering fludarabine and cyclophosphamide. In some embodiments, the lymphodepleting regimen comprises administering fludarabine, cyclophosphamide, and an anti-CD52 agent (e.g., a CD52 antibody having the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10). In some embodiments, the lymphodepleting regimen further comprises administering Mentha (sodium-2-mercaptoethanesulfonate). In some embodiments, the lymphodepleting regimen further comprises administering at least one corticosteroid. In some embodiments, the corticosteroid is administered immediately before the administration of the anti-CD52 antibody (e.g., an antibody having the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10). In some embodiments, the corticosteroid is methylprednisolone administered at a dose of 1 to 5 mg / kg (e.g., 2 mg / kg). In some embodiments, the corticosteroid is administered at least two days prior to administration of the CAR-T cells. In some embodiments, corticosteroids are not administered for at least two days prior to administration of the CAR-T cells.

[0036] In some embodiments, patients receive pre-medication for infusion-related reactions prior to the lymphodepleting regimen. Pre-medication may include, for example, an antihistamine or acetaminophen.

[0037] In some embodiments, components of a lymphodepleting regimen of fludarabine / cyclophosphamide (FC) or fludarabine / cyclophosphamide / anti-CD52 antibody (FCA) are administered simultaneously; in other embodiments, the components are administered sequentially. In some embodiments, a subject receives an FC regimen prior to a first dose of CAR-T cell therapy and an FCA regimen prior to re-administration of CAR-T cell therapy. In some embodiments, a subject receives an FCA regimen prior to a first dose of CAR-T cell therapy and a second FCA regimen prior to re-administration of CAR-T cell therapy. In some embodiments, a subject receives an FC regimen prior to a first dose of CAR-T cell therapy and an CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) after the first dose of CAR-T cell therapy. In some embodiments, the subject receives an FC regimen prior to the first dose of CAR-T cell therapy and a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) after the first dose of CAR-T cell therapy, and the subject further receives an FC regimen prior to the second / subsequent dose of CAR-T cell therapy and a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) after the second / subsequent dose of CAR-T cell therapy.

[0038] In some embodiments, the method further comprises administering a disease-reducing agent prior to administration of the first dose and / or prior to administration of the second / subsequent dose. In some embodiments, the method comprises administering a chemotherapeutic agent prior to administration of the first dose and / or prior to administration of the second / subsequent dose. In some embodiments, the subject has been previously treated with a chemotherapeutic agent prior to the first dose. In some embodiments, the chemotherapeutic agent is or comprises conditioning chemotherapy that reduces the subject's disease burden prior to the first dose and / or subsequent doses. In some embodiments, administering the chemotherapeutic agent comprises administering the chemotherapeutic agent prior to administration of the first dose, and optionally not before administration of the second / subsequent dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 10 days prior to administration of the first dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 10 days prior to administration of the second / subsequent dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 10 days prior to administration of the first dose and between 2 and 14 days prior to administration of the second / subsequent dose.

[0039] Also provided are the use of, and cells and compositions for use of, allogeneic CAR-T cells and compositions for treating a disease in a subject, such as a tumor or cancer. Also provided are the use of, and cells and compositions for use of, allogeneic CAR-T cells and compositions for the manufacture of a medicament for treating a disease in a subject previously treated with allogeneic CAR-T cells. In some embodiments, the compositions or cells for use or medical application are for use 4 to 24 weeks after the previous treatment. In some embodiments, the compositions or cells for use are formulated for administration of a subsequent dose in an amount sufficient to reduce the disease burden in a subject previously treated with CAR-T cells. In some embodiments, the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).

[0040] In some embodiments of such medical uses, the composition or cells are for uses comprising administering a first dose of allogeneic CAR-T cells to a subject having a disease. In some embodiments, the first dose contains about 1 x 10 total cells, or about 6 x 10 total cells, or about 1 x 10 total cells. In some embodiments, the composition or cells are for uses comprising administering a subsequent dose of allogeneic CAR-T cells to the subject at least about 4 weeks, or more than about 4 weeks, after initiation of administration of the first dose and less than about 24 weeks after initiation of administration of the first dose.

[0041] In some embodiments, allogeneic CAR-T cells are provided for use in methods of treating disease in a subject previously treated with allogeneic CAR-T cells. In some embodiments, the cells are for use between about 4 and 24 weeks after the previous treatment. In some embodiments, the cells for use are formulated for administration of a subsequent dose in an amount sufficient to reduce disease burden in a subject previously treated with allogeneic CAR-T cells. In some embodiments, the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).

[0042] In some embodiments of any such composition or cell for use or medical application, the subject does not exhibit morphological disease and / or the subject does not exhibit more than 5% blast cells in the bone marrow.

[0043] In some embodiments, the composition or cells are for use in a method comprising administering a first dose of allogeneic CAR-T cells to a subject with a disease. In some embodiments, the first dose is about 6×10 5 cells, approximately 6 x 10 6 cells, approximately 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8 cells, or approximately 2.4 x 10 8In some embodiments, the cells are for use in a method comprising administering to the subject a subsequent dose of allogeneic CAR-T cells at least about 5 weeks, or more than about 5 weeks, after initiation of administration of said first dose, and less than about 24 weeks after initiation of administration of said first dose.

[0044] Provided herein is a use of allogeneic CAR-T cells for the manufacture of a medicament for treating a disease in a subject, comprising the cells formulated and / or packaged for administration to the subject in first and subsequent doses. In some embodiments, the treatment comprises administering to the subject cells in first and subsequent doses, wherein the first dose is about 6×10 5 cells, approximately 6 x 10 6 cells, approximately 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8 cells, or approximately 2.4 x 10 8 In some embodiments, the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).

[0045] In some embodiments, the cells for use are formulated and / or distributed for administration to a subject in a first and subsequent doses, and / or the treatment comprises administering the cells to a subject in a first and subsequent doses. In some embodiments, the first dose is about 6×10 5 cells, approximately 6 x 10 6 cells, approximately 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8 cells, or approximately 2.4 x 10 8 Contains cells.

[0046] In some embodiments, the use includes the first and subsequent administrations comprising administering the cells in one or more unit doses, each unit dose containing about 6×10 5 cells, approximately 6 x 10 6 cells, approximately 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8cells, or approximately 2.4 x 10 8 Contains cells.

[0047] In some embodiments, the cells or uses include where a first administration comprises administration of a single unit dose. In some embodiments, the cells or uses include where a subsequent administration comprises administration of two or more unit doses. In some embodiments, the cells or uses include where a subsequent administration comprises administration of a single unit dose.

[0048] In some embodiments, the use of cells for treating a disease is provided, wherein the disease is a tumor or cancer. In some embodiments, the disease is a lymphoid malignancy, such as, but not limited to, acute and chronic leukemia, lymphoma, multiple myeloma, myeloid malignancies (e.g., leukemia, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm), or mixed lineage malignancies. In some embodiments, the composition or cells are provided for use in treating acute lymphoblastic leukemia (ALL). In some embodiments, the ALL is relapsed / refractory ALL. In some embodiments, the composition or cells are provided for use in treating non-Hodgkin's lymphoma, e.g., relapsed or refractory non-Hodgkin's lymphoma. In some embodiments, the composition or cells are for use in treating relapsed or refractory large B-cell or follicular lymphoma.

[0049] In some embodiments, the cells, compositions, or uses include cases where the subsequent dose is formulated for administration of approximately the same number of allogeneic CAR-T cells as the allogeneic CD19-specific CAR-T cells in the previous dose. In some embodiments, the composition containing the cells in the subsequent dose is formulated for administration of an increased number of allogeneic CAR-T cells compared to the first dose or the previous dose. In some embodiments, the cells, compositions, or uses include cases where the subsequent dose is formulated for administration of less than the number of allogeneic CAR-T cells as the allogeneic CD19-specific CAR-T cells in the previous dose.

[0050] Also provided are pharmaceutical kits for treating patients with diseases such as cancer, the kits comprising CAR-T cells and a CD52 antibody. In some embodiments, the CAR-T cells are CD19-specific CAR-T cells, such as UCART19. In some embodiments, the CAR-T cells express the CAR of SEQ ID NO: 1. In some embodiments, the CD52 antibody comprises the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10. In some embodiments, the kit comprises a first container comprising the CAR-T cells and a second container comprising the CD52 antibody. In some embodiments, at least one of the first and second containers is a flexible cell infusion bag. In some embodiments, the kit further comprises a label or package insert comprising instructions for administering the CAR-T cells and the CD52 antibody to a subject.

[0051] Also provided are articles of manufacture for carrying out the methods. In some embodiments, the article of manufacture comprises multiple containers, e.g., sealable containers, each individually containing a unit dose of allogeneic CAR-T cells, packaging material, and / or a label or package insert for administration to a subject. In some embodiments, the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).

[0052] In some embodiments, the unit dose comprises the amount of cells provided in the smallest dose of the method, such as the size of the first dose. In some embodiments, the unit dose comprises about 6 x 10 5 cells, approx. 6 x 106 cells, approx. 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8 cells, or approximately 2.4 x 10 8 Contains cells.

[0053] In some embodiments, the label or package insert includes instructions for administering multiple unit doses to a subject by administering a specified number of such unit doses, e.g., one unit dose, followed by one or more subsequent unit doses, e.g., in a first dose administration. In some embodiments, the instructions specify administering a first administration, wherein said first administration comprises delivering one of said unit doses to the subject to administer a subsequent administration, wherein said subsequent administration comprises administering one or more of said unit doses to the subject. In some embodiments, the instructions specify that the subsequent administration is to be administered between about 5 and about 24 weeks after said first administration. In some embodiments, the container is or comprises a flexible cell infusion bag. In some embodiments, the method is for allogeneic administration. In some embodiments, the label and / or packaging material further includes a subject-specific identifier indicating that the cells are derived from and / or should be specifically administered to the subject. In some embodiments, the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).

[0054] Also provided are methods for generating a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) directed to a target of interest, comprising: (a) isolating peripheral blood mononuclear cells (PBMCs) from a healthy donor; (b) activating T cells in the PBMCs; (c) transducing the activated T cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing a CAR of interest; (d) disrupting TCRαβ and CD-52 gene expression in a subset of T cells; (e) expanding the population of T cells; and (f) enriching the population of T cells for TCRαβ-negative cells, thereby generating a "ready-made" population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells). In some embodiments, the CAR-T cells comprise allogeneic CD19CAR / RQR8+_TCRαβ-_T cells.

[0055] Also provided are allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR for use in a method of treating a subject with refractory and / or relapsed non-Hodgkin's lymphoma, the method comprising administering to the subject at least one dose of the CAR-T cells, wherein the at least one dose is greater than or equal to about 20x10 6 cells / dose ~ approx. 360x10 6 cells / dose.

[0056] Also provided are chimeric antigen receptor (CAR)-T cells (CAR-T cells) for use in a method of treatment comprising: (a) administering a first dose of CAR-T cells to a subject; and (b) administering a subsequent dose of CAR-T cells to a subject at least about 28 days, or more than about 28 days, after initiation of said administering in (a) and less than about 200 days after initiation of said administering.

[0057] Also provided are allogeneic chimeric antigen receptor (CAR)-T cells for use in a method of treatment comprising administering a subsequent dose of allogeneic CAR-T cells to a subject previously administered a first dose of allogeneic CAR-T cells, wherein the subsequent dose of cells is administered at least about 5 weeks or more than about 5 weeks after initiation of the first dose and less than about 24 weeks after initiation of the first dose. Optionally, at the time of administration, the subject does not exhibit a detectable adaptive host immune response specific for the CAR-T cells.

[0058] Also provided are allogeneic chimeric antigen receptor (CAR)-T cells for use in a method of treatment comprising administering to a subject a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells, wherein prior to said administering, the subject has received a previous dose of CAR-T cells in an amount sufficient to show a clinical benefit in the subject, and at the time of administration, the subject does not exhibit a detectable adaptive host immune response specific to the CAR-T cells, and / or the time between said previous dose and the subsequent dose is more than about 5 weeks and less than about 24 weeks.

[0059] Also provided are allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR for use in a method of treating an adult subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia, wherein the method comprises administering to the subject at least one dose of CAR-T cells, wherein the at least one dose is about 6x10 5 cells / dose, approximately 6x10 6 cells / dose, approximately 6-8x10 7 cells / dose, and approximately 1.8-2.4x10 8 cells / dose.

[0060] Also included is administering to the subject at least one dose of CAR-T cells comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 2-8 x 10 7 Also provided are cells / doses of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR for use in methods of treating pediatric subjects with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia.

[0061] Also provided are allogeneic chimeric antigen receptor (CAR)-T cells for use in treating a disease in a subject previously treated with CAR-T cells, wherein the cells are for use between about 5 and 24 weeks after the previous treatment, and the cells are prescribed for administration of subsequent doses in an amount sufficient to ameliorate the disease in the subject previously treated with CAR-T cells.

[0062] In some embodiments, the therapeutic methods disclosed herein are applied to subjects who have been treated with antibodies for depletion of CD52+ cells (e.g., antibodies comprising SEQ ID NO:8 and SEQ ID NO:10), or who are being treated with such antibodies as part of such therapeutic methods. These therapeutic methods may include the use of CD52-deficient CAR-T cells (e.g., UCART19).

[0063] Also provided is a method for generating a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) directed to a target of interest, the method comprising: (a) providing isolated peripheral blood mononuclear cells (PBMCs) from a healthy donor; (b) activating T cells in PBMCs; (c) transducing the activated T cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing the CAR of interest; (d) disrupting TCRαβ and CD-52 gene expression in a subset of T cells; (e) expanding a population of T cells; and (f) enriching the population of T cells for TCRαβ negative cells; This generates a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells). [Brief explanation of the drawings]

[0064] [Figure 1] Figure 1 shows an exemplary schematic illustrating potential steps for a multiple-dose regimen for allogeneic CAR-T cell therapy. [Figure 2A] Figure 2A shows exercise data from two patients who received a second UCART19 infusion (rechallenge study). [Figure 2B] Figure 2B shows exercise data from two patients who received a second UCART19 infusion (rechallenge study). [Figure 3A] Figure 3A shows an exemplary schematic diagram illustrating the steps of an administration regimen for allogeneic CAR-T cell therapy. [Figure 3B] Figure 3B shows an exemplary schematic diagram illustrating the steps of the administration regimen for allogeneic CAR-T cell therapy. [Figure 4A] FIG. 4A shows an exemplary flow diagram of allogeneic CAR-T cell manufacturing. [Figure 4B]FIG. 4B shows an exemplary flow diagram of allogeneic CAR-T cell manufacturing. [Figure 5] Figure 5 shows an exemplary engineered allogeneic anti-CD19CAR T cell product (CD19CAR / RQR8+_TCRαβ-_T cells). [Figure 6A] Figure 6A shows the study design for the use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in adult patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 6B] Figure 6B shows the study design for the use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in adult patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 7A] Figure 7A shows the study status after initiation of use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in adult patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 7B] Figure 7B shows response, duration of remission, and re-administration of UCART19 in adult patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 8A] FIG. 8A shows the flow cytometry PK profile and UCART19 kinetics in an adult patient with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 8B] Figure 8B shows the flow cytometry PK profile and UCART19 kinetics in an adult patient with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 9] Figure 9 shows the study design for the use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 10] Figure 10 shows the study status after initiation of use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 11] FIG. 11 shows the response and duration of remission (antileukemic activity) of UCART19 in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 12] FIG. 12 shows cell kinetics following the use of UCART19 in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 13A] FIG. 13A shows chimerism data in blood following use of UCART19 in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 13B] FIG. 13B shows chimerism data in blood following use of UCART19 in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 13C] FIG. 13C shows chimerism data in blood following use of UCART19 in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. [Figure 14] FIG. 14 shows the response to rechallenge of patient #18, as described in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0065] General Technology The practice of the methods and compositions described in this disclosure will generally employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques include Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993~1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988–1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

[0066] definition As used herein, "allogeneic" means that the cells or cell population used to treat a patient do not originate from said patient, but rather from a donor, provided that the cells or cell population do not originate from a human leukocyte antigen (HLA)-matched donor.

[0067] As used herein, "autologous" means that the cells, cell lines, or cell populations used to treat a patient originate from said patient or from a human leukocyte antigen (HLA)-matched donor.

[0068] As used herein, "immune cell" refers to a cell of hematopoietic origin that is functionally involved in the initiation and / or execution of innate and / or adaptive immune responses.

[0069] As used herein, "chimeric antigen receptor" or alternatively "CAR" refers to a molecule in an immune effector cell that, upon engagement with a target, provides the cell with specificity for its target and intracellular signal generation.

[0070] As used herein, the term "dosing regimen" refers to the total course of treatment administered to a patient, e.g., treatment with CAR-T cells.

[0071] As used herein, "treatment" refers to an approach for obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction in (or destruction of) tumor or cancer cell proliferation, inhibition of metastasis of tumor cells, reduction or decrease in tumor size, remission of a disease (e.g., cancer), reduction in symptoms resulting from a disease (e.g., cancer), improvement in the quality of life of a person suffering from a disease (e.g., cancer), reduction in the dosage of other drugs required to treat a disease (e.g., cancer), slowing the progression of a disease (e.g., cancer), curing a disease (e.g., cancer), and / or prolonging survival of a patient with a disease (e.g., cancer). "Reduced incidence" refers to either a reduction in severity, which may include a reduction in the need for and / or amount (e.g., exposure) of other drugs and / or therapies commonly used for the disease.

[0072] "Ameliorate" or "ameliorating" means a lessening or improvement in one or more symptoms compared to not administering a treatment. "Ameliorate" also includes a shortening or reduction in the duration of a symptom.

[0073] As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during disease development. An effective dosage can be administered in one or more administrations. For purposes of this disclosure, an effective dosage of a drug, CAR-T cell, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents; a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result is likely or is achieved.

[0074] An "individual" or "subject" is a mammal, such as a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0075] As used herein, "refractory" refers to a disease (e.g., cancer) that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer is also referred to herein as a resistant cancer.

[0076] As used herein, "recurrent" refers to the return of a disease (e.g., cancer), or signs and symptoms of a disease such as cancer, after a period of improvement, e.g., after prior treatment with a therapy, e.g., cancer therapy.

[0077] As used herein, "minimal residual disease" or "MRD" refers to low-level disease detected in various clinical settings. In some embodiments, MRD refers to molecularly defined relapse after remission.

[0078] As used herein, "vector" refers to a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0079] As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0080] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, located in the variable region of the immunoglobulin molecule through at least one antigen recognition site. As used herein, the term "antibody" encompasses intact polyclonal or monoclonal antibodies, as well as any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof, fusion proteins comprising the antibody, and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site, including, but not limited to, scFv, single-domain antibodies (e.g., shark antibodies and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23(9):1126-1136). Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclass thereof), and antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0081] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., a tumor antigen). The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab, Fab', F(ab'), an Fd fragment consisting of the VH and CH1 domains, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a single-domain antibody (dAb) fragment (Ward et al., 1989, Nature 341:544-546), and isolated complementarity-determining regions (CDRs).

[0082] The term "scFv" includes at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are closely linked, e.g., via a synthetic linker, e.g., via a short flexible linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv may have the VL and VH variable regions in either order, e.g., relative to the N- and C-termini of the polypeptide, which may comprise VL-linker-VH or VH-linker-VL.

[0083] The term "monoclonal antibody" (Mab) refers to an antibody that is derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. In some embodiments, the monoclonal antibodies of the present disclosure exist in a homogeneous or substantially homogeneous population.

[0084] "Humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Generally, humanized antibodies are human immunoglobulins (acceptor antibodies) in which residues from the complementarity-determining regions (CDRs) of the receptor are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0085] As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to that of an antibody that can be produced by a human, and / or an antibody produced using any of the human antibodies known to those of skill in the art or techniques for producing human antibodies disclosed herein. This definition of a human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that comprises a murine light chain and a human heavy chain polypeptide. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, human antibodies are selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be produced by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies may be prepared by immortalizing human B lymphocytes that produce antibodies directed against a target antigen (such B lymphocytes may be harvested from a subject or immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., 1991, J. Immunol., 147(1):86-95, and U.S. Patent No. 5,750,373.

[0086] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions, which contribute to the formation of the antigen-binding site of an antibody. When a variant of a subject variable region is desired, particularly for substitutions of amino acid residues outside the CDR regions (i.e., in the framework regions), appropriate amino acid substitutions, often conservative amino acid substitutions, can be identified by comparing the subject variable region with variable regions of other antibodies containing CDR1 and CDR2 sequences of the same standard family as the subject variable region (Chothia and Lesk, J Mol Biol 196(4): 901-917, 1987). When selecting FRs adjacent to the subject CDRs, for example, when humanizing or optimizing an antibody, FRs from antibodies containing CDR1 and CDR2 sequences of the same standard family are preferred.

[0087] The "CDRs" of a variable domain are those amino acid residues in the variable region identified according to the Kabat, Chothia, or both Kabat and Chothia definitions, the AbM, contact, and / or conformational definitions, or any method of CDR determination known in the art.

[0088] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any material that, when combined with an active ingredient, retains biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. A preferred diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or saline (0.9%). Compositions containing such carriers are formulated by known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st Ed., Mack Publishing, 2005).

[0089] References herein to "about" a value or parameter include (and describe) embodiments that inherently direct that value or parameter. For example, a description referring to "about X" includes a description of "X" and a numerical range includes the numbers defining the range.

[0090] It is understood that for any embodiment described herein using the term "comprising," other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0091] When aspects or embodiments of the present disclosure are described in terms of a Markush group or other alternative grouping, the present disclosure encompasses not only the entire group recited as a whole, but also each individual member of the group, and all possible subgroups of the main group, as well as the main group absent one or more of the group members. The present disclosure also envisions the explicit exclusion of one or more of the group members recited in the claims.

[0092] Ranges: Throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the width of the range.

[0093] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless otherwise defined. In case of conflict, the present specification, including definitions, controls. Throughout this specification and claims, variations such as "comprise" or "comprises" are understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group. Unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include the singular.

[0094] Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions described herein, exemplary methods and materials are described herein. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0095] CAR-T cell treatment Provided herein are methods, compositions, and articles of manufacture for use in cell therapy for the treatment of diseases, including various cancers and tumors. The methods include administering allogeneic chimeric antigen receptor (CAR)-T cells that specifically bind to an antigen associated with the disease and generate a response, such as an immune response, against such molecules based on binding to such antigens.

[0096] The method can be used for the treatment of, for example, hematological neoplasms (WHO classification, 2008) and solid tumors.Hematological neoplasms include, for example, lymphoid malignancies (acute and chronic leukemia, lymphoma, multiple myeloma), myeloid malignancies (leukemia, myelodysplastic syndrome, myelodysplastic or myeloproliferative neoplasms), and mixed lineage malignancies.Solid tumors include, for example, solid tumors that express the antigen targeted by CAR-T cells used in treatment.

[0097] The methods are suitable for treating adult and pediatric populations, including all age subsets, and can be used as any line of treatment, including first-line or subsequent lines.

[0098] In some embodiments, the disease is a tumor, which in some embodiments is a cancer, malignancy, neoplasm, or other proliferative disease or disorder, such as a leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL (e.g., relapsed / refractory ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma. In some embodiments, the disease is selected from the group consisting of DLBCL, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer (e.g., small cell and non-small cell lung cancer), liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, head and neck squamous cell carcinoma (HNSCC), and / or mesothelioma. In some embodiments, the disease is leukemia or lymphoma. In some embodiments, the disease is acute lymphoblastic leukemia. In some embodiments, the disease is relapsed or refractory acute lymphoblastic leukemia. In some embodiments, the disease is non-Hodgkin's lymphoma (NHL).

[0099] In some embodiments, the disease is acute lymphoblastic leukemia (ALL). In some embodiments, the disease is childhood acute lymphoblastic leukemia (ALL).

[0100] In some embodiments, the disease is an aggressive lymphoid malignancy such as B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the disease is refractory B-ALL, e.g., adult refractory B-ALL. In some embodiments, the disease is relapsed B-ALL, e.g., adult relapsed B-ALL.

[0101] In some embodiments, the disease is non-Hodgkin's lymphoma, such as large B-cell and / or follicular lymphoma. Large B-cell lymphoma can be any large B-cell lymphoma as diagnosed using histologic or cytologic methods according to the 2018 WHO revision of the lymphoma classification: diffuse large B-cell lymphoma (DLBCL)—not otherwise specified (NOS) (germinal center B-cell [GCB], and non-GCB), coexistence of DLBCL with any grade of follicular lymphoma, intravascular large B-cell lymphoma, DLBCL associated with chronic inflammation, anaplastic lymphoma kinase-positive (ALK+) DLBCL, Epstein-Barr virus-positive (EBV+) DLBCL-NOS, T-cell / histiocytocyte-rich large B-cell lymphoma, DLBCL with IRF4 / MUM1 rearrangements, high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 translocations (double / triple hit), primary cutaneous DLBCL—limbic type, transformation of follicular lymphoma to DLBCL, and primary mediastinal B-cell lymphoma. Possible parameters for treatment include, for example, kinetic parameters of disease assessment (adapted for disease targeting), and the health status of the subject administered CAR-T.

[0102] The treatment method may include several steps including lymphodepletion (optionally with prior drug administration), treatment and optional re-treatment.

[0103] Lymphatic depletion In some embodiments, the subject is administered a lymphodepleting (LD) regimen prior to the first and / or subsequent doses of CAR-T cells. In some embodiments, the subject is administered a lymphodepleting regimen simultaneously with the first and / or subsequent doses of CAR-T cells. In some embodiments, the subject is administered a lymphodepleting regimen before, during, and / or after the first and / or subsequent doses of CAR-T cells.

[0104] Suitable LD regimens are described herein and / or known in the art. In some embodiments, LD is initiated prior to, concurrent with, or after CAR-T infusion. The dose and timing of LD administration can be adapted for the first or subsequent dose of CAR-T. In some embodiments, the duration of LD is about 3-5 days. In some embodiments, the time window between the end of LD and the start of CAR-T administration is between about 2 days and about 2 weeks. In some embodiments, LD is initiated about 15-7 days prior to administration of a dose of CAR-T cells. In some embodiments, LD is initiated about 19-5 days prior to administration of a dose of CAR-T cells. In some embodiments, LD is initiated about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days prior to administration of a dose of CAR-T cells. In some embodiments, the duration of the LD regimen is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the dose of CAR-T cells is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the end of the LD.

[0105] In some embodiments, the LD regimen comprises administration of one or more chemotherapy agents.

[0106] In some embodiments, the LD regimen involves the administration of an anti-CD52 antibody, such as an antibody that recognizes the human cluster of differentiation (CD)52 antigen, a cell surface glycoprotein expressed on most lymphoid cells. As used herein, a CD52 monoclonal antibody is one that is directed against the 21-28 kD cell surface glycoprotein CD52. CD52 is an abundant molecule (approximately 5 x 10 per cell). 5CD52 antibodies (antibody binding sites) are present on at least 95% of all human peripheral blood lymphocytes and monocytes / macrophages. Exemplary CD52 antibodies for use in the methods and compositions described herein include, for example, alemtuzumab. In some embodiments, the CD52 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as shown in Table 1 below. TIFF2025188073000002.tif48146

[0107] In some embodiments, the CD52 antibody comprises a VH and / or VL comprising a sequence shown in Table 2 below. TIFF2025188073000003.tif219170 TIFF2025188073000004.tif168170

[0108] In some embodiments, the CD52 antibody comprises a VH having SEQ ID NO: 8 or a sequence having 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%, or at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the CD52 antibody comprises a VL having SEQ ID NO: 10 or a sequence having 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%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the CD52 antibody comprises a VH having the sequence of SEQ ID NO: 8 and a VL having the sequence of SEQ ID NO: 10. In some embodiments, the CD52 antibody comprises a VH encoded by the DNA sequence of SEQ ID NO: 9 and a VL encoded by the DNA sequence of SEQ ID NO: 11.

[0109] In some embodiments, the anti-CD52 antibody is a recombinant humanized IgG1 kappa monoclonal antibody (mAb). In some embodiments, the anti-CD52 antibody is alemtuzumab. Alemtuzumab is a recombinant DNA-derived humanized monoclonal antibody directed against the 21-28 kD cell surface glycoprotein, CD52. See, e.g., Saif et al., Pediatr Transplant 2015 Mar;19(2):211-8. In some embodiments, the anti-CD52 antibody comprises one or more CDR sequences isolated from or derived from the CDRs of alemtuzumab. In some embodiments, the anti-CD52 antibody comprises the sequence of SEQ ID NO:8 or a sequence having 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%, or at least 99% sequence identity to SEQ ID NO:8. In some embodiments, the anti-CD52 antibody comprises the sequence of SEQ ID NO: 10, or a sequence having 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%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CD52 antibody comprises an HCDR1 comprising the sequence of SEQ ID NO: 2, an HCDR2 comprising the sequence of SEQ ID NO: 3, an HCDR3 comprising the sequence of SEQ ID NO: 4, an LCDR1 comprising the sequence of SEQ ID NO: 5, an LCDR1 comprising the sequence of SEQ ID NO: 6, and / or an LCDR3 comprising the sequence of SEQ ID NO: 7.In some embodiments, the anti-CD52 antibody comprises an HCDR1 comprising the sequence of SEQ ID NO: 2, an HCDR2 comprising the sequence of SEQ ID NO: 3, an HCDR3 comprising the sequence of SEQ ID NO: 4, an LCDR1 comprising the sequence of SEQ ID NO: 5, an LCDR1 comprising the sequence of SEQ ID NO: 6, and an LCDR3 comprising the sequence of SEQ ID NO: 7, wherein the anti-CD52 antibody comprises the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10, or a sequence having 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%, or at least 99% sequence identity to SEQ ID NO: 8 and / or SEQ ID NO: 10.

[0110] In some embodiments, the LD includes a combination of therapies. In some embodiments, the combination includes fludarabine (about 90-150 mg / m ) with or without an anti-CD52 agent (e.g., an anti-CD52 antibody, such as an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) (total dose of about 0.3 to about 1 mg / kg, or a flat dose of about 30 mg to about 40 mg, about 25 to about 60 mg, or about 100 mg to about 120 mg). 2 total dose), and cyclophosphamide (total dose range approximately 1000 to 4000 mg / m 2 In some embodiments, the combination includes fludarabine (about 30 mg / m ), with or without an anti-CD52 agent (e.g., a CD52 antibody) (total dose of about 0.3 to about 1 mg / kg, or a flat dose of about 30 mg to about 40 mg, about 25 to about 60 mg, or about 100 mg to about 120 mg). 2 ), and cyclophosphamide (range total dose approximately 500–600 mg / m 2 In some embodiments, the combination includes fludarabine (about 30 mg / m), with or without an anti-CD52 agent (e.g., a CD52 antibody) (total dose of about 0.3 to about 1 mg / kg, or a flat dose of about 30 mg to about 40 mg, about 25 mg to about 60 mg, or about 100 mg to about 120 mg). 2), and cyclophosphamide (approximately 300 mg / m 2 In some embodiments, the combination includes fludarabine (about 90 mg / m 2 ), cyclophosphamide (approximately 1500 mg / m 2 In some embodiments, the combination includes fludarabine (about 150 mg / m) with or without an anti-CD52 agent (e.g., an anti-CD52 antibody, about 1 mg / kg). In some embodiments, the combination includes fludarabine (about 150 mg / m) with or without an anti-CD52 agent (e.g., a CD52 antibody, total dose of about 0.3 to about 1 mg / kg, or a flat dose of about 30 mg to about 40 mg, about 25 to about 60 mg, or about 100 mg to about 120 mg). 2 ), and cyclophosphamide (about 130 mg / kg). In some embodiments, the combination includes fludarabine (about 150 mg / m), with or without an anti-CD52 agent (e.g., an anti-CD52 antibody), at a total dose of about 0.3 to about 1 mg / kg, or a flat dose of about 30 mg to about 40 mg, about 25 to about 60 mg, or about 100 mg to about 120 mg. 2 ), and cyclophosphamide (about 120 mg / kg or about 130 mg / kg). In some embodiments, the combination includes fludarabine (about 30 mg / m 2 / day), and cyclophosphamide (approximately 300 mg / m 2 In some embodiments, the combination includes fludarabine (about 30 mg / m) with or without an anti-CD52 agent (e.g., an anti-CD52 antibody, about 10 mg / day). 2 / day), and cyclophosphamide (approximately 300 mg / m 2 / day). In some embodiments, these above doses are administered over the course of a single day. In some embodiments, these above doses are administered over multiple days.

[0111] In some embodiments, fludarabine and cyclophosphamide are administered on day 1, and the anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) is administered on day 2. In some embodiments, fludarabine and cyclophosphamide are administered prior to administration of the CAR-T cells on day 1, and the anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) is administered on day 2, wherein day 2 is the same day the CAR-T cells are administered or day 2 is after the CAR-T cells are administered. In some embodiments, fludarabine and cyclophosphamide are administered on day 1, the CAR-T cells are administered on day 2, and the anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) is administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks after day 2. In some embodiments, fludarabine and cyclophosphamide are administered prior to administration of the CAR-T cells, and the anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) is administered at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks after administration of the CAR-T cells.

[0112] In some embodiments, the lymphodepleting regimen comprises administration of fludarabine and cyclophosphamide (FC). In some embodiments, the lymphodepleting regimen comprises administration of fludarabine and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:10) (FA). In some embodiments, the lymphodepleting regimen comprises administration of cyclophosphamide and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:10) (CA). In some embodiments, the lymphodepleting regimen comprises administration of fludarabine, cyclophosphamide, and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:10) (FCA).

[0113] The selection of a specific lymphodepletion regimen agent and dose prior to the first or second / subsequent dose of CAR-T cells can be determined based on the patient's hematological analysis and hematological recovery. In the case of re-administration, the second lymphodepletion regimen can be more or less intensive compared to the first lymphodepletion regimen (e.g., based on lymphocyte, neutrophil, and viral reactivation after the first dose). For example, if lymphocyte and neutrophil levels are high at the time of re-administration, a strong or aggressive lymphodepletion regimen can be used. Alternatively, if lymphocyte levels are low at the time of re-administration, a weaker or less aggressive lymphodepletion regimen can be used. In some embodiments, if the blast count at the time of re-administration is high, a strong or aggressive lymphodepletion regimen is used. In some embodiments, if the blast count at the time of re-administration is low, a weaker or less aggressive lymphodepletion regimen is used.

[0114] In some embodiments, an increase in the intensity of the LD regimen may be applied upon re-administration (with or without an anti-CD52 agent), hi some embodiments, a decrease in the intensity of the LD regimen may be applied, for example, in the case of grade 3-4 lymphopenia upon re-administration (with or without an anti-CD52 agent).

[0115] In some embodiments, components of a lymphodepleting regimen of fludarabine / cyclophosphamide (FC) or fludarabine / cyclophosphamide / anti-CD52 antibody (FCA) are administered simultaneously; in other embodiments, the components are administered sequentially. In some embodiments, a subject receives an FC regimen prior to a first dose of CAR-T cell therapy and an FCA regimen prior to re-administration of CAR-T cell therapy. In some embodiments, a subject receives an FCA regimen prior to a first dose of CAR-T cell therapy and a second FCA regimen prior to re-administration of CAR-T cell therapy.

[0116] Exemplary LD regimens are provided in Tables 3A, 3B, 3C, 3D, and 3E. In Tables 3A-3E, the timing shown in the schedules relates to the timing of administration of the dose of CAR-T cells on a daily basis (D0). Negative numbers indicate the number of days prior to administration of CAR-T cells (day D0). TIFF2025188073000005.tif45167

[0117] TIFF2025188073000006.tif52167

[0118] TIFF2025188073000007.tif48164

[0119] TIFF2025188073000008.tif42167

[0120] TIFF2025188073000009.tif44165

[0121] In some embodiments, the LD regimen may further comprise treatment with Mentha (sodium 2-mercaptoethanesulfonate).

[0122] Premedication In some embodiments, the method may include a pre-medication for infusion-related reactions prior to the lymphodepletion regimen. In some embodiments, the method may include a pre-medication administered prior to treatment with an anti-CD52 antibody. In some embodiments, the pre-medication may include treatment with high-dose corticosteroids. For example, the pre-medication may include treatment with 2 mg / kg methylprednisolone. In some embodiments, the pre-medication is administered immediately prior to infusion with the anti-CD52 antibody. In some embodiments, the pre-medication is discontinued at least one day or at least two days prior to any CAR-T cell (e.g., UCART19) infusion.

[0123] In some embodiments, the prior drug administration can include treatment with one or more of an antihistamine, cimedidine, ranitidine, and acetaminophen. The antihistamine can be administered, for example, about 1 day, about 1 hour, or about 0.5 hours before administration of the anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:10). The acetaminophen can be administered, for example, about 1 day, about 1 hour, or about 0.5 hours before administration of the anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:10).

[0124] treatment In some embodiments, the CAR-T cells are administered by intravenous infusion, hi some embodiments, the intravenous infusion lasts for about 1 minute, about 3 minutes, about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 3 hours, about 6 hours, about 12 hours, or about 24 hours.

[0125] In some embodiments, a method of treating an adult subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 6x10 5 cells / dose, approximately 6x10 6 cells / dose, approximately 6-8x10 7 cells / dose, and approximately 1.8-2.4x10 8 In some embodiments, the CAR-T cells are selected from the group consisting of: cells / dose. In some embodiments, the CAR-T cells express the CAR of SEQ ID NO: 1. In some embodiments, the CAR-T cells are UCART19 (CD19)CAR / RQR8+_TCRαβ-_T cells. In some embodiments, the CAR-T cells are CD-52 deficient. In some embodiments, the CAR-T cells are a mixture of CD52 deficient and CD52 positive cells. In some embodiments, the CAR-T cells express a safety switch such as RQR8.

[0126] In some embodiments, a method of treating a pediatric subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 2-8 x 10 7 In some embodiments, the CAR-T cells express the CAR of SEQ ID NO: 1. In some embodiments, the CAR-T cells are UCART19 (CD19)CAR / RQR8+_TCRαβ-_T cells. In some embodiments, the CAR-T cells are CD-52 deficient. In some embodiments, the CAR-T cells are a mixture of CD52 deficient and CD52 positive cells. In some embodiments, the CAR-T cells express a safety switch such as RQR8.

[0127] In some embodiments, a method of treating a subject with non-Hodgkin's lymphoma (e.g., refractory and / or relapsed large B-cell lymphoma or follicular lymphoma) includes administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising (e.g., an anti-human CD19 4-1BB / CD3 zeta CAR), wherein the at least one dose is greater than or equal to about 20x10 6 cells / dose ~ approx. 360x10 6 In some embodiments, at least one dose is about 20x10 cells / dose. 6 cells / dose, approximately 40x10 6 cells / dose, approximately 80x10 6 cells / dose, approximately 120x10 6 cells / dose, 240x10 6 cells / dose, and approximately 360x10 6In some embodiments, the CAR-T cells are selected from the group consisting of: cells / dose. In some embodiments, the CAR-T cells express the CAR of SEQ ID NO: 1. In some embodiments, the CAR-T cells are UCART19 (CD19)CAR / RQR8+_TCRαβ-_T cells. In some embodiments, the CAR-T cells are CD-52 deficient. In some embodiments, the CAR-T cells are a mixture of CD52 deficient and CD52 positive cells. In some embodiments, the CAR-T cells express a safety switch, such as the RQR8 safety switch described herein.

[0128] In some embodiments, CAR expression is detectable in the subject by at least 14 days or at least 28 days after CAR administration. In some embodiments, the subject exhibits a CR (complete response) or Cri (complete response with incomplete recovery of blood counts) for at least 1 month, at least 1.3 months, at least 1.4 months, at least 1.6 months, at least 1.8 months, at least 2 months, at least 2.3 months, at least 1.4 months, at least 1.6 months, at least 1.8 months, at least 3 months, at least 3.3 months, at least 3.4 months, at least 3.6 months, at least 3.8 months, at least 6 months, at least 12 months, or at least 36 months after CAR-T (e.g., UCART19) administration.

[0129] Retreatment with CAR-T cells Also provided herein are methods for re-treatment (re-administration) with CAR-T cells. In particular, the methods involve administering one or more subsequent doses of cells to a subject who has received a first dose, and / or administering a first and one or more subsequent doses. The doses are generally administered in specific amounts and according to specific timing parameters. In some embodiments, the methods generally involve administering a first dose of cells, thereby reducing disease burden, followed by a subsequent dose administered within a specific time window relative to the first dose, or a subsequent dose to the subject who received such a first dose. In some embodiments, additional subsequent doses are then administered, e.g., within the same or similar time window relative to the subsequent dose. In some embodiments, the number of cells administered and the timing of multiple doses are designed to improve one or more outcomes, such as reducing the likelihood or degree of toxicity to the subject, improving subject exposure and / or persistence of the administered cells, and / or improving therapeutic efficacy. Also provided are articles of manufacture containing the cells and designed for administration following such dosing regimens.

[0130] In some embodiments, the subject being treated with a subsequent dose of one or more cells is in relapse, meaning that the patient was in CR (complete response) or Cri (complete response with incomplete recovery of blood counts) at a first time point after the initial administration, but relapsed at a second, subsequent time point. Relapse may be, for example, a morphological relapse characterized by 5% or more blasts in the bone marrow, the presence of extramedullary disease associated with the presence of blasts in the bone marrow, or the presence of minimal residual disease (MRD), defined as 3-10 or more blasts in the bone marrow.

[0131] In some embodiments, a subject treated with a subsequent dose of one or more cells exhibits a suboptimal response after administration of the first dose, e.g., the subject may exhibit the presence of minimal residual disease (MRD) about 7 days, about 14 days, about 28 days, about 60 days, about 90 days, about 120 days, or about 365 days after administration of the first dose.

[0132] In some embodiments, a subject treated with one or more subsequent doses of cells exhibits non-persistence of the CAR after administration of the initial dose. For example, a subject may exhibit a detectable CAR after the initial dose (DO, CAR expansion), but no detectable CAR after DO. The lack of a detectable CAR occurs in some embodiments even if the subject exhibits some signs of CAR-T cell activity (such as cytokine release syndrome).

[0133] In some embodiments, subjects treated with one or more subsequent doses of cells do not exhibit substantial proliferation of CAR-T cells. In some embodiments, about 7 days, about 14 days, about 28 days, about 60 days, about 90 days, about 120 days, or about 365 days after administration of the first dose, the subject has no detectable CAR proliferation and no response. In some embodiments, about 7 days, about 14 days, about 28 days, about 60 days, about 90 days, about 120 days, or about 365 days after administration of the first dose, the subject has no detectable CAR proliferation and has some signs of a therapeutic response (e.g., an MRD-positive subject). In some embodiments, about 7 days, about 14 days, about 28 days, about 60 days, about 90 days, about 120 days, or about 365 days after administration of the first dose, the subject has no detectable CAR proliferation and has a complete response (e.g., an MRD-negative subject).

[0134] In some embodiments, the provided methods include administering subsequent doses of cells in approximately the same number, and therefore similar, doses as the first dose. As demonstrated herein, administering subsequent doses of cells can be advantageous compared to a single administration. In some embodiments, about 6×10 5 cells, approximately 6 x 10 6 cells, approximately 6 x 10 7 cells, approximately 8 x 10 7 cells, approximately 1.8 x 10 8 cells, or approximately 2.4 x 10 8Administration of subsequent doses of cells, such as cells, is associated with increased overall survival in subjects, particularly in subjects who exhibit morphological disease prior to treatment. In some embodiments, the methods involve administering a first dose of cells that are capable of proliferating in the presence of disease-associated antigens and reducing symptoms associated with the disease, but without the same degree of toxic consequences that may be associated with higher doses.

[0135] In some embodiments, the first dose is also generally large enough to be effective in reducing disease burden. In some cases, the first dose is large enough to reduce disease burden, where the cell dose is sufficient to reduce in vivo proliferation and disease. In some embodiments, the first dose of cells thereby debulks or reduces disease burden, e.g., tumor size, without causing severe undesirable consequences. In some cases, the first dose is an amount of cells effective to reduce tumor burden, e.g., by reducing disease from a morphological setting to minimal residual disease (MRD) and / or clinical remission or complete remission. In some aspects, the first dose is a low dose. In some aspects, for example, in situations of relatively low disease burden, the first dose may be higher.

[0136] In some embodiments, a risk-based dosing regimen may be used to determine the appropriate number or amount or relative number or amount of cells or CAR-T cells in a dose. For example, in some aspects, prior to infusion of CAR-T cells, the subject's disease burden is determined, e.g., based on the observations described above and elsewhere herein that higher doses of recombinant receptor-expressing cells (e.g., CAR-expressing, such as CAR-expressing T cells) may be associated with toxic outcomes, such as severe neurotoxicity, in subjects with morphological disease burdens that need not be observed or are observed to a lesser extent in subjects with relatively lower disease burdens, and a first dose (e.g., a low dose or a high dose) of CAR-T cells that may minimize toxicity and maximize efficacy is selected based on the disease burden. For example, it has been observed that subjects with a high bone marrow tumor burden before treatment, which in some cases may be associated with greater CAR T cell expansion after treatment, have a higher risk of developing severe neurotoxicity that may require ICU care.

[0137] In some embodiments, the subject is assessed for response after the first dose of allogeneic CAR-T cells. Such assessment can occur about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks after administration of the first or previous dose of allogeneic CAR-T cells. In some embodiments, the subject is assessed for response about 1, about 2, or about 3 months after administration of the first dose of allogeneic CAR-T cells.

[0138] In some embodiments, expression of the antigen targeted by CAR-T may be confirmed before administering a subsequent dose of CAR-T cells. For example, antigen expression may be assessed by flow cytometry in blood, other matrices, or solid tumors. In some embodiments, the subject may be assessed for the absence of anti-HLA antibodies developed by the subject in response to a previous CAR-T administration. In some embodiments, the subject may be assessed for the absence of anti-scFv antibodies developed by the subject after a previous CAR-T administration. In some embodiments, the subject may be assessed for the absence of any significant safety issues after any course of CAR-T (first or subsequent course). In some embodiments, the subject may be assessed for suitability for subsequent lymphodepletion associated with CAR-T administration. In some embodiments, the absence of donor-specific anti-HLA, anti-CAR scFv, and / or anti-TALEN antibodies after administration of CAR-T cells indicates the subject's suitability for administration of a subsequent dose of CAR-T cells.

[0139] In some embodiments, a lack of persistence of the CAR-T cells beyond the day of the first assessment of response after administration of the CAR-T cells indicates the subject's suitability for administration of subsequent doses of CAR-T cells. In some embodiments, a subject can be evaluated for a lack of persistence of the CAR-T cells beyond the day of the first assessment of response after administration of the CAR-T cells. Evaluation can be performed according to disease, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks after administration, regardless of the response evaluated. Parameters for kinetic assessment include, for example, AUC0-timepoint of response assessment, Cmax (peak proliferation of transgene CAR-T, time of last observed quantitative transgene, and levels of CAR-T cell proliferation in any other tissues or fluids. Whether a suboptimal response after the first CAR-T, CAR-T persistence, or administration of CAR-T, can include one or more of the following: (a) complete response (CR) with minimal detectable residual disease (e.g., in leukemia patients), or complete response with incomplete recovery of blood counts (CRi), (b) absence of cytogenetic response, (c) complete bone marrow response, (d) partial response, and / or (e) stable response.

[0140] In some embodiments, subjects may be evaluated for adverse events beyond the day of assessment of the first response after administration of a dose of CAR-T cells. Evaluation may be performed, for example, at about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks after administration. In some embodiments, evaluation may be performed, for example, within the first 4 weeks (up to day 28) after CAR-T administration. Exemplary adverse events are summarized in Table 4.

[0141] TIFF2025188073000010.tif68167

[0142] In some embodiments, disease recurrence may refer to recurrence after achieving an optimal CR after a first CAR-T administration. In some embodiments, disease recurrence may refer to recurrence after one or more CRs have been achieved with CAR-T, regardless of when the recurrence occurred.

[0143] Subsequent CAR-T doses may originate from the same or a different donor (the donor of the healthy cells used in CAR-T manufacturing). The same or different (higher or lower) doses of CAR-T cells may be applied to the subsequent doses.

[0144] CAR-T cells may be administered as a single dose or as divided doses. For example, the dose can be divided into two or more doses within a period of time, such as, for example, 7 days. In some embodiments, the divided dose comprises a first and a second dose within a period of time. In some embodiments, the same number of cells are administered in each of the first and second doses. In some embodiments, fewer cells are administered in the first dose than in the second dose. In some embodiments, a greater number of cells are administered in the first dose than in the second dose. In some embodiments, the divided dose comprises a first, second, and third dose within a period of time. In some embodiments, the same number of cells are administered in each of the first, second, and third doses. In some embodiments, fewer cells are administered in the first dose than in the second and third doses. In some embodiments, a greater number of cells are administered in the first dose than in the second and third doses. In some embodiments, fewer cells are administered in the second dose than in the first and third doses. In some embodiments, a greater number of cells are administered during the second administration than during the first and third administrations. In some embodiments, fewer cells are administered during the third administration than during the first and second administrations. In some embodiments, a greater number of cells are administered during the third administration than during the first and second administrations.

[0145] In some embodiments, the split dose is administered over the first and second days. In some embodiments, half of the dose is administered on the first day and half of the dose is administered on the second day. In some embodiments, one-third of the dose is administered on the first day and two-thirds of the dose is administered on the second day. In some embodiments, two-thirds of the dose is administered on the first day and one-third of the dose is administered on the second day. In some embodiments, each sub-dose of the split dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of each other. In certain embodiments, each sub-dose of the split dose is administered within 7 days of each other. In some embodiments, each sub-dose is administered on consecutive days.

[0146] In some embodiments, the divided doses are administered over the first, second, and third days. In some embodiments, one-third of the dose is administered on the first day, one-third of the dose is administered on the second day, and one-third of the dose is administered on the third day. In some embodiments, each sub-dose of the divided dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of each other. In certain embodiments, each sub-dose of the divided dose is administered within 7 days of each other. In some embodiments, each sub-dose is administered on consecutive days.

[0147] In some embodiments, the divided doses are administered over a period of at least 3 days, eg, at least 4 days, at least 5 days, at least 6 days, or at least 7 days.

[0148] Dosing regimen In some embodiments, allogeneic CAR-T cells are administered using a flat dose. In other embodiments, allogeneic CAR-T cells are administered using dose banding. For example, dose banding may be used to avoid the risk of broad CAR-T cell exposure. In some embodiments, weight bands may be used. For example, but not limited to, subjects under 66 kg may be administered dose X, and subjects over 66 kg may be administered doses of approximately 1.33X. In some embodiments, subjects over 50 kg may be administered one dose, and subjects under 50 kg may be administered different doses.

[0149] Exemplary dose levels for the first dose of UCART19 for use in adult patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia are provided in Table 5a. TIFF2025188073000011.tif44165

[0150] Exemplary dose levels for the first dose of UCART19 for use in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia are provided in Table 5b. TIFF2025188073000012.tif69170

[0151] Exemplary dosage forms of the first dose of UCART19 for use in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia are provided in Table 5c. TIFF2025188073000013.tif45133

[0152] Exemplary dosages for the first dose of UCART19 for use in adult patients with relapsed / refractory non-Hodgkin's lymphoma, such as large B-cell and / or follicular lymphoma, are provided in Table 5d. TIFF2025188073000014.tif46148

[0153] In some embodiments, the allogeneic CAR-T cell dosing regimen comprises about 1x10 5 ~5x10 8 cells, or approximately 1 x 10 4 cells / kg ~ approx. 3x10 6 In some embodiments, the allogeneic CAR-T cell dosing regimen comprises administering a first dose of CAR T cells in the range of between about 6x10 cells / kg. 6 In some embodiments, the allogeneic CAR-T cell dosing regimen comprises administering a first dose of CAR T cells of about 1x10 cells (flat dose). 4 , about 2x10 4 , about 3x10 4 , about 4x10 4 , about 5x10 4 , about 6x10 4 , approximately 7x10 4 , about 8x10 4 , approximately 9x10 4 , about 10x10 4 , approximately 1x10 5 , about 2x10 5 , about 3x10 5 , about 4x10 5 , about 5x10 5 , about 6x10 5 , approximately 7x10 5 , about 8x10 5 , approximately 9x10 5 , approximately 1x10 6 , about 2x10 6 , about 3x10 6 , about 4x10 6 , about 5x10 6 , about 6x10 6 , approximately 7x10 6 , about 8x10 6 , approximately 9x10 6 , approximately 1x10 7 , about 2x10 7 , about 3x10 7 , about 4x10 7 , about 5x10 7 , about 6x10 7 , approximately 7x10 7 , about 8x10 7 , approximately 9x10 7 , approximately 1x10 8 , about 2x10 8, about 3x10 8 , about 4x10 8 , about 5x10 8 , about 6x10 8 , approximately 7x10 8 , about 8x10 8 , or about 9x10 8 In some embodiments, the administration regimen comprises administering a first dose of CAR-T cells. 4 cells, approximately 6x10 6 cells, approximately 7x10 6 cells (weight-banded), or approximately 1.8 x 10 8 In some embodiments, the administration regimen comprises administering a first dose of cells (weight-banded). 6 cells / dose, approximately 40x10 6 cells / dose, approximately 80x10 6 cells / dose, approximately 120x10 6 cells / dose, approx. 240x10 6 cells / dose, or approximately 360x10 6 In some embodiments, if the subject weighs 50 kg or less, the dose is about 20 x 10 6 cells / dose, approximately 80x10 6 cells / dose, and approximately 240x10 6 In some embodiments, if the subject weighs more than 50 kg, the dose is selected from the group consisting of about 20x10 cells / dose. 6 cells / dose, approximately 40x10 6 cells / dose, approximately 120x10 6 cells / dose, and approximately 360x10 6 cells / dose.

[0154] In some embodiments, the dosing regimen is about 1x10 4 , about 2x10 4 , about 3x10 4 , about 4x10 4 , about 5x10 4 , about 6x10 4 , approximately 7x10 4 , about 8x10 4 , approximately 9x10 4, about 10x10 4 , approximately 1x10 5 , about 2x10 5 , about 3x10 5 , about 4x10 5 , about 5x10 5 , about 6x10 5 , approximately 7x10 5 , about 8x10 5 , approximately 9x10 5 , approximately 1x10 6 , about 2x10 6 , about 3x10 6 , about 4x10 6 , about 5x10 6 , about 6x10 6 , approximately 7x10 6 , about 8x10 6 , approximately 9x10 6 , approximately 1x10 7 , about 2x10 7 , about 3x10 7 , about 4x10 7 , about 5x10 7 , about 6x10 7 , approximately 7x10 7 , about 8x10 7 , approximately 9x10 7 , approximately 1x10 8 , about 2x10 8 , about 3x10 8 , about 4x10 8 , about 5x10 8 , about 6x10 8 , approximately 7x10 8 , about 8x10 8 , or about 9x10 8 In some embodiments, the dosing regimen comprises administering a subsequent dose of CAR-T cells. 4 cells, approximately 6x10 6 cells, approximately 7x10 6 cells, or approximately 1.8x10 8 In some embodiments, the allogeneic CAR-T cell administration regimen comprises administering a subsequent dose of cells. 6 In some embodiments, the administration regimen comprises administering a subsequent dose of CAR-T cells (flat dose). 6 cells / dose, approximately 40x10 6cells / dose, approximately 80x10 6 cells / dose, approximately 120x10 6 cells / dose, approx. 240x10 6 cells / dose, or approximately 360x10 6 In some embodiments, if the subject weighs 50 kg or less, the dose is about 20x10 6 cells / dose, approximately 80x10 6 cells / dose, and approximately 240x10 6 In some embodiments, if the subject weighs more than 50 kg, the dose is selected from the group consisting of about 20x10 cells / dose. 6 cells / dose, approximately 40x10 6 cells / dose, approximately 120x10 6 cells / dose, and approximately 360x10 6 cells / dose.

[0155] In some embodiments, the allogeneic CAR-T cell dosing regimen comprises about 6x10 6 a first dose of CAR T cells, and approximately 6x10 6 The administration of a subsequent dose of CAR T cells comprises administering a subsequent dose of cells. In some embodiments, the subsequent dose of CAR-T cells has about the same number of cells as the previous dose of CAR-T cells. In some embodiments, the subsequent dose of CAR-T cells has more cells than the previous dose of CAR-T cells. In some embodiments, the subsequent dose of CAR-T cells has fewer cells than the previous dose of CAR-T cells.

[0156] In some embodiments, the subsequent dose of allogeneic CAR-T cells may be administered within a specific time window relative to the first or previous dose of allogeneic CAR-T cells. In some embodiments, the subsequent dose of allogeneic CAR-T cells is administered about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks after administration of the first or previous dose of allogeneic CAR-T cells. In some embodiments, the subsequent dose of allogeneic CAR-T cells is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after administration of the first or previous dose of allogeneic CAR-T cells. In some embodiments, a subsequent dose of allogeneic CAR-T cells is administered within about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 days later.

[0157] In some embodiments, the time between administration of the first dose (initial dose), e.g., initiation of administration of the first or previous dose, and initiation of administration of the subsequent dose (re-administration, e.g., initiation of administration of the subsequent dose) is more than about 4 weeks, e.g., more than about 5, 6, 7, 8, or 9 weeks, e.g., more than about 20 weeks, e.g., between about 9 weeks and about 35 weeks, between about 14 weeks and about 28 weeks, between 15 weeks and 27 weeks, or between 16 weeks and about 18 weeks, and / or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 weeks. In some embodiments, the administration of a subsequent dose (e.g., its initiation) is more than about 5 weeks and less than about 24 weeks after the administration of the first or previous dose (e.g., its initiation). In some embodiments, the administration of a subsequent dose begins 17 weeks after the initiation of the first dose. In some embodiments, the interval between the administration of a first dose and a subsequent dose (e.g., its initiation), or between the administration of a previous dose and the next subsequent dose, is more than about 5 weeks and less than about 24 weeks, e.g., between 10 and 24 weeks, e.g., about 17 weeks. In some embodiments, the time between the administration of the first dose and the administration of a subsequent dose (e.g., its initiation) is about 17 weeks.

[0158] In some embodiments, subjects are re-administered 28 days to 9 months after the first CAR-T infusion. The same lymphodepletion regimen as for the first CAR-T administration may be applied, or the regimen may be adapted to the patient's condition and the degree of recovery of the host immune system.

[0159] In some embodiments, if a subject's disease recurs after administration of a subsequent dose of allogeneic CAR-T cells, a further subsequent dose of allogeneic CAR-T cells may be administered. In some embodiments, if a subject's disease recurs after administration of a subsequent dose of allogeneic CAR-T cells and a lack of CAR-T persistence is observed, a further subsequent dose of allogeneic CAR-T cells may be administered. In some embodiments, two or more subsequent doses of allogeneic CAR-T cells may be administered after the first dose. Various regimens for subsequent administration can be used to extend the durability of the allogeneic CAR-T response. For example, subsequent doses of CAR-T can be administered, for example, every other month (after the first dose of CAR-T) or every three months. Any number of subsequent infusions may be administered. The timing of re-administration can be adapted to the patient's health status. An exemplary administration regimen is shown in FIG. 1.

[0160] In some embodiments, the dosing regimen is about 6x10 6 a first dose of CAR-T cells, and then about 99 days after administration of the first dose, about 6x10 6 In other embodiments, the dosing regimen comprises administering a subsequent dose of about 6x10 CAR-T cells. 6 A first dose of CAR-T cells of approximately 6x10 6 A maintenance dose of CAR-T cells of about 6x10 is administered every 4 weeks until molecular remission is achieved. In other embodiments, the administration regimen is about 6x10 6 A first dose of CAR-T cells of approximately 6x10 6 Maintenance doses of CAR-T cells will be administered every 8 weeks until molecular remission is achieved.

[0161] In other embodiments, the dosing regimen is about 6x10 6 In other embodiments, the dosing regimen comprises administering a bimonthly dose of about 6x10 CAR-T cells. 6 The method includes administering a dose of CAR-T cells.

[0162] However, depending on the pharmacokinetic decay pattern that the practitioner wants to achieve, other dosing regimens may be useful.The progress of this therapy can be easily monitored by conventional techniques and assays.In a preferred embodiment, the first dose and the first subsequent dose and the additional subsequent dose are separated in time by at least 4 weeks from each other.Dosing regimens can change over time.

[0163] Generally, for administration of allogeneic CAR-T cells, the initial candidate dose is about 1.1 to about 2.3 x 10 5 For purposes of this disclosure, a typical flat dose of CAR-T cells can be anywhere from about 1x10 cells / kg, depending on the factors discussed above. 4 ~1x10 5 ~1x10 6 ~1x10 7 , 1x10 7 , 1x10 8 It can range from 10 to 10 cells, for example, about 0.3 x 10 4 cells, approx. 0.5x10 4 cells, approximately 1x10 4 cells, approximately 1.5x10 4 cells, approximately 2 x 10 4 cells, approximately 2.5x10 4 cells, approximately 3x10 4 cells, approximately 3.5x10 4 cells, approximately 4 x 10 4 cells, approximately 4.5x10 4 cells, approximately 5x10 4 cells, approximately 5.5x10 4 cells, approximately 6x10 4 cells, approximately 6.5x10 4 cells, approximately 7x10 4 cells, approximately 7.5x10 4 cells, approximately 8x10 4 cells, approximately 8.5x10 4 cells, approximately 9 x 10 4 cells, approx. 9.5x10 4 cells, approximately 1x10 5 cells, approximately 1.5x10 5 cells, 2x10 5 cells, approximately 2.5x10 5 cells, approximately 3x105 Cells, approximately 3.5 x 10 5 Cells, approximately 4 × 10 5 Cells, approximately 4.5 x 10 5 Cells, approximately 5 x 10 5 Cells, approximately 5.5 x 10 5 Cells, approximately 6 x 10 5 Cells, approximately 6.5 x 10 5 Cells, approximately 7 x 10 5 Cells, approximately 7.5 x 10 5 Cells, approximately 8 x 10 5 Cells, approximately 8.5 x 10 5 Cells, approximately 9 × 10 5 Cells, approximately 9.5 x 10 5 Cells, approximately 1 × 10 6 Cells, approximately 1.5 x 10 6 Cells, 2x10 6 Cells, approximately 2.5 x 10 6 Cells, approximately 3 x 10 6 Cells, approximately 3.5 x 10 6 Cells, approximately 4 × 10 6 Cells, approximately 4.5 x 10 6 Cells, approximately 5 x 10 6 Cells, approximately 5.5 x 10 6 Cells, approximately 6 x 10 6 Cells, approximately 6.5 x 10 6 Cells, approximately 7 x 10 6 Cells, approximately 7.5 x 10 6 Cells, approximately 8 x 10 6 Cells, approximately 8.5 x 10 6 Cells, approximately 9 × 10 6 Cells, approximately 9.5 x 10 6 Cells, approximately 1 × 10 7 Cells, approximately 1.5 x 10 7 Cells, 2x10 7 Cells, approximately 2.5 x 10 7 Cells, approximately 3 x 10 7 Cells, approximately 3.5 x 10 7 Cells, approximately 4 × 10 7 Cells, approximately 4.5 x 10 7 Cells, approximately 5 x 10 7 Cells, approximately 5.5 x 10 7 Cells, approximately 6 x 10 7 Cells, approximately 6.5 x 10 7 Cells, approximately 7 x 10 7 Cells, approximately 7.5 x 10 7cells, approximately 8x10 7 cells, approximately 8.5x10 7 cells, approximately 9 x 10 7 cells, approx. 9.5x10 7 cells, approximately 1 x 10 8 cells, approximately 1.5x10 8 cells, 2x10 8 cells, approximately 2.5x10 8 cells, approximately 3x10 8 cells, approximately 3.5x10 8 cells, approximately 4 x 10 8 cells, approximately 4.5x10 8 cells, approximately 5x10 8 cells, approximately 5.5x10 8 cells, approximately 6x10 8 cells, approximately 6.5x10 8 cells, approximately 7x10 8 cells, approximately 7.5x10 8 cells, approximately 8x10 8 cells, approximately 8.5x10 8 cells, approximately 9 x 10 8 cells, approx. 9.5x10 8 cells, or approximately 1x10 9 Doses of 100 or more cells may be used. For subsequent administrations over several weeks or longer, depending on the disease, treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved, e.g., until molecular remission is achieved.

[0164] An exemplary dosing regimen is about 0.3x10 4 cells, approx. 0.5x10 4 cells, approximately 1 x 10 4 cells, approximately 1.5x10 4 cells, approximately 2 x 10 4 cells, approximately 2.5x10 4 cells, approximately 3x10 4 cells, approximately 3.5x10 4 cells, approximately 4 x 10 4 cells, approximately 4.5x10 4 cells, approximately 5x10 4 cells, approximately 5.5x10 4 cells, approximately 6x10 4 cells, approximately 6.5x10 4 cells, approximately 7x10 4Cells, approximately 7.5 x 10 4 Cells, approximately 8 x 10 4 Cells, approximately 8.5 x 10 4 Cells, approximately 9 × 10 4 Cells, approximately 9.5 x 10 4 Cells, approximately 1 × 10 5 Cells, approximately 1.5 x 10 5 Cells, 2x10 5 Cells, approximately 2.5 x 10 5 Cells, approximately 3 x 10 5 Cells, approximately 3.5 x 10 5 Cells, approximately 4 × 10 5 Cells, approximately 4.5 x 10 5 Cells, approximately 5 x 10 5 Cells, approximately 5.5 x 10 5 Cells, approximately 6 x 10 5 Cells, approximately 6.5 x 10 5 Cells, approximately 7 x 10 5 Cells, approximately 7.5 x 10 5 Cells, approximately 8 x 10 5 Cells, approximately 8.5 x 10 5 Cells, approximately 9 × 10 5 Cells, approximately 9.5 x 10 5 Cells, approximately 1 × 10 6 Cells, approximately 1.5 x 10 6 Cells, 2x10 6 Cells, approximately 2.5 x 10 6 Cells, approximately 3 x 10 6 Cells, approximately 3.5 x 10 6 Cells, approximately 4 × 10 6 Cells, approximately 4.5 x 10 6 Cells, approximately 5 x 10 6 Cells, approximately 5.5 x 10 6 Cells, approximately 6 x 10 6 Cells, approximately 6.5 x 10 6 Cells, approximately 7 x 10 6 Cells, approximately 7.5 x 10 6 Cells, approximately 8 x 10 6 Cells, approximately 8.5 x 10 6 Cells, approximately 9 × 10 6 Cells, approximately 9.5 x 10 6 Cells, approximately 1 × 10 7 Cells, approximately 1.5 x 10 7 Cells, 2x10 7 Cells, approximately 2.5 x 10 7 Cells, approximately 3 x 107 cells, approximately 3.5x10 7 cells, approximately 4 x 10 7 cells, approximately 4.5x10 7 cells, approximately 5x10 7 cells, approximately 5.5x10 7 cells, approximately 6x10 7 cells, approximately 6.5x10 7 cells, approximately 7x10 7 cells, approximately 7.5x10 7 cells, approximately 8x10 7 cells, approximately 8.5x10 7 cells, approximately 9 x 10 7 cells, approx. 9.5x10 7 cells, approximately 1 x 10 8 cells, approximately 1.5x10 8 cells, approximately 2 x 10 8 cells, approximately 2.5x10 8 cells, approximately 3x10 8 cells, approximately 3.5x10 8 cells, approximately 4 x 10 8 cells, approximately 4.5x10 8 cells, approximately 5x10 8 cells, approximately 5.5x10 8 cells, approximately 6x10 8 cells, approximately 6.5x10 8 cells, approximately 7x10 8 cells, approximately 7.5x10 8 cells, approximately 8x10 8 cells, approximately 8.5x10 8 cells, approximately 9 x 10 8 cells, approx. 9.5x10 8 cells, or approximately 1 x 10 9In some embodiments, the subsequent doses are administered about every three months. In some embodiments, the subsequent doses are administered about every four months. In some embodiments, the subsequent doses are administered about every five months. In some embodiments, the subsequent doses are administered about every six months. In some embodiments, the subsequent doses are administered about every seven months. In some embodiments, the subsequent doses are administered about every eight months. In preferred embodiments, the first dose and the first subsequent dose and the additional subsequent doses are separated in time from each other by at least about four weeks. In some embodiments, the subsequent doses are administered every two months.

[0165] In some embodiments, the dosing regimen is to administer about 6x10 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 1 x 10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 2x10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 3x10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 4x10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6In some embodiments, the dosing regimen comprises administering to the subject about 5x10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 7x10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 8x10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 1 x 10 subsequent doses of CAR-T cells between D30 and about D110. 6 and administering a first dose of CAR-T cells of about 9x10 at D0. 6 In some embodiments, the dosing regimen comprises administering to the subject about 1 x 10 subsequent doses of CAR-T cells between D30 and about D110. 7 and administering a first dose of CAR-T cells of about 6x10 at D0. 6 and administering a subsequent dose of CAR-T cells between D30 and about D110.

[0166] In some embodiments, the dosing regimen comprises administering to the subject a lymphodepleting regimen on D-7 to D-2, followed by about 6x10 lymphocytes on D0. 6 a first dose of CAR-T cells, then any lymphodepletion regimen on D60-D100, followed by 6x10 on D63-D104 6 In some embodiments, the dosing regimen comprises administering to the subject a lymphodepleting regimen on D-7 to D-2, followed by about 6x10 subsequent doses of CAR-T cells on D0. 6 A first dose of CAR-T cells of 6x10 from D60 to D90, followed by an optional lymphodepletion regimen from D63 to D104. 6In some embodiments, the dosing regimen comprises administering to the subject a lymphodepleting regimen (LD) on D-7, followed by about 6x10 CAR-T cells on D0. 6 at D70, followed by a reduced intensity LD (compared to the previous LD) at D99, followed by 6x10 6 In some embodiments, the dosing regimen comprises administering to the subject a lymphodepleting regimen on D-15, followed by about 6x10 subsequent doses of CAR-T cells on D0. 6 a first dose of CAR-T cells, then lymphodepletion regimen on D60, followed by 6x10 on D75 6 In some embodiments, the dosing regimen comprises administering to the subject a lymphodepleting regimen on D-7 to D-2, followed by about 6x10 subsequent doses of CAR-T cells on D0. 6 A first dose of CAR-T cells of 6x10 from D60 to D90, followed by an optional lymphodepletion regimen from D63 to D104. 6 In some embodiments, the dosing regimen comprises administering to the subject a lymphodepleting regimen on D-7 to D-2, followed by about 6x10 subsequent doses of CAR-T cells on D0. 6 A first dose of CAR-T cells of 6x10 from D60 to D90, followed by an optional lymphodepletion regimen from D63 to D104. 6 and administering a subsequent dose of CAR-T cells.

[0167] In some embodiments, the dosing regimen involves administering a lymphodepleting regimen to a subject on D-5 to D-3, followed by about 20x10 6 cells, approximately 40x10 6 cells, approximately 80x10 6 cells, approx. 120x10 6 cells, approximately 240 x 10 6 cells, or approximately 360 x 10 6 In some embodiments, the lymphodepleting regimen comprises administering a first dose of CAR-T cells, followed by optional subsequent doses of CAR-T cells. 2 Fludarabine, administered at a dose of approximately 300 mg / m 2and optionally a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10) administered at a dose of about 10 to about 13 mg.

[0168] However, depending on the pharmacokinetic decay pattern that the practitioner wants to achieve, other dosing regimens may be useful.The progress of this therapy is easily monitored by conventional techniques and assays.In some preferred embodiments, the first dose and the first subsequent dose and the additional subsequent dose are separated in time by at least 4 weeks from each other.Dosing regimens may change over time.

[0169] Allogeneic CAR-T cells and additional agents Exemplary CAR-T cells for use in accordance with the present disclosure are described.

[0170] The methods include administering CAR-T cells that specifically bind to an antigen associated with the disease and generate a response, such as an immune response, to such a molecule based on binding to such an antigen. Unless specifically stated that the method involves a specific type of allogeneic CAR-T cell (e.g., UCART19 cell), any allogeneic CAR-T cell may be used in conjunction with the methods described herein. In some embodiments, the methods and compositions of the present disclosure are practiced using allogeneic CAR-T cells specific for a tumor-specific antigen.

[0171] In some embodiments, the methods and compositions of the present disclosure are carried out using allogeneic CAR-T cells specific for CD19. In some embodiments, the CAR-T cells express a CAR comprising a CD19-binding domain. In some embodiments, the CD19-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv is derived from an anti-CD19 antibody (e.g., 4G7 antibody) and is an anti-CD19 antibody. In some embodiments, the scFv comprises a VH and a VL isolated from or derived from an anti-CD19 antibody (e.g., 4G7 antibody). In some embodiments, the scFv comprises HCDR1, HCDR2, HCDR3, LCDR1, LDCR2, and LCDR3 isolated from or derived from an anti-CD19 antibody (e.g., 4G7 antibody).

[0172] In some embodiments, the CAR-T cells express a CAR comprising a CD19 binding domain, a 4-1BB domain, and / or a CD3 zeta domain.

[0173] In some embodiments, the CAR-T cells further express a "safety switch." The safety switch may include an epitope that allows for the selection of transduced cells and for the detection and / or deletion of cells expressing the polypeptide. In some embodiments, the safety switch includes a mimitope. Exemplary safety switches are disclosed, for example, in International Patent Publication No. 2013 / 153391, incorporated herein by reference in its entirety. In some embodiments, the safety switch binds to rituximab. In some embodiments, the safety switch is expressed in trans with the CAR (e.g., as a separate polypeptide on the same cell). In some embodiments, the safety switch is RQR8. In some embodiments, the RQR8 containing the safety switch is expressed in trans. The full length and component sequences of RQR8 are shown in Table 6 below.

[0174] TIFF2025188073000015.tif199170

[0175] In some embodiments, the safety switch is R2.

[0176] In some embodiments, the CAR-T cells do not contain a safety switch.

[0177] In some embodiments, the sequence encoding the safety switch is provided on the same vector as the sequence encoding the CAR, or in some embodiments, the sequence encoding the safety switch is provided on a different vector than the vector comprising the sequence encoding the CAR.

[0178] In some embodiments, the sequence encoding the safety switch is provided on the same polypeptide as the CAR (i.e., provided in cis), while in some embodiments, the safety switch is provided on a different polypeptide than the CAR (i.e., provided in trans).

[0179] Thus, in some embodiments, the safety switch is expressed in trans with the CAR (e.g., as a separate polypeptide on the same cell). In some embodiments, the safety switch is RQR8. In some embodiments, RQR8 containing the safety switch is expressed in trans.

[0180] In some embodiments, the safety switch is expressed in cis with the CAR and comprises an antibody-specific mimotope, as described in WO 2013 / 153391.

[0181] In some embodiments, the CAR-T cells further comprise an inactivated CD52 gene. In some embodiments, the CAR-T cells are CD-52 deficient. In some embodiments, the CAR-T cells for administration to a patient comprise a mixture of CD52 deficient and CD52 positive cells.

[0182] In some embodiments, a composition of CAR-T cells for administration to a patient comprises a mixture of cells with different genotypes. In some embodiments, the composition comprises CAR-T cells, wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the cells have an inactivated CD52 gene. In some embodiments, the composition comprises CAR-T cells, wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the cells have an inactivated TRAC gene. In some embodiments, the composition comprises CAR-T cells, wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the cells have an inactivated CD52 gene, and further wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the cells have an inactivated TRAC gene.

[0183] In some embodiments, the CAR-T cells are UCART19 CAR-T cells. UCART19 is an allogeneic engineered human T-cell medicinal product developed for the treatment of CD19-expressing B-cell leukemia, including young adult, adult, and pediatric acute lymphoblastic leukemia (B-ALL). Published information related to UCART19 includes the following: Qasim et al., "Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells," Sci Transl Med 2017 Jan 25;9(374); Gouble et al., "In Vivo Proof of Concept of Activity and Safety of UCART19, an Allogeneic "Off-the-Shelf" Adoptive T-Cell Immunotherapy Against CD19 + B-Cell Leukemias,” Blood 2014, 124(21) page 4689. and U.S. Patent Application No. 14 / 891,296, published May 2, 2016 as US 20160145337, each of which is incorporated by reference in its entirety. UCART19 expresses a CAR directed against human CD19 with the safety switch RQR8, which combines epitopes from both the CD34 and CD20 antigens. UCART19 contains an anti-human CD19 4-1BB / CD3 zeta CAR made from one or more of the peptides shown in Table 7 and / or described in U.S. Patent Application Publication No. 2016 / 0145337, which is incorporated by reference in its entirety.

[0184] TIFF2025188073000016.tif249165 TIFF2025188073000017.tif172169

[0185] UCART19 further undergoes simultaneous disruption of the TRAC and CD52 genes, followed by depletion of residual TCRαβ+ cells. UCART19 contains a single-chain variable fragment (scFv) derived from the mouse anti-human CD19 4G7 hybridoma, the CD8 hinge and transmembrane domain, the 4-1BB costimulatory domain, and the CD3 zeta signaling domain. The drug substance (DS) of UCART19 is defined as allogeneic genetically modified CD19CAR / RQR8+_TCRαβ-_T-cells that (i) contain an integrated self-inactivating (SIN) recombinant lentiviral delivery vector expressing an anti-CD19 CAR that redirects T cells to CD19+ tumor cells and eliminates them, (ii) express the RQR8 safety switch that confers sensitivity to rituximab, (iii) are TCRαβ-negative via depletion of the TRAC gene using mRNA-based TALEN® gene editing during the manufacturing process, followed by depletion of remaining TCRαβ+ cells, and (iv) are a mix of CD52 knockout and CD52-positive cells via disruption of the CD52 gene using mRNA-based TALEN® gene editing, allowing UCART19 to be administered to patients who have been previously treated or are currently being treated with an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:10).

[0186] An exemplary flow chart for UCART19 production is shown in Figures 4A and 4B.

[0187] As used herein, UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells) refers to UCART19 cells expressing a CD19 CAR and an RQR8 safety switch. The cells also contain an inactivated TRAC gene. Optionally, the cells contain an inactivated CD52 gene.

[0188] As used herein, UCART19 (CD19CAR / R2+_TCRαβ-_T cells) refers to UCART19 cells expressing a CD19 CAR and an R2 safety switch. The cells also contain an inactivated TRAC gene. Optionally, the cells contain an inactivated CD52 gene.

[0189] As used herein, UCART19 (CD19CAR / TCRαβ-_T cells) refers to UCART19 cells that express CD19CAR. The cells also contain an inactivated TRAC gene. Optionally, the cells contain an inactivated CD52 gene.

[0190] In some embodiments, the CAR-T cells are allogeneic CAR-T cells that express the amino acid sequence set forth in SEQ ID NO:1.

[0191] For all methods described herein, reference to a CAR-T cell composition also includes compositions containing one or more additional agents. These compositions may further include suitable excipients, such as pharmaceutically acceptable excipients, including buffers, as are well known in the art. The compositions and methods disclosed herein can be used alone or in combination with other conventional therapeutic methods.

[0192] CAR-T cells can be administered to a subject via any suitable route. It will be apparent to those skilled in the art that the examples described herein are not intended to be limiting, but rather to illustrate available techniques. Thus, in some embodiments, CAR-T cells are administered to a subject by known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intraspinal, transdermal, subcutaneous, intra-articular, sublingual, intrasynovial, via insufflation, intrathecal, oral, inhalation, or topical. Administration can be systemic, e.g., intravenous, or local. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution.

[0193] Various formulations of CAR-T cells may be used for administration. In some embodiments, CAR-T cells and pharmaceutically acceptable excipients may be various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of pharmacologically effective substances. For example, excipients can provide form or consistency or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting agents, emulsifiers, salts of various osmolality, encapsulating agents, buffers, and skin penetration promoters.

[0194] These agents may be combined with a pharmaceutically acceptable vehicle, such as saline, Ringer's solution, dextrose solution, and the like. The particular administration regimen, i.e., dosage, timing, and repetition, will depend on the particular individual and their medical history.

[0195] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, e.g., phosphate, citric acid, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl, or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., For example, the surfactant may include serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0196] Pharmaceutical Kits and Articles of Manufacture Also provided are pharmaceutical kits for treating patients suffering from diseases such as cancer. In some embodiments, the pharmaceutical kit comprises CAR-T cells and a CD52 antibody. In some embodiments, the pharmaceutical kit comprises UCART19 cells and an antibody. In some embodiments, the pharmaceutical kit comprises a first container comprising CAR-T cells (e.g., UCART19 cells) and a second container comprising a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO:8 and / or SEQ ID NO:10). In some embodiments, the CAR-T cells (e.g., UCART19 cells) are CD52-deficient. In some embodiments, the kit comprises multiple containers comprising CAR-T cells, wherein the amount of CAR-T cells in each container is the same or different. In some embodiments, the first and / or second container is a flexible cell infusion bag. In some embodiments, the first and / or second container is a vial or tube (e.g., a glass or plastic vial or tube). The pharmaceutical kit may further comprise a label or package insert containing instructions for administering the CAR-T cells and the CD52 antibody to a subject.

[0197] Also, a unit dose of allogeneic chimeric antigen receptor (CAR)-T cells for administration to a subject, said unit dose being about 1 x 10 6 ~Approx. 5×10 8Also provided are articles of manufacture comprising a plurality of sealable containers, each individually comprising a unit dose comprising cells; packaging material; and a label or package insert comprising instructions for administering a plurality of said unit doses to a subject by performing a first administration and subsequent administrations, wherein said first administration comprises delivering one of said unit doses to a subject, and said subsequent administration comprises administering one or more of said unit doses to a subject. In some embodiments, the article of manufacture specifies that following said first administration, said subsequent administrations are to be performed at a time between about 30 and 150 days, optionally at about day 30, about day 60, about day 90, or about day 99. In some embodiments, the container is or comprises a flexible cell infusion bag. In some embodiments, the container is or comprises a vial or tube (e.g., a glass or plastic vial or tube).

[0198] Numbered Embodiments of the Disclosure The disclosure and invention described herein can be defined by reference to the following numbered exemplary embodiments.

[0199] 1. A method of treating a subject with refractory and / or relapsed non-Hodgkin's lymphoma, the method comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 20x10 6 cells / dose ~ approx. 360x10 6 cells / dose.

[0200] 2. The method of embodiment 1, wherein the non-Hodgkin's lymphoma is large B-cell lymphoma.

[0201] 3. The method of embodiment 1, wherein the non-Hodgkin's lymphoma is follicular lymphoma.

[0202] 4. At least one dose is approximately 20x10 6 cells / dose, approximately 40x106 cells / dose, approximately 80x10 6 cells / dose, approximately 120x10 6 cells / dose, 240x10 6 cells / dose, and approximately 360x10 6 4. The method according to any one of embodiments 1 to 3, wherein the cell / dose is selected from the group consisting of:

[0203] 5. If the subject weighs less than 50 kg, at least one dose is administered in a dose of approximately 20 x 10 6 cells / dose, approximately 80x10 6 cells / dose, and approximately 240x10 6 5. The method according to any one of embodiments 1 to 4, wherein the cell / dose is selected from the group consisting of:

[0204] 6. If the subject weighs more than 50 kg, at least one dose is administered in a dose of about 20x10 6 cells / dose, approximately 40x10 6 cells / dose, approximately 120x10 6 cells / dose, and approximately 360x10 6 5. The method according to any one of embodiments 1 to 4, wherein the cell / dose is selected from the group consisting of:

[0205] 7. The method of any one of embodiments 1 to 6, wherein the CAR-T cells are CD52-deficient.

[0206] 8. The method of any one of embodiments 1 to 6, wherein the CAR-T cells comprise a mixture of CD52-deficient cells and CD52-positive cells.

[0207] 9. The method of any one of embodiments 1 to 8, wherein the CAR-T cells comprise a CAR of SEQ ID NO: 1.

[0208] 10. The method of any one of embodiments 1-9, wherein the CAR-T cells comprise UCART19(CD19)CAR / RQR8+_TCRαβ-_T cells.

[0209] 11. The method of any one of embodiments 1 to 9, wherein the CAR-T cells do not express a safety switch.

[0210] 12. The method of any one of embodiments 1-11, wherein CAR expression is detectable in the subject for at least 14 days after administration of the CAR-T cells.

[0211] 13. The method of any one of embodiments 1-12, wherein CAR expression is detectable in the subject for at least 28 days after administration of the CAR-T cells.

[0212] 14. The method of any one of embodiments 1 to 13, wherein the subject exhibits a CR or Cri state for at least one month after CAR-T administration.

[0213] 15. The method of any one of embodiments 1 to 14, wherein the subject exhibits a CR or Cri state for at least 2 months after CAR-T administration.

[0214] 16. The method of any one of embodiments 1-15, wherein the subject exhibits a CR or Cri state for at least 6 months after CAR-T administration.

[0215] 17. The method of any one of embodiments 1-16, wherein the subject exhibits a CR or Cri state for at least 12 months after CAR-T administration.

[0216] 18. The method of any one of embodiments 1-17, wherein the subject undergoes a first lymphodepleting regimen prior to administration of at least one dose.

[0217] 19. The method of embodiment 18, wherein the first lymphodepleting regimen comprises administration of fludarabine and cyclophosphamide.

[0218] 20. The method of embodiment 18, wherein the first lymphodepleting regimen comprises administration of fludarabine, cyclophosphamide, and an anti-CD52 antibody.

[0219] 21. The method of embodiment 19 or 20, wherein the first lymphodepletion regimen further comprises administering Mentha (sodium-2-mercaptoethanesorbonate).

[0220] 22. The method of any one of embodiments 19-21, wherein the first lymphodepletion regimen further comprises administering at least one corticosteroid.

[0221] 23. The method of embodiment 22, wherein the corticosteroid is administered immediately before administration of the anti-CD52 antibody.

[0222] 24. The method of embodiment 22 or 23, wherein the corticosteroid is methylprednisolone.

[0223] 25. The method of embodiment 24, wherein methylprednisolone is administered at a dose of 2 mg / kg.

[0224] 26. The method of any one of embodiments 1-25, wherein the patient receives pre-medication for infusion-related reactions prior to the lymphodepleting regimen.

[0225] 27. The method of embodiment 26, wherein the pre-medication comprises at least one antihistamine.

[0226] 28. The method of embodiment 26 or 27, wherein the pre-medication comprises acetaminophen.

[0227] 29. Fludarabine is approximately 30 mg / m 2 / day dose, with cyclophosphamide at approximately 300 mg / m 2 29. The method of any one of embodiments 19-28, wherein the CD52 antibody is administered at a dose of about 10 to about 13 mg / day.

[0228] 30. Fludarabine is approximately 30 mg / m 2 / day dose, with cyclophosphamide at approximately 300 mg / m 2 29. The method of any one of embodiments 20-28, wherein the CD52 antibody is administered at a dose of about 10 to about 13 mg / day.

[0229] 31. The method of any one of embodiments 18-30, wherein the first lymphodepletion regimen is initiated between about 1 and 15 days prior to administration of at least one dose.

[0230] 32. The method of any one of embodiments 18-31, wherein the first lymphodepletion regimen is administered over the course of 1, 2, 3, 4, or 5 days.

[0231] 33. The method of any one of embodiments 1 to 32, wherein the subject receives a subsequent dose of CAR-T cells.

[0232] 34. The method of embodiment 33, wherein the subject exhibits a suboptimal response upon administration of the subsequent dose.

[0233] 35. A suboptimal response is (a) complete response (CR) with minimal detectable residual disease (in leukemia patients) and absence of cytogenetic response; complete response with incomplete recovery of blood counts (CRi); (b) complete bone marrow response; (c) partial response, or (d) The method of embodiment 34, comprising a stable response.

[0234] 36. The method of any one of embodiments 33-35, wherein the subsequent dose comprises approximately the same number of cells as the first dose.

[0235] 37. The method of any one of embodiments 33-35, wherein the subsequent dose comprises an increased number of cells compared to the first dose.

[0236] 38. The method of any one of embodiments 33-35, wherein the subsequent dose comprises a reduced number of cells compared to the first dose.

[0237] 39. The method of any one of embodiments 33-38, wherein the subsequent dose is administered at least 14, at least 28, at least 42, or at least 56 days after the first dose.

[0238] 40. A pharmaceutical kit for treating a patient suffering from cancer, the kit comprising: (a) anti-CD19 CAR-T cells; and (b) A pharmaceutical kit comprising a CD52 antibody.

[0239] 41. The pharmaceutical kit of embodiment 40, wherein the CAR-T cells are UCART19 cells.

[0240] 42. A pharmaceutical kit according to embodiment 40 or 41, wherein the CAR-T cells express the CAR of sequence number 1.

[0241] 43. A pharmaceutical kit according to any one of embodiments 40 to 42, wherein the CD52 antibody comprises the sequence of SEQ ID NO: 8 and / or SEQ ID NO: 10.

[0242] 44. A pharmaceutical kit according to any one of embodiments 40 to 43, wherein the kit comprises a first container containing CAR-T cells and a second container containing a CD52 antibody.

[0243] 45. The pharmaceutical kit of embodiment 44, wherein at least one of the first and second containers is a flexible cell infusion bag.

[0244] 46. ​​A pharmaceutical kit described in any one of embodiments 40 to 45, wherein the kit further comprises a label or package insert comprising instructions for administering the CAR-T cells and the CD52 antibody to a subject.

[0245] 47. A method of treatment comprising: (a) administering a first dose of chimeric antigen receptor (CAR)-T cells (CAR-T cells) to a subject having a disease; and (b) administering to the subject a subsequent dose of CAR-T cells at least about 28 days, or more than about 28 days, after initiation of said administering in (a) and less than about 200 days after initiation of said administering in (a).

[0246] 48. The method of embodiment 47, wherein the CAR-T cells are allogeneic.

[0247] 49. The first dose is approximately 1 x 10 4 ~Approx. 5×10 8 49. The method of embodiment 47 or 48, comprising a total of:

[0248] 50. The first dose is approximately 6 x 10 5 Total cells, approximately 6 x 10 6 Total cells, approximately 6 x 10 7 Total cells, approximately 8 x 10 7 Total cells, approximately 1.8 x 10 8 Total cells, approximately 2.4 x 10 8 total cells, or approximately 5 x 10 8 50. The method of embodiment 49, comprising total cells.

[0249] 51. The first dose is 1 x 10 per kilogram of subject's body weight. 4 ~2x10 7 49. The method of embodiment 47 or 48, comprising cells.

[0250] 52. The first dose is about 1 x 10 per kilogram of the subject's body weight. 4 , approximately 1x10 5 , approximately 1x10 6 , about 3x10 6 , or about 9x10 6 52. The method of embodiment 51, comprising cells.

[0251] 53. The method of any one of embodiments 47-52, wherein the subsequent dose of CAR-T cells is administered about 28 days, about 60 days, or about 99 days after the initiation of said administration in (a).

[0252] 54. The method of embodiment 53, wherein the subsequent dose of CAR-T cells is administered approximately 99 days after the initiation of said administration in (a).

[0253] 55. A method according to any one of embodiments 47 to 54, wherein after step (a) and before step (b), the subject is subjected to an interim lymphodepletion regimen.

[0254] 56. The method of any one of embodiments 47 to 55, wherein the interim lymphodepletion regimen comprises administering fludarabine, cyclophosphamide, and a CD52 antibody to the subject for approximately 0 to 14 days prior to step (b).

[0255] 57. Fludarabine is approximately 90-150 mg / m 2 and cyclophosphamide at a dose of approximately 1000–4000 mg / m 2 and the CD52 antibody is administered at a dosage of about 0.3 to 1 mg / kg.

[0256] 58. The method of any one of embodiments 47-57, wherein the subject is subjected to a first lymphodepletion regimen.

[0257] 59. The method of embodiment 47, wherein the first lymphodepleting regimen comprises administration of fludarabine, cyclophosphamide, and a CD52 antibody to the subject.

[0258] 60. Fludarabine is approximately 90-150 mg / m 2 and cyclophosphamide at a dose of approximately 1000–4000 mg / m 2 and the CD52 antibody is administered at a dosage of about 0.3 to 1 mg / kg.

[0259] 61. A method according to any one of embodiments 58 to 60, wherein the first lymphodepletion regimen is initiated between about 15 and 5 days prior to step (a).

[0260] 62. A method according to any one of embodiments 58 to 60, wherein the first lymphodepletion regimen is completed between about 2 and 10 days prior to step (a).

[0261] 63. The method of any one of embodiments 47-62, wherein the response is assessed between step (a) and step (b).

[0262] 64. The method of embodiment 63, wherein the response is assessed before interim lymphodepletion.

[0263] 65. Subsequent doses are approximately 1 x 10 5 ~Approx. 5×10 8 65. The method of any one of embodiments 47-64, comprising total cells.

[0264] 66. Subsequent doses are approximately 6 x 10 6 66. The method of any one of embodiments 47-65, comprising total cells.

[0265] 67. A method according to any one of embodiments 47 to 66, wherein said administration in (a) results in an improvement of the subject's disease, as indicated by a reduction in one or more symptoms of the disease after administration in (a).

[0266] 68. The method of embodiment 67, wherein the subject has relapsed at the time of administration in (b).

[0267] 69. The method of any one of embodiments 47-68, wherein subsequent doses of cells comprise a quantity of cells sufficient to ameliorate the subject's disease.

[0268] 70. The method of any of embodiments 47 to 69, wherein the administration in (b) results in further improvement of the subject's disease.

[0269] 71. The method of any of embodiments 47-70, wherein administration of the subsequent dose results in an improvement of the disease in the subject compared to immediately before the start of administration of the subsequent dose.

[0270] 72. The method of any of embodiments 47-71, wherein the method results in improvement of the disease to a greater extent and / or for a longer duration compared to a method comprising another administration regimen, wherein the subject is administered a single dose of the cells in (a) and the cells in (b).

[0271] 73. The method of any of embodiments 47-72, wherein the disease persists after administration of the first dose and / or administration of the first dose is not sufficient to eradicate the disease in the subject.

[0272] 74. The method of any one of embodiments 47 to 73, wherein the subject exhibits an absence of CAR-T cell persistence upon administration in (b).

[0273] 75. The method of any one of embodiments 47-74, wherein the subject exhibits one or more symptoms of the disease at the time of administration in (b).

[0274] 76. The method of any one of embodiments 47-75, wherein the subject exhibits a suboptimal response upon administration in (b).

[0275] 77. A suboptimal response is (a) complete response (CR) with minimal detectable residual disease (in leukemia patients) and absence of cytogenetic response; complete response with incomplete recovery of blood counts (CRi); (b) complete bone marrow response; (c) partial response, or (d) The method of embodiment 76, comprising a stable response.

[0276] 78. The method of any of embodiments 47-77, wherein following administration in (b), on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, the subject has undetectable cytokine release syndrome (CRS)-related outcomes or is reduced by about 20% to about 99% compared to a method comprising an alternative administration regimen, wherein the subject does not receive the first dose and receives the cells in (b).

[0277] 79. The method of any one of embodiments 47-78, wherein the subsequent dose comprises approximately the same number of cells as the first dose.

[0278] 80. The method of any of embodiments 47-78, wherein the subsequent dose comprises an increased number of cells compared to the first dose.

[0279] 81. The method of embodiment 80, wherein the subsequent dose comprises at least about 5% more cells than the number of cells in the first dose.

[0280] 82. The method of any of embodiments 47-78, wherein the subsequent dose comprises a reduced number of cells compared to the first dose.

[0281] 83. The method of embodiment 82, wherein the subsequent dose contains at least about 5% fewer cells than the number of cells in the first dose.

[0282] 84. The method of any one of embodiments 47 to 83, wherein the disease is a tumor or cancer.

[0283] 85. The method of embodiment 84, wherein the cancer is leukemia or lymphoma.

[0284] 86. The method of embodiment 84, wherein the tumor or cancer is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), HNSCC, non-Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancy, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, or mesothelioma.

[0285] 87. The method of embodiment 86, wherein the ALL is relapsed or refractory ALL.

[0286] 88. 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 least about 5 weeks, or more than about 5 weeks, after initiation of the first dose and less than about 24 weeks after initiation of the first dose.

[0287] 89. 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, the subject has received a previous dose of (CAR)-T cells in an amount sufficient to show a clinical benefit in the subject, and at the time of administration, the subject does not exhibit a detectable adaptive host immune response specific for the CAR-T cells, and / or the time between said previous dose and the subsequent dose is more than about 5 weeks and less than about 24 weeks.

[0288] 90. The method of embodiment 88, wherein the number of cells administered in the subsequent dose is the same as the number of cells administered in the first dose.

[0289] 91. The method of embodiment 88, wherein the number of cells administered in the subsequent dose is greater than the number of cells administered in the first dose.

[0290] 92. Use of a composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for the manufacture of a medicament for the treatment of a disease in a subject previously treated with CAR-T cells, wherein the composition is for use between about 5 and 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 the subject previously treated with CAR-T cells.

[0291] 93. A composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for use in treating a disease in a subject previously treated with CAR-T cells, wherein the cells are for use between about 5 and 24 weeks after the previous treatment, and the cells are formulated for administration of subsequent doses in an amount sufficient to ameliorate the disease in the subject previously treated with CAR-T cells.

[0292] 94. The use of embodiment 92, or a composition for use according to embodiment 93, wherein a dose of CAR-T cells in a previous treatment improved one or more symptoms of the subject's disease before use of a subsequent dose.

[0293] 95. Use of allogeneic chimeric antigen receptor (CAR)-T cells in the manufacture of a medicament for use in a method for treating a disease, the method comprising administering a first dose of CAR-T cells to a subject having the disease, the first dose being about 1 x 10 6 ~Approx. 5×10 8 and administering to the subject a subsequent dose of CAR-T cells at least about 4 weeks or more than about 4 weeks after initiation of said administering in (a) and less than about 24 weeks after initiation of said administering in (a).

[0294] 96. The use according to embodiment 92, 94, or 95, or the composition for use according to embodiment 93, wherein the disease is leukemia or lymphoma.

[0295] 97. The method according to any one of embodiments 47 to 83, the use according to any one of embodiments 92 or 94 to 95, or the composition for use according to embodiment 93, wherein the CAR-T cells are tumor antigen-specific CAR-T cells.

[0296] 98. The method according to any one of embodiments 47 to 83, the use according to any one of embodiments 92 or 94 to 95, or the composition for use according to embodiment 93, wherein the CAR-T cells are UCART19 cells.

[0297] 99. An article of manufacture, A unit dose of allogeneic chimeric antigen receptor (CAR)-T cells for administration to a subject, said unit dose being about 1 x 10 6 ~Approx. 5×10 8 An article of manufacture comprising a plurality of sealable containers, each individually comprising: a unit dose comprising cells; packaging material; and a label or package insert comprising instructions for administering a plurality of said unit doses to a subject by performing a first administration and subsequent administrations, wherein said first administration comprises delivering one of said unit doses to the subject, and said subsequent administrations comprise administering one or more of said unit doses to the subject.

[0298] 100. The article of manufacture of embodiment 99, wherein the instructions specify that following said first administration, said subsequent administration is to be administered at a time between about 30 and 150 days, optionally at about day 30, about day 60, about day 90, or about day 99.

[0299] 101. An article of manufacture described in embodiment 99 or 100, wherein the container is or comprises a flexible cell infusion bag.

[0300] 102. A method of treating an adult subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 6x10 5 cells / dose, 6x10 6 cells / dose, approximately 6-8x10 7 cells / dose, and approximately 1.8-2.4x10 8 The method of claim 1, wherein the cell / dose is selected from the group consisting of:

[0301] 103. A method of treating a pediatric subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 2-8 x 10 7 cells / dose, method.

[0302] 104. The method of embodiment 102 or 103, wherein the CAR-T cells are CD52-deficient.

[0303] 105. The method of embodiment 102 or 103, wherein the CAR-T cells comprise a mixture of CD52-deficient cells and CD52-positive cells.

[0304] 106. The method of any one of embodiments 102 to 105, wherein the CAR-T cells express a CAR of sequence number 1.

[0305] 107. The method of any one of embodiments 102-106, wherein the CAR-T cells comprise UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells).

[0306] 108. The method of any one of embodiments 102 to 107, wherein CAR expression is detectable in the subject for at least 42 days after administration of the CAR-T cells.

[0307] 109. The method of any one of embodiments 102 to 108, wherein CAR expression is detectable in the subject for at least 56 days after administration of the CAR-T cells.

[0308] 110. The method of any one of embodiments 102-109, wherein the subject exhibits a CR or Cri state for at least 1.3 months after administration of UCART19.

[0309] 111. The method of any one of embodiments 102-110, wherein the subject exhibits a CR or Cri state for at least 1.8 months after administration of UCART19.

[0310] 112. The method of any one of embodiments 102-111, wherein the subject exhibits a CR or Cri state for at least 3.6 months after administration of UCART19.

[0311] 113. The method of any one of embodiments 102-112, wherein the subject exhibits a CR or Cri status for at least 12.4 months after administration of UCART19.

[0312] 114. The method of any one of embodiments 102-113, wherein the subject undergoes a first lymphodepleting regimen prior to administration of at least one dose.

[0313] 115. The method of embodiment 114, wherein the first lymphodepleting regimen comprises administration of fludarabine and cyclophosphamide.

[0314] 116. The method of embodiment 114, wherein the first lymphodepleting regimen comprises administration of fludarabine, cyclophosphamide, and an anti-CD52 antibody.

[0315] 117. Fludarabine is approximately 30-150 mg / m 2 and cyclophosphamide at a dose of approximately 300-4000 mg / m 2 and the CD52 antibody is administered at a dosage of about 0.3 to 1 mg / kg.

[0316] 118. The method of any one of embodiments 114-117, wherein the first lymphodepletion regimen is initiated between about 1 and 15 days prior to administration of at least one dose.

[0317] 119. The method of any one of embodiments 114 to 118, wherein the subject receives a subsequent dose of CAR-T cells.

[0318] 120. The method of embodiment 119, wherein the subject exhibits a suboptimal response upon administration of the subsequent dose.

[0319] 121. A suboptimal response is (a) complete response (CR) with minimal detectable residual disease (in leukemia patients) and absence of cytogenetic response; complete response with incomplete recovery of blood counts (CRi); (b) complete bone marrow response; (c) partial response, or (d) The method of embodiment 120, comprising a stable response.

[0320] 122. The method of embodiment 119, wherein the subsequent dose contains approximately the same number of cells as the first dose.

[0321] 123. The method of embodiment 119, wherein the subsequent dose contains an increased number of cells compared to the first dose.

[0322] 124. The method of embodiment 119, wherein the subsequent dose contains a reduced number of cells compared to the first dose.

[0323] 125. A method for generating a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) directed to a target of interest, comprising: Isolating peripheral blood mononuclear cells (PBMCs) from healthy donors; Activating T cells in PBMCs and transducing the activated T cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing the CAR of interest; and Disrupting TCRαβ and CD-52 gene expression in subsets of T cells; Expanding the T cell population and enriching the population of T cells for TCRαβ negative cells; thereby generating a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells).

[0324] 126. The method of embodiment 125, wherein step (c) is performed about 3 to 4 days after step (b).

[0325] 127. The method of embodiment 125 or 126, wherein step (d) is performed about 2 days after step (c).

[0326] 128. The method of any one of embodiments 125-127, wherein step (f) is performed about 10-12 days after step (d).

[0327] 129. The method of any one of embodiments 125 to 128, wherein the CAR-T cells are CD52-deficient.

[0328] 130. The method of any one of embodiments 125 to 129, wherein the CAR-T cells comprise a mixture of CD52-deficient cells and CD52-positive cells.

[0329] 131. The method of any one of embodiments 125 to 130, wherein the CAR-T cells express a CAR of sequence number 1.

[0330] 132. The method of any one of embodiments 125-131, wherein the CAR-T cells comprise UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells).

[0331] 133. A method according to any one of embodiments 125 to 132, wherein the lentiviral vector of step (c) further expresses a safety switch.

[0332] 134. The method of embodiment 133, wherein the safety switch is RQR8. [Example]

[0333] The following examples are meant to illustrate the methods and materials of the present disclosure. Suitable modifications and adaptations of the described diseases and parameters commonly encountered in the art, as would be apparent to those skilled in the art, are within the spirit and scope of the present disclosure. [Example]

[0334] Example 1: Allogeneic CAR-T Dosing Regimen This example demonstrates multi-dose treatment with allogeneic CAR-T cells.

[0335] Prior to administration of CAR-T cells, the diseased subject is subjected to a lymphodepletion regimen. Such regimens include, for example, fludarabine (approximately 30-150 mg / m ) with an anti-CD52 agent (e.g., a CD52 antibody, such as an antibody comprising the sequences of SEQ ID NO: 8 and SEQ ID NO: 10) (total dose ranging from approximately 0.3-1 mg / kg). 2 total dose ranging from 100 to 4000 mg / m 2 This may include treatment with a total dose ranging from 100 mg / kg to 150 mg / kg.

[0336] The target is approximately 1 x 10 4 ~Approx. 5×10 8 The first dose of CAR-T cells is administered on DO.

[0337] Between about D24 and D62, the response to the first dose of CAR-T cells is assessed. The number of CAR-T cells present in the peripheral blood of treated subjects is measured by performing flow cytometry of cell samples for surface expression of CAR-specific markers, CD4 and / or CD8. Loss of positive MRD and / or persistence suggests subsequent administration of CAR-T cells.

[0338] Prior to subsequent administration with CAR-T cells, subjects with MRD are subjected to a lymphodepletion regimen. Such regimens include, for example, fludarabine (approximately 30-150 mg / m ), with or without anti-CD52 (total dose ranging from approximately 0.3-1 mg / kg). 2total dose ranging from 100 to 4000 mg / m 2 For 2-14 days after lymphodepletion, subjects may receive approximately 1 x 10 4 ~Approx. 1×10 8 A dose of CAR-T cells is administered. The response to the dose of CAR-T cells is assessed, and the number of CAR-T cells present in the treated subject's peripheral blood is determined. Positive MRD and / or persistent loss of MRD suggests subsequent administration of CAR-T cells. Multiple doses of allogeneic CAR-T may be administered until MRD negativity is achieved. [Example]

[0339] Example 2: Use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in adult patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia

[0340] This study was conducted to address the treatment of B-ALL, which is curable in approximately 60% of adult patients. In relapse, the prognosis is very poor (overall survival less than 10%). Standard therapy involves combination chemotherapy ± allogeneic SCT.

[0341] Subjects received allogeneic, universal, or adoptive T-cell therapy targeted to CD19+ malignancies.

[0342] Transgene expression was achieved using lentiviral transduction. UCART19 contains an anti-CD19 scFv and an intracellular domain containing CD3ζ + 4-1BB. The CAR-T cells further contain a safety-switched RQR8 (CD20 mimotope) expressed in trans. CAR-T cells were further knocked out using TALEN-based technology. Specifically, TRAC was knocked out to prevent TCR-mediated recognition of the patient's HLA antigens. CD52 was knocked out to allow the use of CD52 antibodies in lymphodepletion. An exemplary UCART19 cell line (CD19CAR / RQR8+_TCRαβ-_T cells) as used here is provided in Figure 5.

[0343] This study had at least three objectives, and a schematic overview of the study design is shown in Figures 6A and 6B (BMA = bone marrow aspirate).

[0344] Objective #1: Evaluate the safety and tolerability of UCART19 at different doses and determine the maximum tolerated dose (MTD). (Figure 3B)

[0345] Objective #2: Evaluate anti-leukemic activity

[0346] Objective #3: Evaluate the growth and persistence of UCART19.

[0347] Figure 3B shows a study design with the following characteristics: A dose-escalation, open-label, non-comparative study to evaluate up to four dose levels (DL) of UCART19 and determine the maximum tolerated dose (MTD) in adult patients with R / R (relapsed / refractory) B-ALL. Dose escalation, followed by a safety extension portion, patients dosed at the MTD or recommended dose (RD) Lymphodepletion (LD) regimen was started on D-7 before UCART19 infusion and consisted of cyclophosphamide 1500 mg / m without (FC) or with (FCA) 1 mg / kg CD52 antibody. 2 and fludarabine 90 mg / m 2 Combine with. During the expansion phase, the role of CD52 antibodies can be investigated in two cohorts of patients (LD with FC or FCA) At D0, UCART19 will be administered as a single, undivided dose by slow IV infusion (5 minutes). Dose-limiting toxicity assessment will be performed 28 days after infusion (D28) Bone marrow aspiration / biopsy will be performed on D-1, D28 and D84 before LD. Minimal residual disease (MRD) is defined by fewer than 104 blast cells in the bone marrow, assessed by flow cytometry (FLC) and / or qPCR. At study completion (D84 post-infusion), patients will be rolled over to a long-term follow-up study (LTFU) for a period of 15 years.

[0348] The main eligibility criteria for this study were: Age 16 or older, with the oldest person under 70 (male or female) CD19 according to the National Comprehensive Cancer Network guidelines (NCCN, 2017) + Patients with relapsed or refractory (relapsed / refractory; R / R) B-ALL Morphological or MRD + (1x10 by flow cytometry and / or qPCR) -3 (End) Individuals who have exhausted available treatment options Not previously treated with an investigational gene or cell therapy product No clinically suspected extramedullary involvement Adequate kidney, liver, lung and heart function No active infection No active CNS leukemia

[0349] The data presented herein are 6x10 6 Total cells (approximately 1x10 5 Six patients at the first dose level of 10 cells / kilogram, and 6–8 x 10 7 Total cells (approximately 1x10 6 Of the 12 treated patients, 6 patients were at the second dose level of 1.8-2.4 x 10 cells / kilogram. 8 None had been treated at the third and final dose level of total cells. Reflecting European clinical practice, 10 of the 12 patients enrolled had received prior blinatumomab and 7 had received prior allogeneic stem cell transplantation (SCT), with the majority having received three or more prior lines of therapy.

[0350] Patient characteristics are shown in Table 8 below. TIFF2025188073000018.tif100161

[0351] safety All 12 enrolled patients, 10 who received a CD52 antibody (FCA regimen) and 2 who did not, received UCART19 at the target cell dose following lymphodepleting chemotherapy consisting of cyclophosphamide and fludarabine. Table 6 below summarizes adverse events associated with UCART19 infusions and those associated with lymphodepleting regimens by grade. Grade 1 indicates mild toxicity, Grade 2 indicates moderate toxicity, Grade 3 indicates severe toxicity, and Grade 4 indicates life-threatening toxicity. Grade 5 toxicity indicates toxicity leading to death.

[0352] TIFF2025188073000019.tif126170

[0353] The most common UCART19-associated adverse event was CRS, reported in 11 patients (two patients experienced significant cases of CRS, one grade 3 and one grade 4). Tocilizumab was administered in six of the 11 patients. CRS correlated with increased serum cytokines (IL-6, IL-10, and IFN-gamma) and proliferation of UCART19 in the blood in all patients.

[0354] Viral reactivation (CMV and / or adenovirus) occurred in four patients (G1–G3).

[0355] Three patients developed prolonged cytopenias, defined as persistent cytopenias beyond day 42 after UCART19 infusion.

[0356] Three patients experienced mild or grade 1 neurotoxic events.

[0357] One patient experienced a cutaneous grade 1 GvHD adverse event that resolved with topical steroids.

[0358] Two dose-limiting toxicities were reported. The first case occurred at the first dose level and was a grade 4 CRS related to UCART19, associated with lymphodepletion and grade 5 neutropenic sepsis related to UCART19. Death occurred on day 15 after UCART19 infusion. The second case, a grade 4 prolonged cytopenia, occurred at the second dose level and was reported to be related to both lymphodepletion and UCART19. This patient underwent allogeneic SCT and had an unrelated grade 5 pulmonary hemorrhage on day 19 after allogeneic SCT or in the setting of an infection on day 82 after UCART19 infusion. Grade 5 adverse events have been reported in other autologous anti-CD19 CAR T-cell therapy trials, partially due to advanced disease stage and associated confounding conditions.

[0359] Two additional non-UCART19-related deaths have also been reported: one patient died from progressive disease and one from allogeneic SCT-related complications. Transplant-related mortality occurs in approximately 20-30% of patients following allogeneic SCT.

[0360] Effectiveness Figure 7A shows the study status. Figure 7B shows the response, duration of remission, and rechallenge of UCART19 in this study (antileukemic activity).

[0361] MRD-CR occurs when a patient achieves CR and has no evidence of intrathecal ALL cells when using sensitive tests such as polymerase chain reaction or flow cytometry. While the rate of CR or CRi is the typical regulatory criterion, studies in both children and adults with ALL have shown a strong correlation between minimal residual disease (MRD+) and the risk of relapse.

[0362] Of the 12 patients who received UCART19, two were unevaluable (one died on day 15 as described above, and one did not reach evaluation on day 28, the time of data collection). Eight of the 10 evaluable patients achieved a CR, defined as the absence of any evidence or symptoms of cancer or as a CR with incomplete recovery of blood counts (Cri). Seven patients achieved an MRD-CR.

[0363] Two patients with refractory disease did not have UCART19 amplification.

[0364] Six patients proceeded to allogeneic SCT, including four patients after the first dose of UCART19 and two patients after the second dose. At the time of the data collected here, four patients remained in MRD-CR (molecular remission) at 12.4, 3.6, 1.8, and 1.3 months after UCART19 infusion.

[0365] CAR T cell proliferation was detected in the blood beginning 7 days after UCART19 infusion, reaching peak proliferation between days 10 and 17. One patient at the second dose level showed the highest peak, leading to long-term persistence at day 42 and still progressing at data cutoff. At dose level 2, the longest persistence observed at data cutoff was 56 days.

[0366] Two patients in the FC regimen showed no evidence of CAR-T cell proliferation.A similar lack of CAR-T cell proliferation was seen in two of 10 patients in the FCA regimen.

[0367] Figures 8A and 8B show flow cytometry PK profiles and UCART19 kinetics in adult patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia. Specifically, referring to Figures 8A and 8B, preliminary flow cytometry data at DL1 and DL2 indicate that UCART19 was detectable in the blood between D3 and D14, with a proliferation peak between D10 and D17. One patient at DL2 showed the highest peak, leading to the longest persistence. Among patients with proliferation, at DL1, one patient demonstrated UCART19 persistence up to D42; at DL2, two patients demonstrated persistence up to D42 and continued persistence at D56. Preliminary data indicate that UCART19 proliferation levels do not correlate with response at D28; instead, MRD-CR at D28 was observed even with low levels of UCART19 proliferation. After the first dose of UCART19, no proliferation was observed in 2 of 10 patients who received LD with FCA and in 2 of 2 patients who received FC.

[0368] The role of CD52 antibodies in UCART19 proliferation is also being evaluated. [Example]

[0369] Example 3: Re-administration of UCART19 in patients with relapsed / refractory CD19-positive B-ALL This example illustrates treatment with UCART19, where the treatment includes subsequent doses of UCART19 administered after a first dose of UCART19, as administered in Example 2.

[0370] Two patients from the study above in Example 2 received a second dose of UCART19 and both achieved MRD-CR.

[0371] Patient #4: A 23-year-old male patient with relapsed / refractory CD19-positive B-ALL received a single dose of UCART19 (6 × 10 6Total cells, dose DL1) were received on day 0 (D0) of the study. Lymphodepleting regimen (fludarabine 122 mg / m 2 , cyclophosphamide 1230 mg / m 2 , and CD52 antibody 1 mg / kg) was administered for a period of 6 days prior to UCART19 infusion. No major toxicity was observed. The patient had a recurrence of CD19+ disease on D61 following the first dose (LD with FCA). The second dose (LD with FC) allowed the patient to achieve MRD- on D28. The patient then underwent a reduced-intensity lymphodepleting regimen (fludarabine and cyclophosphamide without CD52 antibody) for 6 days. 99 days after the initial UCART19 infusion, the patient was re-administered UCART19 at the same dose (6 x 10 6 total cells).

[0372] The data are presented graphically in Figure 2 A. Immediately prior to administration of subsequent doses of UCART19, UCART19 was undetectable in the patient's body (Figure 2 A).

[0373] The patient achieved MRD negativity on Day 28 after the infusion of the second dose of UCART19. Thus, administration of UCART19 after relapse following molecular remission after treatment with the first dose of UCART19 was effective in achieving MRD negativity. The patient proceeded to allogeneic stem cell transplantation 6 weeks after the second dose.

[0374] Patient #13: A male patient, 31 years old, was diagnosed with refractory B-ALL. Fludarabine (90 mg / m 2 total dose) and cyclophosphamide (1500 mg / m 2 After lymphodepletion combined with total dose (FC), patients received a first infusion of UCART19 at a dose of 8 × 10 7Total cells (dose level 2). After the first dose: UCART19 was undetectable (<1 cell / μL by flow cytometry). At day 28 (28 days after UCART19 infusion), no anti-leukemic activity was observed (89% blasts in bone marrow). No mechanism of action-related toxicity was reported within 28 days after UCART19 infusion. The patient was withdrawn from the study and received a second UCART19 infusion (total dose 8×10) under compassionate use ("specialized" in the UK). 7 Total cells) (48 days after the first dose). Lymphodepletion was achieved with fludarabine (90 mg / m 2 total dose), cyclophosphamide (1500 mg / m 2 The patient received UCART19 in combination with a CD52 antibody (1 mg / kg total dose) (FCA). The patient demonstrated low-level UCART19 proliferation (10 cells / µL) on Day 10, and UCART19 was undetectable after Day 14. Low-grade CRS was reported. Anti-leukemic activity was observed with negative minimal residual disease (MRD-negative) by flow cytometry and qPCR on Days 14 and 28. The patient underwent allogeneic transplantation (63 days after the second dose). He ultimately relapsed on August 15, 2018 (Day 112 after transplantation) with low-level MRD detected (molecular relapse). The patient is alive 9.3 months after the first dose of UCART19 and 7.8 months after the second dose of UCART19. The data are shown in Figure 2B.f.

[0375] Both patients then proceeded to allogeneic SCT

[0376] Patient #18: A male patient, 22 years old, diagnosed with B-ALL. Fludarabine (90 mg / m 2 total dose), cyclophosphamide (1500 mg / m 2 After lymphodepletion with a combination of CD52 antibody (40 mg total dose) and CD52 antibody (40 mg total dose) (FCA), the patient received 1.8 x 10 8The patient received a first infusion of UCART19 cells (dose level 3). UCART19 was undetectable, and no mechanism of action-related toxicity was reported. At day 28 (28 days after UCART19 infusion), no antileukemic activity was observed (intramedullary biopsy showed 15% blasts). The patient was refractory and received an intensive lymphodepleting regimen with fludarabine (120 mg / m 2 total dose), cyclophosphamide (1500 mg / m 2 Patient #18 received a second UCART19 infusion (75 days after the first dose) along with a CD52 antibody (1 mg / kg total dose = 65 mg) and a CD52 antibody (1 mg / kg total dose = 65 mg) (FCA). The patient remained on study because rechallenge was permitted by the modified protocol. UCART19 proliferation peak was observed on day 7, UCART19 was detectable on day 10 (10 cells / μL), and no UCART19 was detectable from day 14 onwards. 28 days after the second dose, the patient did not respond and progressed. The response of patient #18 to rechallenge is shown in Figure 14.

[0377] These results indicate that rechallenge with allogeneic CAR-T after relapse effectively achieves MRD negativity after relapse. [Example]

[0378] Example 4: Use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in high-risk pediatric patients with CD19+ relapsed / refractory B-cell acute lymphoblastic leukemia

[0379] B-cell acute lymphoblastic leukemia (B-cell leukemia) is the most common malignant disease in children, accounting for 80% of childhood leukemias. The most common age group is 2-10 years (peaking at 3-4 years), and the survival rate is approximately 90%. Relapse may occur in 10% of children, and approximately 40%-50% survive unless they are at high risk, in which case the survival rate is approximately 10%-30%.

[0380] The same UCART19 provided in Example 2 was used in this study.

[0381] This study had at least the following goals: Evaluating the safety and tolerability of fixed-dose UCART19 · Evaluation of the ability of UCART19 to induce molecular remission prior to the initiation of D28, D56, D84, and / or allo-SCT conditioning regimens. Assessment of remission rate, duration of remission, time to remission, disease-specific survival, and progression-free survival. · Assessment of the proportion of patients who underwent allogeneic SCT. · Evaluation of UCART19 phenotype, trafficking, growth, and persistence.

[0382] The main eligibility criteria for this study were: Ages 6 months to under 18 (male or female) CD19+R / R B-ALL morphological or MRD +(1x10 -3 or more by flow cytometry and / or qPCR) Individuals who have exhausted available treatment options Eligible for allogeneic SCT with an available suitable donor Not previously treated with an investigational gene or cell therapy product No active infection No active CNS leukemia

[0383] Figure 9 shows the study design.

[0384] The lymphodepletion regimen was initiated on D-7 (during the week before UCART19 infusion) and combined the following: cyclophosphamide (C) (60 mg / kg / day for 2 days), fludarabine (F) (30 mg / m 2 / day for 5 days), and w / woCD52 antibody (A) (0.2 mg / kg / day for 5 days). On D0, a flat dose of UCART19 (1.1–2.3 x 10 6 2x10 cells / kg equivalent 7 Total cells) was administered as a single, undivided dose by slow IV infusion over 5 minutes.

[0385] Safety assessments were performed on D28 after UCART19 administration. BMAs were performed at baseline, D-1, D14 (optional), D28, D56, D84, or prior to initiation of allogeneic SCT conditioning regimens or at rest visits (at the investigator's discretion). During the 12-month follow-up period, BMAs were performed at M1, M2, M3, M6, and M12 after allogeneic transplantation for disease assessment. For refractory patients, BMAs were performed at the investigator's discretion. result

[0386] Six patients were enrolled. The six patients had a mean age of 1.1–2.3 × 10 6 Patients were treated with weight-banded cell doses equivalent to cells / kg. Five patients had received three or more prior lines of therapy, and three had received four or more prior lines of therapy. Two patients had undergone prior allogeneic SCT. Patient characteristics are shown in Table 10 below. TIFF2025188073000020.tif108151

[0387] safety All six enrolled patients received lymphodepleting chemotherapy consisting of cyclophosphamide and fludarabine followed by UCART19 at the target cell dose. Five patients also received a CD52 antibody. Table 11 below summarizes adverse events associated with UCART19 cell infusion and those associated with the lymphodepleting regimen and / or UCART19 by grade. [Grade 1 indicates mild toxicity, Grade 2 indicates moderate toxicity, Grade 3 indicates severe toxicity, and Grade 4 indicates life-threatening toxicity. Grade 5 toxicity indicates toxicity leading to death.] TIFF2025188073000021.tif136170

[0388] The most frequent adverse event associated with UCART19 was CRS in all treated patients, with one patient experiencing grade 3 CRS. Mild to moderate neurotoxic events occurred in three of six treated patients. Three patients experienced prolonged cytopenias, reported to be related to lymphodepletion and, in some cases, possibly related to UCART19. Viral reactivation due to cytomegalovirus (CMV), adenovirus, BK virus, and metapneumovirus was attributed to lymphodepletion. One patient experienced transient grade 1 cutaneous GvHD. Two patients died from disease relapse after allogeneic SCT, and one patient died from complications of allogeneic SCT. There were no treatment-related deaths. This is shown in Table 12. TIFF2025188073000022.tif46156

[0389] Effectiveness Figure 10 shows the study status. Figure 11 shows the response, duration of remission (antileukemic activity).

[0390] All patients completed the 28-day evaluation period and were evaluable for anti-leukemic activity. Five of the six patients achieved MRD-CR, and all five underwent allogeneic SCT. Two patients were in remission for more than 13 months after UCART19 infusion at the time of data cutoff; however, three patients died after allogeneic SCT: two due to disease relapse and one due to transplant-related complications. One patient withdrew from the study due to lack of response and received subsequent treatment with blinatumomab off-study. This was the only patient who received the FC regimen.

[0391] Cellular kinetics were assessed and are shown in Figure 12. UCART19 vector copy number (VCN) was measured in blood and bone marrow by qPCR. Preliminary data showed that UCART19 was detectable in the blood by D7 in 5 of 6 patients, with a proliferation peak observed around D14. UCART19 was not detectable in 1 patient who subsequently relapsed. UCART19 persisted in the blood until D28 in 3 of 5 patients. UCART19 was still detectable in the blood at D42 in 2 of 5 patients. Persistence beyond D42 was not measured because UCART19 was eliminated by the conditioning regimen of allogeneic SCT.

[0392] Cytokine kinetics were evaluated, and two of the six patients had elevated IL-6 and IFNγ. No cytokine elevation was observed in four of the six patients. All six patients experienced CRS. CRS Grade 3 was observed in patient 1, and CRS Grade 2 was observed in patient 8. The time to onset of the first CRS symptom ranged between Days 5 and 9.

[0393] T cell donor chimerism was assessed (Figures 13A, 13B, and 13C). UCART19 was detectable in the blood from D7 to at least D42 in all patients, except one, who was detectable by the molecular signature of T cell donor chimerism. [Example]

[0394] Example 5: Use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in patients with relapsed / refractory large B-cell and / or follicular lymphoma

[0395] If untreated, diffuse large B-cell lymphoma (DLBCL), which comprises 20%–30% of B-cell non-Hodgkin's lymphomas (B-NHL), is fatal. Primary mediastinal large B-cell lymphoma (PMBCL) and transformed follicular lymphoma (FL) are typically treated along the DLBCL paradigm. DLBCL comprises a molecularly diverse group of aggressive lymphomas that differ not only in chromosomal alterations but also in signaling pathway activation and clinical outcome. The disease itself may be de novo or arise from the transformation of an indolent B-lymphoma, such as transformed follicular lymphoma. In the United States, the annual incidence of DLBCL is 6.9 per 100,000, making it the most common form of B-NHL.

[0396] Approximately half of all patients with aggressive B-NHL have relapsed or refractory disease, with an estimated 10%–15% of DLBCL patients having primary refractory disease, and an additional 20–30% relapse after an initial objective response (Chaganti et al., 2016). High-grade B-cell lymphomas with abnormalities in MYC, BCL2, and / or BCL6, including "double-hit" and "triple-hit," are associated with a poor prognosis, even in the newly diagnosed setting (Rosenthal and Younes, 2017).

[0397] This is a single-arm, open-label, multicenter, Phase 1 / 2 study evaluating the safety, efficacy, cytokinetics, and pharmacodynamics of UCART19 in adult patients with relapsed / refractory large B-cell lymphoma or follicular lymphoma. The study is divided into a dose-escalation (Phase 1) and a dose-expansion (Phase 2) phase. Phase 1 of the study will use CAR-T cells containing the RQR8 safety switch (CD19CAR / RQR8+_TCRαβ-_T cells). Phase 2 of the study will allow one or more patients to receive CAR-T cells without the RQR8 safety switch (CD19CAR / TCRαβ-_T cells or CD19CAR / R2+_TCRαβ-_T cells).

[0398] Inclusion criteria for this study may include one or more of the following: 1. Histologic or cytologic diagnosis of large B-cell lymphoma as defined in the 2018 WHO revision of the lymphoma classification: diffuse large B-cell lymphoma (DLBCL) - not otherwise specified (NOS) (germinal center B-cell [GCB] and non-GCB), coexistence of DLBCL with any grade of follicular lymphoma, intravascular large B-cell lymphoma, DLBCL associated with chronic inflammation, anaplastic lymphoma kinase-positive (ALK+) DLBCL, Epstein-Barr virus-positive (EBV+) DLBCL-NOS, T-cell / histiocytocyte-rich large B-cell lymphoma, DLBCL with IRF4 / MUM1 rearrangements, high-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 translocations (double / triple hit), primary cutaneous DLBCL - limb type, transformation of follicular lymphoma to DLBCL, primary mediastinal B-cell lymphoma cell lymphoma (PMBCL), and follicular lymphoma. 2. Relapsed or refractory aggressive large B-cell lymphoma or follicular lymphoma, as defined by: Primary refractory, defined as a best response of progressive or stable disease after at least four cycles of first-line therapy (e.g., four cycles of RCHOP) with a duration of stable disease of 6 months or less from the most recent dose of therapy Refractory to second-line or later therapy (defined as stable disease or best response of progressive disease) Relapse ≤ 1 year after autologous stem cell transplant (SCT) 3. Patients must have received at least two lines of prior therapy, including an anthracycline and an anti-CD20 monoclonal antibody. 4. Male or female patients ≥ 18 years of age 5. Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1 6. Estimated life expectancy ≥ 12 weeks after ALLO-501 infusion (as determined by the investigator) 7. Absence of donor-specific anti-HLA antibodies. 8. Adequate hematological function, including: Absolute neutrophil count (ANC) ≥ 1,500 / mm 3 or ≥ 1.5 × 10 9 / L Absolute lymphocyte count (ALC) ≥ 200 / mm 3 or ≥ 0.2 x 10 9 / L · Platelet count ≧50,000 / mm 3 or ≥ 50 × 10 9 / L Hemoglobin ≥ 9 g / dL 9. Adequate renal function, including: Estimated creatinine clearance ≥ 60 mL / min, calculated using institutional method standards and not dialysis dependent. If in doubt, a 24-hour urine collection study can be used to more accurately estimate creatinine clearance. 10. Adequate liver function, including: · Total bilirubin ≤ 2.0 mg / dL, except for patients with Gilbert syndrome, who must have a total bilirubin < 3.0 mg / dL. Aspartate and alanine aminotransferases (AST and ALT) ≤3 x ULN, or ≤5.0 x ULN if there is liver involvement due to neoplasia; · Alkaline phosphatase ≤2.5 × ULN (≤5 × ULN for bone metastases). 11. Normal blood oxygen saturation (SpO2) of 91% or greater (on room air) 12. Left ventricular ejection fraction (LVEF) ≥ 45% and no hematologically significant pericardial effusion at screening. 13. Resolution of acute effects of any prior therapy to baseline severity or CTCAE grade ≤ 1, excluding adverse events (AEs) that do not constitute a safety risk as judged by the investigator. 14. Seronegative for hepatitis B antigen; a positive hepatitis B test can be further evaluated by a confirmatory test, and if the confirmatory test is negative, the patient may be enrolled. 15. Seronegative for hepatitis C antibodies, except for antigen negativity. If the hepatitis C antibody test is positive, the patient should be tested for the presence of antigen by RT-PCR and be HCV RNA negative. 16. Negative serum pregnancy test (for women of childbearing potential) at screening. 17. Female patients who are unable to conceive must meet at least one of the following criteria: · Achieved postmenopausal state, defined as the cessation of regular menstruation for at least 12 consecutive months without another pathological or physiological cause, can be confirmed by serum follicle-stimulating hormone (FSH) levels, which confirm postmenopausal state. · Underwent a confirmed hysterectomy and / or bilateral oophorectomy. · Have medically confirmed ovarian failure. All other female patients (including those with tubal ligation) are considered to be of fertile potential. 18. Evidence of a personally signed and dated informed consent document stating that the patient has been informed of all relevant aspects of the study. 19. Intends to and is able to adhere to scheduled visits, treatment plans, laboratory tests, and other procedures.

[0399] Phase 1 and phase 2 studies are divided into different periods, including screening, lymphodepletion, treatment, and follow-up. A single cycle is defined as the combination of one lymphodepletion and one treatment period.

[0400] Screening begins after the informed consent form is signed. Patients are checked for eligibility criteria. The screening period lasts for a maximum of 28 days. At the end of the screening period, patients who meet all eligibility criteria enter the lymphodepletion period.

[0401] Lymphodepletion begins on day -5 and ends on UCART19 infusion day 0. Prior to treatment with UCART19, all patients received fludarabine (30 mg / m ) in an outpatient setting on days -5, -4, and -3. 2 / day), cyclophosphamide (300 mg / m 2 Patients will undergo intravenous lymphodepletion with UCART19 (10 mg / day) and a CD52 antibody (13 mg / day). The CD52 antibody is administered over approximately 4 hours, and patients must be closely monitored until 2 hours after the infusion is complete. Premedication, including high-dose corticosteroids, is required prior to CD52 antibody administration. If CD52 antibody-related dose-limiting toxicities (DLTs) are observed at the starting dose level, a lower total dose of 30 mg (10 mg / day administered for 3 days) will be evaluated. At the end of the lymphodepletion period, eligibility criteria for UCART19 administration will be evaluated to ensure patient safety.

[0402] The treatment period, which begins on Day 0, will end on Day 56 after UCART19 infusion. Patients will receive UCART19 as an intravenous infusion over approximately 5 minutes on Day 0. To closely manage UCART19-related toxicities, patients will be hospitalized for a minimum of 5 days from Day 0 or until UCART19-related non-hematologic toxicities have resolved to Grade ≤1, or will receive UCART19 in the outpatient setting of a clinical trial site that allows direct patient admission.

[0403] A dosing strategy for UCART19 using two different weight bands will be implemented, as shown in Table 13 below: TIFF2025188073000023.tif44154

[0404] Follow-up will continue from day 56 to month 9. Patients will be monitored throughout the follow-up period until the EOS visit at month 9 after the first UCART19 infusion or after early withdrawal from the study. Patients will then immediately roll over to 15 years of long-term follow-up under a separate long-term follow-up (LTFU) protocol.

[0405] After cytokinetic and / or disease evaluation, patients may receive one optional retreatment with UCART19. Retreatment with UCART19 may be administered at the highest dose level deemed safe at the RP2D in Phase 1 or Phase 2. Retreatment may use a different batch and / or donor than the initial dose. Each retreatment with UCART19 must be separated by at least 4 weeks from the initial dose after planned tumor assessments at Day 56, Month 4, and Month 6, and must be followed by lymphodepletion with Flu / Cy or Flu / Cy and CD52 antibody. Patients with undetectable UCART19 in whole blood at D14 may be retreated with subsequent lymphodepletion beginning after completion of the DLT observation window at Day 28 in Phase 1 or Phase 2. Patients eligible for retreatment will begin a new cycle beginning at lymphodepletion (Day 5) and up to 2 months after rechallenge (Day 56) and will follow the same schedule of visits and evaluations as described for the initial UCART19 infusion. At the end of the treatment period after the last UCART19 infusion (Day 56), patients will continue the follow-up period as scheduled in the study protocol. Retreatment cannot occur after Month 6 (6 months following the initial Day 0).

[0406] While the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be understood that various changes and modifications can be made without departing from the teachings herein and the scope of the claims that follow. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, those skilled in the art will readily appreciate that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

[0407] All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, to the extent they have not already been referenced, are incorporated herein by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, this application will control, including, but not limited to, with respect to defined terms, term usage, described techniques, or the like.

[0408] The foregoing description and examples detail certain embodiments of the invention and describe the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears in text, it will be understood that the invention may be practiced in many ways and that the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims

1. 1. A method of treating a subject with refractory and / or relapsed non-Hodgkin's lymphoma, the method comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 20x10 6 cells / dose ~ approx. 360x10 6 The method is cells / dose.

2. 2. The method of claim 1, wherein the non-Hodgkin's lymphoma is large B-cell lymphoma.

3. 10. The method of claim 1, wherein the non-Hodgkin's lymphoma is follicular lymphoma.

4. The at least one dose is about 20x10 6 cells / dose, approximately 40x10 6 cells / dose, approximately 80x10 6 cells / dose, approximately 120x10 6 cells / dose, 240x10 6 cells / dose, and approximately 360x10 6 The method of any one of claims 1 to 3, wherein the cell / dose is selected from the group consisting of:

5. If the subject weighs 50 kg or less, the at least one dose is about 20 x 10 6 cells / dose, approximately 80x10 6 cells / dose, and approximately 240x10 6 The method of any one of claims 1 to 4, wherein the cell / dose is selected from the group consisting of:

6. If the subject weighs more than 50 kg, the at least one dose is about 20x10 6 cells / dose, approximately 40x10 6 cells / dose, approximately 120x10 6 cells / dose, and approximately 360x10 6 The method of any one of claims 1 to 4, wherein the cell / dose is selected from the group consisting of:

7. 7. The method of any one of claims 1 to 6, wherein the CAR-T cells are CD52-deficient.

8. 7. The method of any one of claims 1 to 6, wherein the CAR-T cells comprise a mixture of CD52-deficient cells and CD52-positive cells.

9. 9. The method of any one of claims 1 to 8, wherein the CAR-T cells comprise a CAR of SEQ ID NO:

1.

10. 10. The method of any one of claims 1 to 9, wherein the CAR-T cells comprise UCART19 (CD19) CAR / RQR8+_TCRαβ-_T cells.

11. The method of any one of claims 1 to 9, wherein the CAR-T cells do not express a safety switch.

12. 12. The method of any one of claims 1-11, wherein CAR expression is detectable in the subject for up to at least 14 days after administration of the CAR-T cells.

13. 13. The method of any one of claims 1-12, wherein CAR expression is detectable in the subject for up to at least 28 days after administration of the CAR-T cells.

14. 14. The method of any one of claims 1 to 13, wherein the subject exhibits a CR or Cri state for at least one month after CAR-T administration.

15. 15. The method of any one of claims 1 to 14, wherein the subject exhibits a CR or Cri state for at least two months after CAR-T administration.

16. 16. The method of any one of claims 1 to 15, wherein the subject exhibits a CR or Cri status for at least 6 months after CAR-T administration.

17. 17. The method of any one of claims 1 to 16, wherein the subject exhibits a CR or Cri status for at least 12 months after CAR-T administration.

18. 18. The method of any one of claims 1 to 17, wherein the subject undergoes a first lymphodepleting regimen prior to administration of the at least one dose.

19. 19. The method of claim 18, wherein the first lymphodepleting regimen comprises administering fludarabine and cyclophosphamide.

20. 19. The method of claim 18, wherein the first lymphodepleting regimen comprises administering fludarabine, cyclophosphamide, and an anti-CD52 antibody.

21. 21. The method of claim 19 or 20, wherein the first lymphodepletion regimen further comprises administering Mentha (sodium-2-mercaptoethanesorbonate).

22. 22. The method of any one of claims 19 to 21, wherein the first lymphodepletion regimen further comprises administering at least one corticosteroid.

23. 23. The method of claim 22, wherein the corticosteroid is administered immediately before administration of the anti-CD52 antibody.

24. 24. The method of claim 22 or 23, wherein the corticosteroid is methylprednisolone.

25. 25. The method of claim 24, wherein the methylprednisolone is administered at a dose of 2 mg / kg.

26. 26. The method of any one of claims 1 to 25, wherein the patient receives pre-medication for infusion-related reactions prior to the lymphodepleting regimen.

27. 27. The method of claim 26, wherein the premedication comprises at least one antihistamine.

28. 28. The method of claim 26 or 27, wherein the premedication comprises acetaminophen.

29. Fludarabine at approximately 30 mg / m 2 / day dose, with cyclophosphamide at approximately 300 mg / m 2 29. The method of any one of claims 19 to 28, wherein the CD52 antibody is administered at a dose of about 10 to about 13 mg / day.

30. Fludarabine at approximately 30 mg / m 2 / day dose, with cyclophosphamide at approximately 300 mg / m 2 29. The method of any one of claims 20-28, wherein the CD52 antibody is administered at a dosage of about 10 to about 13 mg / day.

31. 31. The method of any one of claims 18 to 30, wherein the first lymphodepletion regimen is initiated between about 1 and 15 days prior to administration of at least one dose.

32. 32. The method of any one of claims 18-31, wherein the first lymphodepletion regimen is administered over the course of 1, 2, 3, 4, or 5 days.

33. 33. The method of any one of claims 1-32, wherein the subject receives a subsequent dose of the CAR-T cells.

34. 34. The method of claim 33, wherein the subject exhibits a suboptimal response upon said administration of said subsequent dose.

35. The suboptimal response is (a) complete response (CR), with minimal detectable residual disease (in leukemia patients) and absence of cytogenetic response; complete response with incomplete recovery of blood counts (CRi); (b) complete bone marrow response; (c) partial response, or (d) The method of claim 34, comprising a stable response.

36. 36. The method of any one of claims 33 to 35, wherein the subsequent dose comprises about the same number of cells as the first dose.

37. 36. The method of any one of claims 33 to 35, wherein the subsequent dose comprises an increased number of cells compared to the first dose.

38. 36. The method of any one of claims 33 to 35, wherein the subsequent dose comprises a reduced number of cells compared to the first dose.

39. 39. The method of any one of claims 33 to 38, wherein the subsequent dose is administered at least 14, at least 28, at least 42, or at least 56 days after the first dose.

40. 1. A pharmaceutical kit for treating a patient suffering from cancer, said kit comprising: (a) anti-CD19 CAR-T cells, and (b) A pharmaceutical kit comprising a CD52 antibody.

41. 41. The pharmaceutical kit of claim 40, wherein the CAR-T cells are UCART19 cells.

42. 42. The pharmaceutical kit of claim 40 or 41, wherein the CAR-T cells express a CAR of SEQ ID NO:

1.

43. The pharmaceutical kit according to any one of claims 40 to 42, wherein the CD52 antibody comprises the sequence of SEQ ID NO: 8 and / or SEQ ID NO:

10.

44. 44. The pharmaceutical kit of any one of claims 40 to 43, wherein the kit comprises a first container comprising the CAR-T cells and a second container comprising the CD52 antibody.

45. 45. The pharmaceutical kit of claim 44, wherein at least one of the first and second containers is a flexible cell infusion bag.

46. 46. ​​The pharmaceutical kit of any one of claims 40 to 45, wherein the kit further comprises a label or package insert comprising instructions for administering the CAR-T cells and the CD52 antibody to the subject.

47. 1. A method of treatment comprising: (a) administering a first dose of chimeric antigen receptor (CAR)-T cells (CAR-T cells) to a subject having a disease; and (b) administering to said subject a subsequent dose of CAR-T cells at least about 28 days, or more than about 28 days, after initiation of said administering in (a) and less than about 200 days after initiation of said administering in (a).

48. 48. The method of claim 47, wherein the CAR-T cells are allogeneic.

49. The first dose is about 1×10 4 ~Approx. 5×10 8 49. The method of claim 47 or 48, comprising a total of:

50. The first dose is about 6×10 5 Total cells, approximately 6 x 10 6 Total cells, approximately 6 x 10 7 Total cells, approximately 8 x 10 7 Total cells, approximately 1.8 x 10 8 Total cells, approximately 2.4 x 10 8 total cells, or approximately 5 x 10 8 50. The method of claim 49, comprising total cells.

51. The first dose is 1 x 10 per kilogram of the subject's body weight 4 ~2x10 7 49. The method of claim 47 or 48, comprising a cell.

52. The first dose is about 1 x 10 per kilogram of the subject's body weight. 4 , approximately 1x10 5 , approximately 1x10 6 , about 3x10 6 , or about 9x10 6 52. The method of claim 51, comprising cells.

53. 53. The method of any one of claims 47-52, wherein the subsequent dose of CAR-T cells is administered about 28 days, about 60 days, or about 99 days after the initiation of said administering in (a).

54. 54. The method of claim 53, wherein the subsequent dose of CAR-T cells is administered about 99 days after the initiation of the administering in (a).

55. 55. The method of any one of claims 47 to 54, wherein after step (a) and before step (b), the subject is administered a temporary lymphodepleting regimen.

56. 56. The method of any one of claims 47-55, wherein the interim lymphodepletion regimen comprises administering to the subject fludarabine, cyclophosphamide, and a CD52 antibody for about 0-14 days prior to step (b).

57. Fludarabine at approximately 90–150 mg / m 2 and cyclophosphamide at a dose of approximately 1000–4000 mg / m 2 and the CD52 antibody is administered at a dosage of about 0.3 to 1 mg / kg.

58. 58. The method of any one of claims 47 to 57, wherein the subject is administered a first lymphodepleting regimen.

59. 48. The method of claim 47, wherein the first lymphodepleting regimen comprises administering to the subject fludarabine, cyclophosphamide, and a CD52 antibody.

60. Fludarabine at approximately 90–150 mg / m 2 and cyclophosphamide at a dose of approximately 1000–4000 mg / m 2 and the CD52 antibody is administered at a dosage of about 0.3 to 1 mg / kg.

61. 61. The method of any one of claims 58-60, wherein the first lymphodepletion regimen is initiated between about 15 and 5 days prior to step (a).

62. 61. The method of any one of claims 58-60, wherein the first lymphodepletion regimen is completed between about 2 and 10 days prior to step (a).

63. 63. The method of any one of claims 47 to 62, wherein the response is assessed between step (a) and step (b).

64. 64. The method of claim 63, wherein the response is assessed prior to interim lymphodepletion.

65. said subsequent dose being about 1×10 5 ~Approx. 5×10 8 65. The method of any one of claims 47 to 64, comprising total cells.

66. said subsequent dose being about 6×10 6 66. The method of any one of claims 47 to 65, comprising total cells.

67. 67. The method of any one of claims 47-66, wherein said administering in (a) results in an improvement of said disease in said subject, as indicated by a reduction in one or more symptoms of said disease after said administering in (a).

68. 68. The method of claim 67, wherein at the time of administering in (b), the subject has relapsed.

69. 69. The method of any of claims 47-68, wherein said subsequent dose of cells comprises a quantity of cells sufficient to ameliorate said disease in said subject.

70. 70. The method of any of claims 47-69, wherein the administering in (b) results in further improvement of the disease in the subject.

71. 71. The method of any of claims 47-70, wherein said administration of said subsequent dose results in an improvement of the disease in the subject compared to immediately before the start of said administration of said subsequent dose.

72. 72. The method of any of claims 47-71, wherein the method results in amelioration of the disease to a greater extent and / or for a longer duration compared to a method comprising an alternative administration regimen, wherein the subject is administered the cells in (a) and the cells in (b) in a single administration.

73. 73. The method of any of claims 47-72, wherein the disease persists after said administration of said first dose and / or said administration of said first dose is not sufficient to eradicate the disease in the subject.

74. 74. The method of any of claims 47-73, wherein the subject exhibits an absence of CAR-T cell persistence upon said administering in (b).

75. 75. The method of any of claims 47-74, wherein the subject exhibits one or more symptoms of the disease at the time of the administering in (b).

76. 76. The method of any of claims 47-75, wherein the subject exhibits a suboptimal response upon said administering in (b).

77. The suboptimal response is (a) complete response (CR), with minimal detectable residual disease (in leukemia patients) and absence of cytogenetic response; complete response with incomplete recovery of blood counts (CRi); (b) complete bone marrow response; (c) partial response, or (d) The method of claim 76, comprising a stable response.

78. 78. The method of any of claims 47-77, wherein following the administration in (b), on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, a cytokine release syndrome (CRS)-related outcome in the subject is undetectable or is reduced by about 20% to about 99% compared to a method comprising an alternative administration regimen, wherein the subject is administered the cells in (b) without receiving the first dose.

79. 79. The method of any of claims 47-78, wherein the subsequent dose comprises about the same number of cells as the first dose.

80. 79. The method of any of claims 47-78, wherein the subsequent dose comprises an increased number of cells compared to the first dose.

81. 81. The method of claim 80, wherein the subsequent dose comprises at least about 5% more cells than the number of cells in the first dose.

82. 79. The method of any of claims 47-78, wherein the subsequent dose comprises a reduced number of cells compared to the first dose.

83. 83. The method of claim 82, wherein the subsequent dose comprises at least about 5% fewer cells than the number of cells in the first dose.

84. The method of any one of claims 47 to 83, wherein the disease is a tumor or cancer.

85. 85. The method of claim 84, wherein the cancer is leukemia or lymphoma.

86. 85. The method of claim 84, wherein the tumor or cancer is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), HNSCC, non-Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, or mesothelioma.

87. 87. The method of claim 86, wherein the ALL is relapsed or refractory ALL.

88. 1. 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 least about 5 weeks or more than about 5 weeks after initiation of the first dose and less than about 24 weeks after initiation of the first dose.

89. 1. A method of treatment comprising administering to a subject a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells, wherein prior to said administration, the subject has received a previous dose of the CAR-T cells in an amount sufficient to show a clinical benefit in the subject, and at the time of administration, the subject does not exhibit a detectable adaptive host immune response specific for the CAR-T cells, and / or the time between the previous dose and the subsequent dose is greater than about 5 weeks and less than about 24 weeks.

90. 90. The method of Claim 88 or Claim 89, wherein the number of cells administered in the subsequent dose is the same as the number of cells administered in the first dose.

91. 90. The method of Claim 88 or Claim 89, wherein the number of cells administered in the subsequent dose is greater than the number of cells administered in the first dose.

92. 1. Use of a composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for the manufacture of a medicament for the treatment of a disease in a subject previously treated with said CAR-T cells, wherein said composition is for use between about 5 and about 24 weeks after said previous treatment and / or said composition is formulated for administration of a subsequent dose in an amount sufficient to ameliorate the disease in said subject previously treated with said CAR-T cells.

93. 1. A composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for use in treating a disease in a subject who has been previously treated with said CAR-T cells, wherein the cells are for use between about 5 and 24 weeks after the previous treatment, and wherein the cells are formulated for administration of a subsequent dose in an amount sufficient to ameliorate the disease in the subject who was previously treated with the CAR-T cells.

94. 94. The use of Claim 92, or the composition for use according to Claim 93, wherein the dose of CAR-T cells in the previous treatment ameliorated one or more symptoms of the disease in the subject prior to use of the subsequent dose.

95. 1. Use of allogeneic chimeric antigen receptor (CAR)-T cells in the manufacture of a medicament for use in a method for treating a disease, said method comprising: (a) administering a first dose of the CAR-T cells to a subject having the disease, wherein the first dose is about 1 x 10 6 ~Approx. 5×10 8 administering, comprising total cells; (b) administering to the subject a subsequent dose of the CAR-T cells at least about 5 weeks, or more than about 5 weeks, after initiation of said administering in (a) and less than about 24 weeks after initiation of said administering in (a).

96. 96. The use of claim 92, 94 or 95, or the composition for use according to claim 93, wherein the disease is leukemia or lymphoma.

97. 94. The method of any of claims 47 to 83, the use of any of claims 92 or 94 to 95, or the composition for use according to claim 93, wherein the CAR-T cells are tumor antigen-specific CAR-T cells.

98. 93. The method of any one of claims 47 to 83, the use of any one of claims 92 or 94 to 95, or the composition for use according to claim 93, wherein the CAR-T cells are UCART19 cells.

99. An article of manufacture comprising: a plurality of sealable containers, each containing a separate unit dose of allogeneic chimeric antigen receptor (CAR-T) cells for administration to a subject, said unit doses comprising approximately 1 x 10 6 ~Approx. 5 x 10 8 a sealable container containing cells; packaging materials, and 1. An article of manufacture comprising: 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, wherein said first administration comprises delivering one of said unit doses to said subject, and said subsequent administrations comprise administering one or more of said unit doses to said subject.

100. 100. The article of manufacture of claim 99, wherein the instructions specify that following the first administration, the subsequent administration is to be administered at a time between about 30 and 150 days, optionally at about day 30, about day 60, about day 90, or about day 99.

101. 101. The article of manufacture of claim 99 or 100, wherein the container is or comprises a flexible cell infusion bag.

102. 1. A method of treating an adult subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose is greater than or equal to about 6x10 5 cells / dose, 6x10 6 cells / dose, approximately 6-8x10 7 cells / dose, and approximately 1.8-2.4x10 8 The method of claim 1, wherein the cell / dose is selected from the group consisting of:

103. 1. A method of treating a pediatric subject with refractory and / or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB / CD3 zeta CAR, wherein the at least one dose comprises about 2-8x10 7 cells / dose, method.

104. The method of claim 102 or 103, wherein the CAR-T cells are CD52-deficient.

105. 104. The method of claim 102 or 103, wherein the CAR-T cells comprise a mixture of CD52-deficient cells and CD-52-positive cells.

106. 106. The method of any one of claims 102 to 105, wherein the CAR-T cells express the CAR of SEQ ID NO:

1.

107. 107. The method of any one of claims 102-106, wherein the CAR-T cells comprise UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells).

108. 108. The method of any one of claims 102-107, wherein CAR expression is detectable in the subject for up to at least 42 days after administration of the CAR-T cells.

109. 109. The method of any one of claims 102-108, wherein CAR expression is detectable in the subject for up to at least 56 days after administration of the CAR-T cells.

110. 110. The method of any one of claims 102-109, wherein the subject exhibits a CR or Cri status for at least 1.3 months after administration of UCART19.

111. 111. The method of any one of claims 102-110, wherein the subject exhibits a CR or Cri status for at least 1.8 months after administration of UCART19.

112. 112. The method of any one of claims 102-111, wherein the subject exhibits a CR or Cri status for at least 3.6 months after administration of UCART19.

113. 113. The method of any one of claims 102-112, wherein the subject exhibits a CR or Cri status for at least 12.4 months after administration of UCART19.

114. 114. The method of any one of claims 102-113, wherein the subject undergoes a first lymphodepleting regimen prior to administration of the at least one dose.

115. 115. The method of claim 114, wherein the first lymphodepleting regimen comprises administering fludarabine and cyclophosphamide.

116. 115. The method of claim 114, wherein the first lymphodepleting regimen comprises administering fludarabine, cyclophosphamide, and an anti-CD52 antibody.

117. Fludarabine at approximately 30–150 mg / m 2 and cyclophosphamide at a dose of approximately 300–4000 mg / m 2 and the CD52 antibody is administered at a dosage of about 0.3 to 1 mg / kg.

118. 118. The method of any one of claims 114-117, wherein the first lymphodepletion regimen is initiated between about 1 and 15 days prior to administration of the at least one dose.

119. 119. The method of any one of claims 114-118, wherein the subject receives a subsequent dose of the CAR-T cells.

120. 120. The method of claim 119, wherein the subject exhibits a suboptimal response upon said administration of said subsequent dose.

121. The suboptimal response is (a) complete response (CR), with minimal detectable residual disease (in leukemia patients) and absence of cytogenetic response; complete response with incomplete recovery of blood counts (CRi); (b) complete bone marrow response; (c) partial response, or (d) The method of claim 120, comprising a stable response.

122. 120. The method of claim 119, wherein the subsequent dose comprises about the same number of cells as the first dose.

123. 120. The method of claim 119, wherein the subsequent dose comprises an increased number of cells compared to the first dose.

124. 120. The method of claim 119, wherein the subsequent dose comprises a reduced number of cells compared to the first dose.

125. 1. A method for generating a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) directed against a target of interest, comprising: (a) isolating peripheral blood mononuclear cells (PBMCs) from a healthy donor; (b) activating said T cells in PBMCs; (c) transducing the activated T cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing a CAR of interest; and (d) disrupting TCRαβ and CD-52 gene expression in said subset of T cells; and (e) expanding said population of T cells; (f) enriching the population of T cells for TCRαβ negative cells; thereby generating a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells).

126. 126. The method of claim 125, wherein step (c) is performed about 3 to 4 days after step (b).

127. 127. The method of claim 125 or 126, wherein step (d) is performed about 2 days after step (c).

128. 128. The method of any one of claims 125-127, wherein step (f) is performed about 10-12 days after step (d).

129. 129. The method of any one of claims 125-128, wherein the CAR-T cells are CD52-deficient.

130. 130. The method of any one of claims 125-129, wherein the CAR-T cells comprise a mixture of CD52-deficient and CD-52-positive cells.

131. 131. The method of any one of claims 125 to 130, wherein the CAR-T cells express the CAR of SEQ ID NO:

1.

132. 132. The method of any one of claims 125-131, wherein the CAR-T cells comprise UCART19 (CD19CAR / RQR8+_TCRαβ-_T cells).

133. 133. The method of any one of Claims 125-132, wherein the lentiviral vector of step (c) further expresses a safety switch.

134. 134. The method of claim 133, wherein the safety switch is an RQR8.

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