RU2776322C2 - Methods and compositions for administration of doses of allogeneic t-cells with chimeric antigen receptor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a method for the treatment of a subject, providing the administration to the subject of at least one dose of allogeneic T-cells with a chimeric antigen receptor (hereinafter – CAR) (CAR-T-cells), containing CAR 4-1BB/CD3-zeta for human CD19, where at least one dose is from about 20×10⁶ cells/dose to about 360×10⁶ cells/dose.

EFFECT: invention is effective for the treatment of refractory and/or recurrent non-Hodgkin’s lymphoma.

39 cl, 14 dwg, 13 tbl, 5 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Application Serial No. 62/750215, filed October 24, 2018, U.S. Provisional Application Serial No. 62/716898, filed August 9, 2018; and U.S. Provisional Application Serial No. 62/579426, filed October 31, 2017, the contents of each of which are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY

[0002] The present invention relates to therapeutic regimens for the treatment of disorders, including proliferative disorders and immune disorders. Therapeutic regimens of the present invention include the administration of one or more doses of immune cells containing allogeneic T cells with a chimeric antigen receptor. The therapeutic regimens of the present invention can be used in the prevention and/or treatment of disorders, including cancer.

DESCRIPTION OF THE TEXT FILE PRESENTED IN ELECTRONIC FORM

[0003] The contents of the text file provided electronically herein are incorporated by reference in their entirety: machine-readable form of the sequence listing (file name: ALGN-012-03WO Sequence Listing.txt, Compiled Date October 30, 2018, file size 39283 bytes).

BACKGROUND OF THE INVENTION

[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 doses of allogeneic CAR-T cells required to effectively treat the disease and achieve remission in human patients are unknown. Provided herein are methods, compositions, and articles of manufacture for dosing and re-dosing allogeneic CAR-T cells to patients. The methods disclosed herein provide a reduction in the risk of toxicity and/or an increase in efficacy, for example, by increasing the exposure of the subject to the administered cells (eg, by improving the increase in the number and/or persistence of the administered cells).

SHORT DESCRIPTION

[0005] The present invention relates to therapeutic regimens for the treatment of a disorder such as cancer. More specifically, the present invention relates to therapeutic regimens for the treatment of a disorder such as cancer by administering allogeneic CAR-T cells. Features of the methods, including timing of doses and numbers of cells administered, provide various advantages, such as improved efficacy and/or lower toxicity, for example due to increased exposure of the subject to the cells administered.

[0006] Methods of administering CAR-T cells to subjects are provided, for example, to treat diseases and/or disorders in the subjects. Typically, methods include administering single or multiple doses of such cells and/or administering a subsequent dose to a subject previously treated with a previous (eg, first) dose of such cells. Cells, compositions and articles for use in such methods are also provided.

[0007] In some embodiments, the methods include treating an adult subject who has refractory and/or recurrent acute lymphoblastic CD19+ B cell leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR -T-cells) containing CAR 4-1 BB/CD3-zeta for human CD19 (e.g., UCART19 (CD19 CAR/RQR8+_TCRαβ-_T-KneTKH), where at least one dose is selected from the group consisting of about 6 x10 ⁵ cells/dose, about 6x10 ⁶ cells/dose, about 6-8x10 ⁷ cells/dose, and about 1.8-2.4x10 ⁸ cells/dose.

[0008] In some embodiments, the methods include treating a pediatric subject who has refractory and/or recurrent acute lymphoblastic CD19+ B cell leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR -T cells) containing CAR 4-1 BB/CD3-zeta for human CD19 (eg, UCART19 (CD19CAR/RQR8+_TCRαβ-_T cells) or UCART19 (CD19CAR/R2+_TCRαβ-_T cells), or UCART19 (CD19CAR/TCRαβ-_T cells), where at least one dose is approximately 2-8 x 10 ⁷ cells/dose.

[0009] In some embodiments, the methods include treating a subject who has refractory and/or recurrent acute non-Hodgkin's lymphoma (e.g., large B-cell lymphoma or follicular lymphoma), the method comprising administering to the subject at least one dose of T cells with allogeneic chimeric antigen receptor (CAR) (CAR-T cells) containing CAR 4-1 BB/CD3-zeta for human CD19 (CD19CAR/RQR8+_TCRαβ-_T cells), where at least one dose is from about 20 ×10 ⁶ cells/dose up to approximately 360×10 ⁶ cells/dose, e.g., approximately 20×10 ⁶ cells/dose, approximately 40×10 ⁶ cells/dose, approximately 80×10 ⁶ cells/dose, approximately 120×10 ⁶ cells/dose, 240×10 ⁶ cells/dose, or approximately 360×10 ⁶ cells/dose.

[0010] In some embodiments, the methods include (a) administering to the subject with the 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 methods are carried out by administering to the subject the subsequent dose or doses as in (b) to the subject who has previously received 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 (eg, 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 leukemia, lymphoma, eg, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL (eg, relapsed/refractory ALL)), non-Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, mantle cell lymphoma, slow-growing B-cell lymphoma, B-cell malignancies, forms of colon, lung (eg, small cell and non-small cell lung cancer), liver, breast, prostate, ovary, skin, melanoma, bone and brain cancer, ovarian cancer, forms of epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical carcinoma, colorectal cancer , glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, si novial sarcoma, head and neck squamous cell carcinoma (HNSCC), and/or mesothelioma. In some embodiments, the disease is a 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 advanced lymphoid malignancy, such as B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the disease is refractory B-ALL, such as refractory B-ALL in adults. In some embodiments, the disease is recurrent B-ALL, such as recurrent B-ALL in adults. In some embodiments, the disease is an extramedullary disease associated with, for example, one or more of the cancers described herein. In some embodiments, the subject has detectable molecular disease and/or minimal residual disease at the time of the next dose. In some embodiments, the subject has one or more symptoms of the disease at the time of the next dose. In some embodiments, the disease is cancer and the subject relapses at the time the next dose is started. In some embodiments, the disease is leukemia or lymphoma, and the subject has more than 5% blast cells in the bone marrow at the time of the next dose.

[0012] In some embodiments, the disease is cancer, and the subject has a morphological disease. In some embodiments, the disease is leukemia or lymphoma and the subject has extramedullary disease (ie, 6 fins outside the marrow) at the time of the next dose. In some embodiments, the disease is leukemia or lymphoma, and the subject has more than 5% blast cells in the bone marrow at the time of the next dose. In other embodiments, the disease is cancer and the subject does not have a morphological disease at the time the next dose is initiated. In some embodiments, the disease is leukemia or lymphoma, and the subject does not have more than 5% blast cells in the bone marrow at the time of the next dose.

[0013] In some embodiments, the methods include (a) administering to a subject suffering from a disease a first dose of allogeneic CAR-T cells. In some embodiments, the first dose contains about 1x10 ⁴ cells, about 5x10 ⁴ cells, about 1x10 ⁵ cells, about 5x10 ⁵ cells, about 1x10 ⁶ cells, about 5x10 ⁶ cells, about 6×10 ⁶ cells, approximately 1×10 ⁷ cells, approximately 6×10 ⁷ cells, approximately 1×10 ⁸ , approximately 1.8×10 ⁸ cells, or approximately 4.8×10 ⁸ cells. In some embodiments, the first dose contains from about 20x10 ⁶ cells to about 360x10 ⁶ cells, for example, about 20x10 ⁶ cells, about 40x10 ⁶ cells, about 80x10 ⁶ cells, about 120x10 ⁶ cells, 240×10 ⁶ cells or approximately 360×10 ⁶ cells. In some embodiments, the methods further comprise (b) administering to the subject a subsequent dose of CAR-T cells at a time point that is at least or greater than about 5 weeks after the start of administration according to (a) to less than about 24 weeks after him.

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

[0015] In some embodiments, the methods further comprise administering additional subsequent or subsequent doses such that the first and subsequent doses are administered, for example, in accordance with the dosage amounts and time schedules indicated for the first and subsequent doses. In some embodiments, the first of one or more subsequent doses is administered at least or more than 5 weeks after the start of the next dose. In some embodiments, administering the first, second, and subsequent doses comprises administering at least three doses over about 5 weeks. In some embodiments, a subsequent dose is administered approximately 16 weeks after the start of the first dose, and an additional follow-up or follow-up dose is administered at week 17 after the start of the first dose. In some embodiments, additional subsequent doses are administered at week 17 and/or week 34 after the start of the first dose.

[0016] In some aspects, the time of administration of the subsequent dose(s) is additional, while the subject does not experience an immune response, for example, there is no detectable adaptive immune response in the host, specific for CAR-T after the specified first ( or previous) dose.

[0017] In some embodiments, the time between the first dose (initial dose), for example, the start of the first or previous dose, and the start of the next dose (for example, the start of the next dose) is more than about 4 weeks, for example, more than about 5, 6, 7, 8, or 9 weeks, such as more than about 20 weeks, such as from about 9 to about 35 weeks, from about 14 to about 28 weeks, from 15 to 27 weeks, or from 16 weeks to approximately 18 weeks; and/or is or is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 weeks. In some embodiments, the next dose (eg, start of administration) is administered more than about 5 weeks after the first or previous dose (eg, start of administration) and less than about 24 weeks thereafter. In some embodiments, the next dose is started 17 weeks after the start of the first dose. In some embodiments, the time between administration of the first and subsequent dose (e.g., start of administration) or the previous and subsequent subsequent dose is from more than about 5 weeks to less than about 24 weeks, such as from 10 to 24 weeks, such as about 17 weeks. In some embodiments, the time between the first and subsequent doses (eg, start of administration) is approximately 17 weeks. In some embodiments, the next dose (eg, start of administration) is administered more than about 7 days after and less than about 365 days after the first or previous dose (eg, start of administration). In some embodiments, the next dose (eg, start of administration) is administered more than about 30 days after and less than about 110 days after the first or previous dose (eg, start of administration).

[0018] In some embodiments, the subject has no persistence of CAR-T cells at the time of the next dose. In some embodiments, the subject has a suboptimal response at the time of the next dose. In some embodiments, the suboptimal response may include any one or more of the following: (i) complete response (CR), complete response with incomplete recovery of blood cell count (CRi) with detectable minimal residual disease (in patients with leukemia) and no cytogenetic response; (ii) complete bone marrow response; (iii) partial response or (iv) stable response.

[0019] In some embodiments, the implementation of the first dose leads to the alleviation of one or more symptoms of the disease in the subject after the first dose. In some embodiments, at the time of administration of the subsequent dose, the subject has relapsed and/or one or more symptoms of the disease have worsened after the initial relief (eg, remission) that occurred after the first dose.

[0020] In some embodiments, the subsequent dose of cells contains CAR-T cells in an amount sufficient to alleviate the disease in the subject. In some embodiments, administration of a subsequent dose results in further alleviation of the disease in the subject. In some embodiments, the administration of the subsequent dose results in an improvement in the disease in the subject as compared to the state immediately prior to the commencement of the subsequent dose. In some embodiments, administration of a subsequent dose results in MRD-negativity. In some embodiments, the method provides relief of the disease to a greater extent and/or for a longer period of time compared to the method with an alternative dosing schedule, where the cells are administered to the subject in the first dose and the cells in the subsequent dose in a single dose. Relief may include reducing the total number of cells, such as tumor cells, reducing disease in the subject, in the subject's organ, in the subject's tissue, or in the subject's body fluid. Relief may include reducing molecular detection by flow cytometry or quantitative PCR, tumor mass or volume, and/or reducing the number and/or extent of metastases. In some embodiments, the relief involves improving the survival of the subject, for example, increasing the time of survival or survival without incident, without progression or without relapse.

[0021] In some embodiments, the disease persists after the first dose is administered and/or the first dose is insufficient to eliminate the disease in the subject. In some embodiments, administration of said subsequent dose results in an improvement in the disease in the subject compared with the state immediately prior to the commencement 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) can be assessed according to the modified classification 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 cause CRS in the subject. In some embodiments, based on clinical data, administration of the first dose does not cause severe CRS in most subjects. In some embodiments, administration of the first dose does not cause CRS comprising a combination of (1) persistent fever (temperature rise to at least 38 degrees Celsius for at least three days) and (2) serum C-reactive protein (CRP) level up to at least or up to about 20 mg/dL, and/or does not cause CRS encompassing 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 cause grade 3 or higher neurotoxicity in the subject. In some embodiments, based on clinical data, administration of the first dose does not cause grade 3 or higher neurotoxicity in the majority of subjects. In some embodiments, symptoms associated with clinical risk for neurotoxicity and/or grade 3 or higher neurotoxicity include confusion, delirium, severe aphasia, pain blunting, myoclonus, lethargy, altered mental status, seizures, seizure-like activity, seizures (optional if confirmed by an electroencephalogram [EEG]), elevated beta-amyloid (AR) levels, elevated glutamate levels, and elevated oxygen radical levels.

[0024] In some embodiments, the subject was treated with a tumor-targeted therapeutic agent prior to the first dose. In some aspects, the subject is refractory or refractory to the specified therapeutic agent at the time of administration of the first dose and/or subsequent dose. In some embodiments, after administration of the first dose and prior to said administration of a subsequent dose, or prior to administration of the first dose, the methods further comprise assessing a serum level of a factor indicative of CRS, a disease symptom, and/or an indicator of the subject's host immune response to the recombinant receptor (e.g., , to CAR), such as humoral or cellular immune response. In some such embodiments, the detected symptom of the disease is or includes the total number of disease-related cells in the subject, in the subject's organ, in the subject's tissue, or in the subject's body fluid, molecular detection by flow cytometry or quantitative PCR, mass or volume of solid tumors or the number or extent of metastases.

[0025] In some embodiments, the methods include assessing a disease symptom prior to administration of a subsequent dose, and based on the result of the assessment, determining a subsequent dose of cells to be administered to the subject. In some embodiments, if the evaluation determines that the subject has a morphological disease, the subject is administered a subsequent dose containing less than, more than, or approximately the same number of allogeneic CAR-T cells as the number of allogeneic CAR-T cells in first dose. In some embodiments, if the evaluation determines that the subject has minimal residual disease, the subject is given a subsequent dose containing an increased amount of allogeneic CAR-T cells compared to the first dose. In some embodiments, the subsequent dose contains approximately the same amount of allogeneic CAR-T cells as the amount in the first dose. In some embodiments, the number of allogeneic CAR-T cells per kilogram administered in the subsequent dose is less than, or equal to, or approximately equal to the number 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 contains an increased number of such cells compared to the first dose, such as at least 2, 5, or 10 times the number in the first dose. In some embodiments, the number of CAR-T cells per kilogram administered in a subsequent dose is at least or about 2 times, or at least or about 3 times, the number of receptor-expressing (e.g., CAR-expressing ) cells per kilogram administered in the first dose.

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

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

[0028] In some embodiments, the implementation of the first and/or subsequent dose is not a fractional dose. For example, in some embodiments, the cells of the first dose are administered in one pharmaceutical composition containing the cells of the first dose, and/or the cells of the next dose are administered in one pharmaceutical composition containing the cells of the next dose. In other embodiments, the first and/or subsequent dose is a fractional dose, for example, when cells from the first dose are administered with a number of compositions, in total, including cells from the first dose, over a time period of no more than three days; and/or the subsequent dose is a fractional dose, where the cells of the subsequent dose are administered with a number of compositions, in total including the cells of the subsequent dose, over a period of time of no more than three days.

[0029] In some embodiments, the methods include 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, a subsequent dose of cells is administered at a time point that is from at least or more than about 5 weeks after the start of the first dose to less than about 24 weeks thereafter. 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 specific for CD19 CAR-T cells (eg, UCART19).

[0030] In some embodiments, the number of cells administered in the first dose is from about 0.5x10 ⁶ cells to 5x10 ⁸ cells, from about 0.75x10 ⁶ cells to 8x10 ⁷ cells, or from about 5×10 ⁶ cells up to 7×10 ⁶ cells, each inclusive. In some embodiments, the number of cells administered in the first dose of CD19-specific CAR-T cells is from about 0.5 x 10 ⁶ cells to 1 x 10 ⁹ cells, from about 1 x 10 ⁵ cells to 3 x 10 ⁸ cells or from about 6×10 ⁵ cells to 2.4×10 ⁸ cells, each inclusive. In some embodiments, the number of cells administered in the first dose of UCART19 cells is about 6 x 10 ⁵ cells, about 6 x 10 ⁶ cells, about 6 x 10 ⁷ cells, about 8 x 10 ⁷ cells, about 1.8 x 10 ⁸ cells or approximately 2.4×10 ⁸ cells. In some embodiments, the number of cells administered in the first dose of UCART19 cells is approximately 6 x 10 ⁶ cells.

[0031] In some embodiments, the number of cells administered in a subsequent dose of CD19-specific CAR-T cells is from about 0.5 x 10 ⁶ cells to 1 x 10 ⁹ cells, from about 1 x 10 ⁵ cells to 3×10 ⁸ cells or from approximately 6×10 ⁵ cells to 2.4×10 ⁸ cells, each inclusive. In some embodiments, the number of cells administered in a subsequent dose of UCART19 cells is about 6x10 ⁵ cells, about 6x10 ⁶ cells, about 6x10 ^{7 cells, about 8x10 7 cells} , about 1.8x10 ⁸ cells or approximately 2.4×10 ⁸ cells. In some embodiments, the number of cells administered in a subsequent dose of UCART19 cells is approximately 6×10 ⁶ cells. In some embodiments, the number of cells administered in the first and subsequent dose of UCART19 cells is approximately 6×10 ⁶ cells.

[0032] In some embodiments, the methods further comprise administering a lymphodepletion regimen prior to the first dose and/or prior to the subsequent dose. In some embodiments, a regimen for lymphodepletion includes cyclophosphamide, fludarabine, and/or a combination thereof. In some embodiments, the lymphodepletion regimen comprises one or more of cyclophosphamide, fludarabine, an anti-CD52 antibody (eg, 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 regimen for lymphodepletion involves administering cyclophosphamide and fludarabine prior to the first dose and an anti-CD-52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) after the first dose. In some embodiments, administering the lymphodepletion regimen comprises administering the lymphodepletion regimen prior to the first dose, and optionally at a different time point than prior to the next dose. In some embodiments, the lymphodepletion regimen is administered 2-10 days prior to the first dose. In some embodiments, the lymphodepletion regimen is administered 2-10 days prior to the second dose. In some embodiments, the lymphodepletion regimen is administered 2-10 days prior to the first dose and 2-14 days prior to the second dose. In some embodiments, the lymphodepletion regimen is administered over a course of 1, 2, 3, 4, or 5 days.

[0033] In some embodiments, the regimen for lymphodepletion includes the administration of about 90-120 mg/m ^{2 of} fludarabine and about 1500 mg/m ^{2 of} cyclophosphamide. In some embodiments, the regimen for lymphodepletion includes the administration of about 150 mg/m ^{2 of} fludarabine and about 120 mg/m ^{2 of} cyclophosphamide.

[0034] In some embodiments, the regimen for lymphodepletion comprises administering fludarabine at a dose of about 30 to 150 mg/m ² ; cyclophosphamide at a dose of about 300 to 4000 mg/m ² or an anti-CD52 antibody (for example, an anti-CD52 antibody containing the sequence under 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 from about 30 to 150 mg/m ² ; cyclophosphamide is administered at a dose of about 300 to 4000 mg/m ² and an anti-CD52 antibody (eg, an anti-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. In some embodiments, fludarabine is administered at a dose of about 30 mg/m ² /day; cyclophosphamide is administered at a dose of about 300 mg/m ² /day, or an anti-CD52 antibody (eg, an anti-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. In some embodiments, fludarabine is administered at a dose of about 30 mg/m ² /day; cyclophosphamide is administered at a dose of about 300 mg/m ² /day, and an anti-CD52 antibody (eg, an anti-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 regimen for lymphodepletion includes the administration of fludarabine and cyclophosphamide. In some embodiments, the regimen for lymphodepletion includes the administration of fludarabine, cyclophosphamide, and an anti-CD52 drug (eg, an anti-CD52 antibody having the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10). In some embodiments, the lymphodepletion regimen further provides for the administration of Mensa (sodium-2-mercaptoethanesulfonate). In some embodiments, the lymphodepletion regimen further comprises administering at least one corticosteroid. In some embodiments, a corticosteroid is administered immediately prior to administration of an anti-CD52 antibody (eg, an antibody comprising 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 1-5 mg/kg (eg, 2 mg/kg). In some embodiments, the implementation of the corticosteroid is administered at least two days before the introduction of CAR-T cells. In some embodiments, the implementation of the corticosteroid is not administered for at least two days before the introduction of CAR-T cells.

[0036] In some embodiments, the patient receives premedication for an infusion-related reaction prior to a regimen for lymphodepletion. Premedication may involve the administration of, for example, an antihistamine or acetaminophen.

[0037] In some embodiments, components of the lymphodepletion regimen fludarabine/cyclophosphamide (FC) or fludarabine/cyclophosphamide/anti-CD52 antibody (FCA) are administered simultaneously; in other embodiments, the components are administered sequentially. In some embodiments, the subject receives an FC regimen prior to the first dose of the CAR-T cell treatment agent and an FCA regimen prior to repeated doses of the CAR-T cell treatment agent. In some embodiments, the subject receives an FCA regimen prior to the first dose of the CAR-T cell treatment agent; and a second FCA regimen prior to re-dosing of the CAR-T cell treatment agent. In some embodiments, the subject receives an FC regimen prior to the first dose of a CAR-T cell treatment agent and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) after the first dose agents for treatment with CAR-T cells. In some embodiments, the subject receives an FC regimen prior to the first dose of a CAR-T cell treatment agent and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) after the first dose agents for treatment with CAR-T cells; and the subject additionally receives an FC regimen prior to the second/subsequent dose of the CAR-T cell treatment agent and an anti-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 the agent for treatment with CAR-T cells.

[0038] In some embodiments, the methods further comprise administering a disease mitigating agent prior to administration of the first dose and/or prior to administration of the second/subsequent dose. In some embodiments, the methods include administering a chemotherapeutic agent prior to a first dose and/or prior to a second/subsequent dose. In some embodiments, the subject has previously received treatment with a chemotherapeutic agent prior to the first dose. In some embodiments, the implementation of the chemotherapeutic agent is or provides for a conditioning chemotherapy that reduces the severity of the disease in the subject before the first dose and/or subsequent dose. In some embodiments, administration of the chemotherapeutic agent comprises administering the chemotherapeutic agent prior to administration of the first dose, and optionally at a different time point than prior to administration of the second/subsequent dose. In some embodiments, the chemotherapeutic agent is administered 2-10 days prior to the first dose. In some embodiments, the chemotherapeutic agent is administered 2-10 days prior to the second/subsequent dose. In some embodiments, the chemotherapeutic agent is administered 2-10 days prior to the first dose and 2-14 days prior to the second/subsequent dose.

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

[0040] In some embodiments of such medical uses, the compositions or cells are for use that includes administering to a subject who has a disease a first dose of allogeneic CAR-T cells. In some embodiments, the first dose contains about 1x10 ⁶ total cells, about 6x10 ⁶ total cells, or about 1x10 ⁷ total cells. In some embodiments, the compositions or cells are for use that includes administering to a subject a subsequent dose of allogeneic CAR-T cells at a time point that is at least or greater than about 4 weeks after the start of the first dose and less than about 24 weeks after him.

[0041] In some embodiments, allogeneic CAR-T cells are provided for use in methods of treating a disease in a subject previously treated with allogeneic CAR-T cells. In some embodiments, the cells are intended for use approximately 4-24 weeks after the previous treatment. In some embodiments, the cells for use are formulated to administer a subsequent dose in an amount sufficient to reduce the severity of the disease in a subject previously treated with allogeneic CAR-T cells. In some embodiments, the CAR-T cells are specific for CD19 CAR-T cells (eg, UCART19).

[0042] In some embodiments of any such compositions or cells for use or medical uses, the subject does not have a morphological disorder and/or the subject does not have more than 5% blast cells in the bone marrow.

[0043] In some embodiments, the compositions or cells are for use in a method comprising administering to a subject having a disease a first dose of allogeneic CAR-T cells. In some embodiments, the first dose is about 6x10 ⁵ cells, about 6x10 ⁶ cells, about 6x10 ^{7 cells, about 8x10 7 cells} , about 1.8x10 ⁸ cells, or about 2.4x10 ⁸ cells. In some embodiments, the cells are for use in a method that includes administering to a subject a subsequent dose of allogeneic CAR-T cells at a time point that is at least or greater than about 5 weeks after the start of the first dose to less than about 24 weeks after it.

[0044] This document also provides options for the use of allogeneic CAR-T cells for the manufacture of a drug intended for the treatment of a disease in a subject, including cells that are formulated and/or packaged for administration to the subject in the first and subsequent doses. In some embodiments, the treatment involves administering cells to the subject at a first and subsequent doses, where the first dose comprises about 6x10 ⁵ cells, about 6x10 ⁶ cells, about 6x10 ⁷ cells, about 8x10 ⁷ cells, about 1, 8×10 ⁸ cells or approximately 2.4×10 ⁸ cells. In some embodiments, the CAR-T cells are specific for CD19 CAR-T cells (eg, UCART19).

[0045] In some embodiments, the cells for use are formulated and/or packaged for administration to the subject in the first and subsequent doses, and/or the treatment involves the administration of the cells to the subject in the first and subsequent doses. In some embodiments, the first dose contains about 6x10 ⁵ cells, about 6x10 ⁶ cells, about 6x10 ^{7 cells, about 8x10 7 cells} , about 1.8x10 ⁸ cells, or about 2.4x10 ⁸ cells.

[0046] In some embodiments, the use includes cases where the first and subsequent injections include the introduction of cells in one or more unit doses, with each standard dose containing about 6x10 ⁵ cells, about 6x10 ⁶ cells, about 6x10 ⁷ cells, approximately 8×10 ⁷ cells, approximately 1.8×10 ⁸ cells or approximately 2.4×10 ⁸ cells.

[0047] In some embodiments, the cell or application includes cases where the first administration involves the administration of a single unit dose. In some embodiments, the cell or application includes cases where the subsequent administration involves the administration of two or more unit doses. In some embodiments, the cell or application includes instances where the subsequent administration involves the administration of a single unit dose.

[0048] In some embodiments, cells are used to treat a disease, where the disease is a tumor or cancer. In some embodiments, the disease is a lymphoid malignancy, such as, for example, without limitation, acute and chronic leukemias, lymphomas, multiple myelomas, myeloid malignancies (e.g., leukemias, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms), or undifferentiated malignancy. In some embodiments, the composition or cells are for use in the treatment of acute lymphoblastic leukemia (ALL). In some embodiments, ALL is relapsed/refractory ALL. In some embodiments, the composition or cells are provided for use in the treatment of non-Hodgkin's lymphoma, such as relapsed or refractory non-Hodgkin's lymphoma. In some embodiments, the composition or cells are for use in the treatment of relapsed or refractory large B-cell or follicular lymphoma.

[0049] In some embodiments, the cell, composition, or use includes cases where the subsequent dose is formulated to administer approximately the same number of allogeneic CAR-T cells as the number of allogeneic CD19-specific CAR-T cells in the previous dose . In some embodiments, the next dose cell composition is formulated to administer an increased amount of allogeneic CAR-T cells compared to the first dose or the previous dose. In some embodiments, the cell, composition, or use includes instances where the subsequent dose is formulated to administer less allogeneic CAR-T cells than the number of allogeneic CD19-specific CAR-T cells in the previous dose.

[0050] Pharmaceutical kits are also provided for the treatment of a patient suffering from a disease, such as cancer, wherein the kit contains CAR-T cells and an anti-CD52 antibody. In some embodiments, the CAR-T cells are C019-specific CAR-T cells, such as UCART19 cells. In some embodiments, the CAR-T cells express the CAR of SEQ ID NO: 1. In some embodiments, the anti-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 containing CAR-T cells and a second container containing an anti-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 containing instructions for administering CAR-T cells and an anti-CD52 antibody to a subject.

[0051] Articles for use in these methods are also provided. In some embodiments, the article of manufacture includes a number of containers, such as sealed containers, each of which separately contains a unit dose of allogeneic CAR-T cells for administration to a subject, packaging material, and/or a label or package insert. In some embodiments, the CAR-T cells are specific for CD19 CAR-T cells (eg, UCART19).

[0052] In some embodiments, the implementation of the standard dose contains the number of cells to be administered at the lowest dose in these methods, for example, in the amount of the first dose. In some embodiments, the unit dose comprises about 6x10 ⁵ cells, about 6x10 ⁶ cells, about 6x10 ^{7 cells, about 8x10 7 cells} , about 1.8x10 ⁸ cells, or about 2.4x10 ⁸ cells.

[0053] In some embodiments, the label or package insert contains instructions for administering to a subject a number of unit doses, for example, by administering a number of such unit doses, such as one unit dose, at the first dose, and then administering a subsequent dose, including one or a number of standard doses. In some embodiments, the instructions describe making a first administration, wherein said first administration comprises delivering one of said unit doses to the subject and making a subsequent administration, said subsequent administration comprising administering to the subject one or more of said unit doses. In some embodiments, they describe that the subsequent administration should be from about 5 to about 24 weeks after said first administration. In some embodiments, the containers are or contain flexible bags for cell infusion. In some embodiments, the methods are for allogeneic administration. In some embodiments, the label and/or packaging material further includes a subject-specific identifier that indicates that the cells were obtained from the subject and/or are to be administered to the subject specifically. In some embodiments, the CAR-T cells are specific for CD19 CAR-T cells (eg, UCART19).

[0054] Also provided is a method for obtaining 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) activation of T cells in PBMCs; (c) transducing activated T cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing the CAR of interest; (d) disruption of TCRαβ and CD-52 gene expression in a subpopulation of T cells; (e) increasing the T cell population; and (f) enriching the T cell population for TCRαβ negative cells; resulting in a population of "ready" allogeneic T-cells with a chimeric antigen receptor (CAR) (CAR-T-cells). In some embodiments, CAR-T cells include allogeneic CD19CAR/RQR8+_TCRαβ-_T cells.

[0055] Also provided are allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) containing CAR 4-1 BB/CD3-zeta for human CD19 for use in a method of treating a subject who has refractory and /or recurrent non-Hodgkin's lymphoma, the method comprising administering to the subject at least one dose of CAR-T cells, where at least one dose is from about 20x10 ⁶ cells/dose to about 360x10 ⁶ cells/dose.

[0056] Also provided are (CAR)-T-KneTKH with chimeric antigen receptor (CAR-T cells) for use in a method of treatment comprising: (a) administering to a subject a first dose of CAR-T cells and (b) administering to a subject a subsequent dose of CAR-T cells at a time point that is from at least or more than about 28 days after the start of the introduction specified in (a) to less than about 200 days after it.

[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 who has previously received a first dose of allogeneic CAR-T cells, wherein the subsequent dose of cells administered at a time point that is at least or more than about 5 weeks after the start of the first dose to less than about 24 weeks thereafter. Optionally, no detectable adaptive host immune response specific for CAR-T cells is observed in the subject at the time of administration.

[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 administration, the subject received a previous dose of CAR-T -cells in an amount sufficient to demonstrate a clinical effect in the subject; and at the time of administration, the subject does not show a detectable adaptive immune response of the body, specific for CAR-T cells; and/or the time between said previous and subsequent doses is greater than about 5 weeks to less than about 24 weeks.

[0059] Allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) containing CAR 4-1 BB/CD3-zeta for human CD19 are also provided for use in a method of treating an adult subject who has a refractory and/or recurrent CD19+ B-cell acute lymphoblastic leukemia, wherein the method comprises administering to the subject at least one dose of CAR-T cells, wherein at least one dose is selected from the group consisting of approximately 6×10 ⁵ cells/dose, 6 ×10 ⁶ cells/dose, approximately 6-8×10 ⁷ cells/dose and approximately 1.8-2.4×10 ⁸ cells/dose.

[0060] Also provided are allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) containing CAR 4-1 BB/CD3-zeta for human CD19 for use in a method of treating a pediatric subject who has a refractory and/or recurrent CD19+B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of CAR-T cells containing CAR 4-1 BB/CD3-zeta to human CD19, wherein at least one dose is approximately 2 -8×10 ⁷ cells/dose.

[0061] Also provided are allogeneic chimeric antigen receptor (CAR) T cells for use in the treatment of disease in a subject previously treated with CAR-T cells, where the cells are intended for use at a time point of about 5 to 24 weeks after previous treatment; and wherein the cells are formulated to administer a subsequent dose in an amount sufficient to alleviate the disease in a subject previously treated with CAR-T cells.

[0062] In some embodiments, the treatments disclosed herein were administered to subjects who were treated with an antibody to deplete CD52+ cells (e.g., an antibody comprising SEQ ID NO: 8 and SEQ ID NO: 10), or who will be treated with said antibody as part of said treatments. These treatments may include the use of CAR-T cells (eg, UCART19) deficient in CD52.

[0063] 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, including:

(a) isolating peripheral blood mononuclear cells (PBMCs) from a healthy donor;

(b) activation of T cells in PBMCs;

(c) transducing activated T cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing the CAR of interest;

(d) disruption of TCRαβ1 and CD-52 gene expression in a subpopulation of T cells;

(e) increasing the population of T cells and

(f) enrichment of the T cell population for TCRαβ negative cells

thereby obtaining a population of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells).

BRIEF DESCRIPTION OF GRAPHICS

[0064] FIG. 1 is an exemplary diagram showing possible steps in a multiple dose regimen for treatment with allogeneic CAR-T cells.

[0065] FIG. 2A and FIG. 2B shows kinetic data from two patients receiving a second infusion of UCART19 (redosing study).

[0066] FIG. 3A is an exemplary diagram showing the steps of a dosing regimen for treatment with allogeneic CAR-T cells.

[0067] FIG. 3B is an exemplary diagram showing the steps of a dosing regimen for treatment with allogeneic CAR-T cells.

[0068] FIG. 4A and FIG. 4B depicts exemplary flowcharts in relation to the production of allogeneic CAR-T cells.

[0069] FIG. 5 shows an exemplary engineered product based on allogeneic CD19+ CAR T cells (CD19CAR/RQR8+_TCRαβ-_T cells).

[0070] FIG. 6A and FIG. 6B shows a study design for the use of a CD19 CAR allogeneic T cell product (UCART19) in adult patients with relapsed/refractory CD19+ B cell acute lymphoblastic leukemia.

[0071] FIG. 7A shows the status of the study following the initiation of a CD19 CAR allogeneic T cell product (UCART19) in adult patients with relapsed/refractory CD19+ B cell acute lymphoblastic leukemia.

[0072] FIG. 7B shows response, duration of remission, and rechallenge of UCART19 in adult patients with relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[0073] FIG. 8A and FIG. 8B depicts the flow cytometry PK profile and UCART19 kinetics in adult patients with relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[0074] FIG. 9 shows a study plan for the use of a CD19 CAR allogeneic T cell product (UCART19) in pediatric patients at high risk for relapsed/refractory CD19+ B cell acute lymphoblastic leukemia.

[0075] FIG. 10 shows the status of the study following the initiation of a CD19 CAR allogeneic T cell product (UCART19) in pediatric patients at high risk for relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[0076] FIG. 11 shows the response and duration of remission (antileukemic activity) of UCART19 in pediatric patients at high risk for relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[0077] FIG. 12 shows the cellular kinetics of UCART19 in pediatric patients at high risk for relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[0078] FIG. 13A, FIG. 13B and FIG. 13C shows blood chimerism data following UCART19 administration in pediatric patients at high risk for relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[0079] FIG. 14 shows the response of patient #18 to repeated dosing as described in Example 3.

DETAILED DESCRIPTION

General Methods

[0080] In the implementation of the methods and compositions described in the present invention, in practice, unless otherwise indicated, traditional methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology, which are within the competence of a specialist in this field of technology. Such techniques are fully explained in literature such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. 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).

Definitions

[0081] As used herein, the term "allogeneic" means that the cells or population of cells used to treat patients do not come from the specified patient, but from a donor, with the exception of a human leukocyte antigen (HLA) compatible donor.

[0082] As used herein, the term "autologous" means that the cells, cell line, or population of cells used to treat patients originate from the specified patient or from a human leukocyte antigen (HLA) compatible donor.

[0083] As used herein, the term "immune cell" refers to a cell of hematopoietic origin functionally involved in the initiation and/or implementation of the innate and/or adaptive immune response.

[0084] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a molecule that, when located in an immune effector cell, provides the cell with target specificity and an intracellular signal when interacting with the target.

[0085] As used herein, the term "dosing regimen" refers to the overall course of treatment administered to a patient, such as treatment with CAR T cells.

[0086] As used herein, "treatment" is an approach to obtain favorable or desired clinical results. For purposes of the present invention, beneficial or desirable clinical results include, but are not limited to, one or more of the following: reduced proliferation (or destruction) of tumor or cancer cells, inhibition of tumor cell metastasis, shrinkage or reduction in tumor size, disease (e.g., cancer) remission, attenuation symptoms resulting from a disease (such as cancer), improving the quality of life of those suffering from the disease (such as cancer), reducing the dose of other drugs needed to treat the disease (such as cancer), delaying the progression of the disease (such as cancer ), curing a disease (eg, cancer), and/or prolonging the survival of patients who have a disease (eg, cancer). "Reduction in incidence" means any 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 to treat said disease.

[0087] "Alleviate" or "alleviate" means the reduction or improvement of one or more symptoms compared to the case when treatment is not prescribed. "Relief" also includes a reduction or reduction in the duration of a symptom.

[0088] As used herein, the term "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve any one or more of the beneficial or desired results. For prophylactic use, beneficial or desirable outcomes include the elimination or reduction of risk, reduction in severity, or delay in onset of the disease, including the biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during the course of the disease. An effective dosage may be administered in one or more administrations. For the purposes of the present invention, an effective dose of a drug, CAR-T cells, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of administering one or more therapeutic agents, and one agent may be considered to be administered in an effective amount if, in combination with one or more other agents, the desired result can be achieved or is achieved.

[0089] An "individual" or "subject" is a mammal, such as a human. Mammals also include, without limitation, farm animals, sports animals, pets, primates, horses, dogs, cats, mice, and rats.

[0090] "Refractory" in the context of this document refers to a disease (eg, cancer) that is not treatable. In embodiments, 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. Refractory cancer is also referred to herein as resistant cancer.

[0091] The term "recurrent" as used herein 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 previous treatment with a treatment agent, e.g. for the treatment of cancer.

[0092] As used herein, the term "minimal residual disease" or "MRD" refers to low-level disease identified in various clinical settings. In some embodiments, MRD refers to a molecularly defined relapse after remission.

[0093] As used herein, the term "vector" means a construct that is capable of delivering and expressing one or more of the gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, without limitation, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0094] As used herein, the term "expression control sequence" means a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence may 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.

[0095] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes not only intact polyclonal or monoclonal antibodies, but also any antigen-binding fragment (i.e., “antigen-binding portion”) or single chain thereof, antibody-containing fusion proteins, and any other modified configuration of an immunoglobulin molecule. , which contains an antigen recognition site, including, for example, scFv, single-domain antibodies (for example, shark and camel antibodies), maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR, and bis-scFv (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23 (9): 1126-1136). An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or a subclass thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the antibody constant region of its heavy chains, immunoglobulins can belong to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subclassed (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, beta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known.

[0096] The term "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (eg, tumor antigen). The antigen-binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include Fab; Fab';F(ab')₂; Fd fragment consisting of VH and CH1 domains; An Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a single domain (dAb) fragment (Ward et al., (1989) Nature 341:544-546) and a complementarity determining region (CDR).

[0097] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are linked to each other. another, eg via a synthetic linker, eg a short flexible polypeptide linker, and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, the scFv used herein may have the VL and VH variable regions in any order, for example, relative to the N-terminal and C-terminal ends of the polypeptide, the scFv may contain a VL-linker-VH, or may contain a VH-linker- VL.

[0098] 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, but not the method by which it is obtained. In some embodiments, the implementation of the monoclonal antibody of the present invention exists in a homogeneous or virtually homogeneous population.

[0099] "Humanized" antibody refers to non-human (e.g., murine) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding antibody subsequences) that contain a minimal sequence derived from a non-human immunoglobulin. Typically, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit, characterized by the desired specificity, affinity and activity.

[00100] As used herein, the term "human antibody" means an antibody having an amino acid sequence corresponding to that of an antibody that can be derived from a human and/or that has been generated using any of the techniques for producing human antibodies known to those skilled in the art. and disclosed in this document. This definition of a human antibody includes antibodies containing at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody containing mouse light chain and human heavy chain polypeptides. Human antibodies can be obtained using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library where human antibodies are expressed in the phage library (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 obtained by immunizing animals in which human immunoglobulin loci have been transgenically introduced in place of endogenous loci, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in US Pat. Nos. 5,545,807; 5545806; 5569825; 5625126; 5,633,425 and 5,661,016. Alternatively, a human antibody can be obtained by immortalizing human B-lymphocytes that produce an antibody directed against the target antigen (such B-lymphocytes can be isolated from a subject or can be immunized in vitro). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77, 1985; Boerner et al., 1991, J. Immunol., 147(l):86-95 and US Pat. No. 5,750,373.

[00101] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is known in the art, each heavy and light chain variable region consists of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions, which contribute to the formation of an antibody antigen-binding site. If variants of a subject's variable region are required, in particular with a substitution at amino acid residues outside the CDR region (i.e., in the framework region), an appropriate amino acid substitution, often a conservative amino acid substitution, can be determined by comparing the subject's variable region with the variable regions of other antibodies. that contain CDR1 and CDR2 sequences in the same canonical class as the subject's variable region (Chothia and Lesk, J Mol Biol 196(4):901-917, 1987). When choosing a FR to flank a CDR of a subject, such as in the case of humanization or optimization of an antibody, FRs from antibodies that contain the CDR1 and CDR2 sequences in the same canonical class are preferred.

[00102] Variable domain "CDRs" are amino acid residues within a variable region that are identified according to definitions according to Kabat, Chothia, accumulation according to both Kabat and Chothia, AbM, contact and/or conformational definitions, or according to any a CDR determination method well known in the art.

[00103] As used herein, the terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" include any material that, when combined with an active ingredient, provides the ingredient with the ability to retain biological activity and not react with the subject's immune system. Examples include, without limitation, any conventional pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as an oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated using well 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 21 ^st Ed. Mack Publishing, 2005).

[00104] Reference to "about" in relation to a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, a description referring to "about X" includes a description of "X". Numeric ranges include numbers that define the range.

[00105] It is clear that whatever embodiments are described herein with the wording "comprising", other similar embodiments described by the terms "consisting of" and/or "consisting in fact of" are also contemplated.

[00106] When aspects or embodiments of this disclosure are described in terms of the Markush group or other group of alternatives, the invention covers not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also in the main group with the absence of one or more group members. The present invention also provides for the express exclusion of any one or more of the claimed members of the group.

[00107] Ranges: Throughout this invention, various aspects of the invention may be provided 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 invention. Accordingly, the description of a range is to be construed as specifically disclosing all possible subranges as well as individual numerical values within a given range. For example, a description of a range such as 1 to 6 should be considered to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 up to 6, and so on, as well as individual numbers within a given 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 anything 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 latitude of the range.

[00108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is generally understood by one of ordinary skill in the art to which the present invention belongs. In the event of a conflict, the present description, including definitions, will take precedence. Throughout the specification and claims, the word "comprise" or variations thereof such as "comprises" or "comprising" should be understood to mean as including the specified integer or group of integers, but not excluding any other integer or group whole numbers. Unless otherwise required by the context, the singular terms will cover the plural forms, and the plural terms will include the singular form.

[00109] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the methods and compositions described herein. These materials, methods and examples are illustrative only and are not intended to be limiting.

Treatment with CAR-T cells

[00110] Provided herein are methods, compositions, and articles of manufacture for use in cell therapy for the treatment of diseases, including various forms of cancer and tumors. The methods include administering allogeneic chimeric antigen receptor (CAR) T cells that specifically bind to disease-associated antigens and result in a response, such as an immune response against such molecules when bound to such antigens.

[00111] The methods of this invention can be used to treat, for example, hematological neoplasms (WHO classification, 2008) and solid tumors. Hematologic neoplasms include, for example, lymphoid malignancies (acute and chronic leukemias, lymphomas, multiple myelomas), myeloid malignancies (leukemias, myelodysplastic syndromes, myelodysplastic or myeloproliferative neoplasms), and undifferentiated malignancies. Solid tumors include, for example, solid tumors expressing the antigen(s) targeted by CAR-T cells used in treatment.

[00112] The methods are suitable for the treatment of adult and pediatric populations, including all age groups, and can be used in any line of treatment, including first line or subsequent lines.

[00113] 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 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 (DLBCL), multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, slow-growing B-cell lymphoma, B-cell malignancies, forms of colon, lung cancer (eg, small cell and non-small cell lung cancer) , liver, breast, prostate, ovary, skin, melanoma, bone and brain cancer, ovarian cancer, forms of epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma , Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma y, head and neck squamous cell carcinoma (HNSCC) and/or mesothelioma. In some embodiments, the disease is a 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).

[00114] In some embodiments, the disease is acute lymphoblastic leukemia (ALL). In some embodiments, the disease is childhood acute lymphoblastic leukemia (ALL).

[00115] In some embodiments, the disease is an advanced lymphoid malignancy, such as B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the disease is refractory B-ALL, such as refractory B-ALL in adults. In some embodiments, the disease is recurrent B-ALL, such as recurrent B-ALL in adults.

[00116] In some embodiments, the disease is non-Hodgkin's lymphoma, such as large B-cell and/or follicular lymphoma. Large B-cell lymphoma may be large B-cell lymphoma that is diagnosed by histological or cytological methods in accordance with the 2018 revision of the WHO classification of lymphomas: Diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS) (B cells terminal center [GCB] and non-GCB), DLBCL coexisting with any grade of follicular lymphoma, intravascular large B-cell lymphoma, DLBCL associated with chronic inflammation, anaplastic lymphomakinase positive (ALK+) DLBCL Epstein-Barr virus positive ( EBV+) DLBCL-NOS, T-cell/histiocyte-rich large B-cell lymphoma, DLBCL with IRF4/MUM1 rearrangement, High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 translocation (double/triple hit), primary cutaneous DLBCL-leg type, follicular lymphoma transformation to DLBCL, primary mediastinal B-cell lymphoma (PMBCL) or follicular lymphoma. Parameters that should be considered for treatment include, for example, the kinetic parameters of the administered CAR-T, the disease score (adapted to the target disease), and the subject's health status.

[00117] Methods of treatment may include several stages, including lymphodepletion (optionally with premedication), treatment, and optional re-treatment.

lymphodepletion

[00118] In some embodiments, a lymphodepletion (LD) regimen is administered to a subject prior to the first and/or subsequent doses of CAR-T cells. In some embodiments, the lymphodepletion (LD) regimen is administered to the subject simultaneously with the first and/or subsequent doses of CAR-T cells. In some embodiments, the lymphodepletion regimen is administered before, during and/or after the first and/or subsequent doses of CAR-T cells.

[00119] Suitable schemes for LD are described herein and/or are known in the art. In some embodiments, LD is started before, at the same time as, or after the CAR-T infusion. The doses and timing of LD administration can be tailored for first or subsequent CAR-T dosing. In some embodiments, the LD duration is from about 3 to 5 days. In some embodiments, the time window between the end of LD and the start of CAR-T administration is from about 2 days to about 2 weeks. In some embodiments, LD is started about 15-7 days prior to dosing with CAR-T cells. In some embodiments, LD is started approximately 19-5 days prior to dosing with CAR-T cells. In some embodiments, LD is started approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days prior to dosing CAR-T -cells. In some embodiments, the duration of the LD regimen is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, a dose of CAR-T cells is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the end of LD.

[00120] In some embodiments, an LD regimen includes administering one or more chemotherapeutic drugs.

[00121] In some embodiments, the regimen for LD comprises administering an anti-CD52 antibody, such as an antibody that recognizes human cluster of differentiation (CD) 52 antigen, a cell surface glycoprotein expressed in most lymphoid cells. As used herein, an anti-CD52 monoclonal antibody is an antibody that is directed against the 21-28 kD CD52 cell surface glycoprotein. CD52 is a ubiquitous molecule (approximately 5 x 10 ⁵ antigen binding sites per cell) present in at least 95% of all human peripheral blood lymphocytes and monocytes/macrophages. Exemplary anti-CD52 antibodies for use in the methods and compositions described herein include, for example, alemtuzumab. In some embodiments, the anti-CD52 antibody comprises the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in Table 1 below.

[00122] In some embodiments, the implementation of the antibody to CD52 contains VH and/or VL containing the sequence presented in table 2 below.

[00123] In some embodiments, the anti-CD52 antibody comprises a VH having the sequence of SEQ ID NO: 8, or a sequence characterized by 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 with SEQ ID NO: 8. In some embodiments, the antibody to CD52 contains a VL having the sequence of SEQ ID NO: 10, or a sequence characterized by 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 with SEQ ID NO: 10. In some embodiments, the CD52 antibody comprises a VH having the sequence under SEQ ID NO: 8, and VL having the sequence under SEQ ID NO: 10. B In some embodiments, the anti-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.

[00124] 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 humanized recombinant DNA monoclonal antibody directed against the 21-28 kDa cell surface glycoprotein CD52. See, for example, 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 characterized by 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 with SEQ ID NO: 8. In some embodiments, the CD52 antibody comprises the sequence under SEQ ID NO: :10 or a sequence characterized by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10. In some embodiments, the implementation of the CD52 antibody contains HCDR1 containing the sequence under SEQ ID NO: 2, HCDR2 containing the sequence under SEQ ID NO: 2 NO: 3, HCDR3 containing a sequence ity under SEQ ID NO: 4, LCDR1 containing the sequence under SEQ ID NO: 5, LCDR1 containing the sequence under SEQ ID NO: 6, and/or LCDR3 containing the sequence under SEQ ID NO: 7. In some embodiments, the antibody to CD52 contains HCDR1 containing the sequence under SEQ ID NO: 2, HCDR2 containing the sequence under SEQ ID NO: 3, HCDR3 containing the sequence under SEQ ID NO: 4, LCDR1 containing the sequence under SEQ ID NO: 5, LCDR1 containing the sequence under SEQ ID NO: 6, and LCDR3 containing the sequence under SEQ ID NO: 7; where the antibody to CD52 contains a sequence under SEQ ID NO: 8 and/or SEQ ID NO: 10 or a sequence characterized by at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8 and/or SEQ ID NO: 10.

[00125] In some embodiments, the LD involves the administration of a combination of therapies. In some embodiments, the combination includes fludarabine (total dose range of about 90 to 150 mg/m ² ) and cyclophosphamide (total dose range of about 1000 to 4000 mg/m ² ) with or without an anti-CD52 drug {e.g., an anti- CD52, e.g., an antibody comprising the sequence under SEQ ID NO: 8 and/or SEQ ID NO: 10) (about 0.3 to about 1 mg/kg total dose, or about 30 mg to about 40 mg fixed dose, about 25 to about 60 mg or about 100 mg to about 120 mg). In some embodiments, the combination includes fludarabine (about 30 mg/m ² ) and cyclophosphamide (total dose range from about 500 to 600 mg/m ² ) with or without an anti-CD52 drug (eg, an anti-CD52 antibody) (total dose from about 0.3 to about 1 mg/kg, or a fixed dose of about 30 mg to about 40 mg, about 25 to about 60 mg, or about 100 mg to about 120 mg). In some embodiments, the combination includes fludarabine (about 30 mg/m ² ) and cyclophosphamide (about 300 mg/m ² ), with or without an anti-CD52 drug (eg, an anti-CD52 antibody) (total dose of about 0.3 to about 1 mg/kg or a fixed dose of about 30 mg to about 40 mg, about 25 mg to about 60 mg, or about 100 mg to about 120 mg). In some embodiments, the combination includes fludarabine (about 90 mg/m ² ), cyclophosphamide (about 1500 mg/m ² ), with or without an anti-CD52 drug (eg, an anti-CD52 antibody, about 1 mg/kg). In some embodiments, the combination includes fludarabine (about 150 mg/m ² ) and cyclophosphamide (about 130 mg/µg) with or without an anti-CD52 drug (e.g., an anti-CD52 antibody, a total dose of about 0.3 to about 1 mg /kg or a fixed dose of about 30 mg to about 40 mg, about 25 to about 60 mg, or about 100 mg to about 120 mg). In some embodiments, the combination includes fludarabine (about 150 mg/m ² ) and cyclophosphamide (about 120 mg/kg or 130 mg/µg) with or without an anti-CD52 drug (eg, an anti-CD52 antibody), a total dose of about 0 3 to about 1 mg/kg, or a fixed dose of about 30 mg to about 40 mg, about 25 to about 60 mg, or about 100 mg to about 120 mg). In some embodiments, the combination includes fludarabine (about 30 mg/m ² /day) and cyclophosphamide (about 300 mg/m ² /day) with or without an anti-CD52 drug (e.g., an anti-CD52 antibody, about 13 mg/day) . In some embodiments, the combination includes fludarabine (about 30 mg/m ² /day) and cyclophosphamide (about 300 mg/m ² /day) with or without an anti-CD52 drug (e.g., an anti-CD52 antibody, about 10 mg/day) . In some embodiments, the above doses are administered within one day. In some embodiments, the above doses are administered over several days.

[00126] In some embodiments, fludarabine and cyclophosphamide are administered on the first day, and an anti-CD52 antibody (eg, an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) is administered on the second day. In some embodiments, fludarabine and cyclophosphamide are administered on the first day prior to CAR-T cell administration, and an anti-CD52 antibody (eg, an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) is administered on the second day; where the second day is the same day on which CAR-T cells are administered, or the second day is the next day after that on which CAR-T cells are administered. In some embodiments, fludarabine and cyclophosphamide are administered on day one, CAR-T cells are administered on day two, and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) is administered via 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 the second day. In some embodiments, fludarabine and cyclophosphamide are administered prior to administration of CAR-T cells, and an 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 CAR-T cell administration.

[00127JB In some embodiments, the lymphodepletion regimen includes the administration of fludarabine and cyclophosphamide (FC). In some embodiments, the lymphodepletion regimen includes the administration of fludarabine and an anti-CD52 antibody (eg, an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) (FA). In some embodiments, the regimen for lymphodepletion involves the administration of cyclophosphamide and an anti-CD52 antibody (eg, an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) (CA). In some embodiments, the lymphodepletion regimen includes the administration of fludarabine, cyclophosphamide, and an anti-CD52 antibody (eg, an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) (FCA).

[00128] The choice of specific drugs for the regimen for lymphodepletion before the first or second/subsequent doses of CAR-T cells can be determined based on the hematological analysis and hematological recovery of the patient. In the case of repeated dosing, the second regimen for lymphodepletion may be more or less intense compared to the first regimen for lymphodepletion (eg, based on the recovery of lymphocytes, neutrophils and viral reactivation after the first dose). For example, during repeated dosing, if the levels of lymphocytes and neutrophils are high, a strong or aggressive schedule for lymphodepletion can be applied. Alternatively, during re-dosing, if lymphocyte levels are low, a weaker or less aggressive regimen for lymphodepletion can be used. In some embodiments, if the number of blasts during repeated dosing is high, a strong or aggressive schedule for lymphodepletion is used. In some embodiments, if the number of blasts during repeated dosing is low, a weaker or less aggressive regimen for lymphodepletion is used.

[00129] In some embodiments, an increased intensity regimen for LD may be applied during repeated dosing (with or without anti-CD52 drug). In some embodiments, a reduced intensity regimen for LD may be applied, for example, in the case of grade 3-4 lymphopenia during repeat dosing (with or without anti-CD52 drug).

[00130] In some embodiments, components of the lymphodepletion regimen fludarabine/cyclophosphamide (FC) or fludarabine/cyclophosphamide/anti-CD52 antibody (FCA) are administered simultaneously; in other embodiments, the components are administered sequentially. In some embodiments, the subject receives an FC regimen prior to the first dose of the CAR-T cell treatment agent and an FCA regimen prior to repeated doses of the CAR-T cell treatment agent. In some embodiments, the subject receives an FCA regimen prior to the first dose of the CAR-T cell treatment agent; and a second FCA regimen prior to re-dosing of the CAR-T cell treatment agent.

[00131] Exemplary schemes for LD are presented in tables 3A, 3B, 3C, 3D and 3E. In tables 3A-3E, the time of administration indicated in the graph refers to the time of dosing of CAR-T cells (D. 0) in days. Negative numbers indicate days before CAR-T cell administration (in D. 0).

[00132] In some embodiments, the LD regimen may further include treatment with Mensa (sodium 2-mercaptoethanesulfonate).

Premedication

[00133] In some embodiments, the methods may include premedication for an infusion-related reaction prior to a regimen for lymphodepletion. In some embodiments, the methods may include premedication administered prior to treatment with an anti-CD52 antibody. In some embodiments, premedication may involve treatment with high doses of corticosteroids. For example, premedication may involve treatment with 2 mg/kg methylprednisolone. In some embodiments, the premedication is administered immediately prior to the anti-CD52 antibody infusion. In some embodiments, the premedication is stopped at least one or at least two days prior to any infusion of CAR T cells (eg, UCART19).

[00134] In some embodiments, premedication may 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 hour before administration of an anti-CD52 antibody (eg, an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10). Acetaminophen can be administered, for example, about 1 day, about 1 hour, or about 0.5 hour before administration of an anti-CD52 antibody (eg, an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10).

Treatment

[00135] In some embodiments, CAR-T cells are administered via intravenous infusion. In some embodiments, the intravenous infusion is 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.

[00136] In some embodiments, a method of treating an adult subject who has refractory and/or recurrent acute lymphoblastic CD19+ B cell leukemia comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR -T-cells) containing CAR 4-1 BB/CD3-zeta for human CD19, where at least one dose is selected from the group consisting of approximately 6×10 ⁵ cells/dose, approximately 6×10 ⁶ cells/dose, approximately 6-8×10 ⁷ cells/dose and approximately 1.8-2.4×10 ⁸ cells/dose. In some embodiments, the CAR T cells express CAR under 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, CAR-T cells express a security switch, such as RQR8.

[00137] In some embodiments, a method of treating a pediatric subject who has refractory and/or recurrent acute lymphoblastic CD19+ B cell leukemia comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR- T-cells) containing CAR 4-1 BB/CD3-zeta for human CD 19, where at least one dose is approximately 2-8×10 ⁷ cells/dose. In some embodiments, the CAR T cells express CAR under 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, CAR-T cells express a security switch, such as RQR8.

[00138] In some embodiments, a method of treating a subject who has non-Hodgkin's lymphoma (e.g., refractory and/or recurrent large B-cell lymphoma or follicular lymphoma) comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor T cells ( CAR) (CAR-T cells) containing, for example, CAR 4-1 BB/CD3-zeta for human CD19, where at least one dose is from about 20x10 ⁶ cells/dose to about 360x10 ⁶ cells /dose. In some embodiments, at least one dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 40x10 ⁶ cells/dose, about 80x10 ⁶ cells/dose, about 120x10 ⁶ cells/dose , 240×10 ⁶ cells/dose and approximately 360×10 ⁶ cells/dose. In some embodiments, the CAR T cells express CAR under 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, CAR-T cells express a security switch, such as the RQR8 security switch described herein.

[00139] In some embodiments, CAR expression is detectable in a subject for a period of at least 14 days or at least 28 days after CAR administration. In some embodiments, the subject has a CR (complete response) or Cri (complete response with incomplete recovery of blood cells) 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 (eg, UCART19) administration.

Retreatment Methods with CAR-T Cells

[00140] Methods for re-treatment (redosing) with CAR-T cells are also provided herein. In particular, the methods include administering one or more subsequent doses of cells to subjects receiving the first dose and/or administering the first and one or more subsequent doses. As a rule, doses are administered in specific amounts and in accordance with specific time parameters. In some embodiments, the methods typically involve administering a first dose of cells, thereby reducing disease severity, followed by a subsequent dose of cells administered within a specific time window relative to the first dose, or administering a subsequent dose to a subject receiving such first dose. In some embodiments, further subsequent doses are then administered, for example, over the same or similar time period relative to the subsequent dose. In some embodiments, the number of cells administered and the time of administration of multiple doses are intended 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 cells administered, and/or improving therapeutic efficacy. Cell-containing articles for administration in accordance with such dosing schedules are also contemplated.

[00141] In some embodiments, the subject being treated with one or more subsequent doses of cells has a relapse. This means that the patient has a CR (complete response) or Cri (complete response with incomplete recovery of blood cells) at the first time point after the initial dose, but at a second, later time point, a relapse is observed. Relapse can be, for example, a morphological recurrence characterized by a bone marrow blast count greater than or equal to 5%, the presence of extramedullary disease associated with the presence of bone marrow blasts, or the presence of minimal residual disease (MRD), defined as the number of blasts in the bone marrow, greater than or equal to 3-10 blasts.

[00142] In some embodiments, a subject being treated with one or more subsequent doses of cells experiences a suboptimal response following the initial dose. For example, a subject may experience minimal residual disease (MRD) at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days after the initial dose.

[00143] In some embodiments, a subject being treated with one or more subsequent doses of cells exhibits variability in CAR persistence after an initial dose. For example, a subject may have CAR detectable after the initial dose (D.0, increase in CAR), but not have detectable CAR after D.0. The absence of detectable CAR in some embodiments occurs even though the subject has some signs of CAR-T cell activity (eg, cytokine release syndrome).

[00144] In some embodiments, the subject being treated with one or more of the subsequent doses of cells does not experience a significant increase in the number of CAR-T cells. In some embodiments, the subject is characterized by no detectable increase in CAR and no response at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days after the initial dose. In some embodiments, the subject is characterized by no detectable increase in CAR and some evidence of response to treatment (e.g., subject positive for MRD) at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days after administration of the initial dose. In some embodiments, the subject is characterized by no detectable increase in CAR and a complete response (e.g., MRD-negative subject) at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days after the initial dose.

[00145] In some embodiments, the methods provided include a subsequent dose of cells that is administered in approximately the same amount, and therefore the same dose, as the first dose of cells. As shown herein, administration of a subsequent dose of cells may be advantageous over administration of a single dose. In some embodiments, administering subsequent dose(s) of cells, e.g., about 6x10 ⁵ cells, about 6x10 ⁶ cells, about 6x10 ^{7 cells, about 8x10 7 cells} , about 1.8x10 ⁸ cells or approximately 2.4×10 ⁸ cells, is associated with increased overall survival in subjects, in particular in subjects who demonstrate morphological disease before treatment. In some embodiments, the methods include administering a first dose of cells that can proliferate in the presence of disease-associated antigens and provide a reduction in symptoms associated with the disease, but without the same degree of toxic outcomes that may be associated with a higher dose.

[00146] In some embodiments, the implementation of the first dose, as a rule, is also large enough to be effective in reducing the severity of the disease. In some cases, the first dose is large enough to lessen the severity of the disease if the cell dose is sufficient to expand in vivo and attenuate the disease. In some embodiments, the implementation of the first dose cells thus provide attenuation or reduction in the severity of the disease, such as tumor size, without causing severe adverse outcomes. In some instances, the first dose is the number of cells that is effective in reducing tumor burden, such as by attenuating the disease from a pathologic state to minimal residual disease (MRD) and/or clinical or complete remission. In some aspects, the first dose is a low dose. In some aspects, for example, in the context of relatively low disease severity, the first dose may be higher.

[00147] In some embodiments, a risk-adapted dosing schedule can be used to determine the appropriate number or number or relative number or number of cells or CAR-T cells per dose. For example, in some aspects, prior to infusion of CAR-T cells, the severity of the disease in a subject is determined and, based on the severity of the disease, a first dose of CAR-T cells (e.g., low or high dose) is selected that can minimize toxicity and maximize efficacy, e.g., Based on the observations described above and elsewhere in this document, in which higher doses of cells expressing recombinant receptors (e.g., CAR-expressing, such as CAR-expressing T cells) may be associated with toxic outcomes, such as severe neurotoxicity , in subjects characterized by a severity of morphological disease that is not necessarily observed or not observed to a significant extent, in subjects with a relatively lower severity of the disease. For example, subjects with a high pre-treatment bone marrow tumor burden, which in some cases may be associated with a greater increase in post-treatment CAR-T cells, were at higher risk of developing severe neurotoxicity that may require treatment in ICU.

[00148] In some embodiments, the subject is assessed for response after the first dose of allogeneic CAR-T cells. This assessment can be made 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 the first or previous dose of allogeneic CAR- T cells. In some embodiments, the subject is assessed for response at about one, about two, or about three months after the first dose of allogeneic CAR-T cells.

[00149] In some embodiments, expression of antigens targeted by CAR-Ts can be confirmed prior to administration of a subsequent dose of CAR-T cells. For example, antigen expression can 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 that the subject develops against previous CAR-T administration. In some embodiments, the subject can be assessed for the absence of anti-scFv antibodies that develop in the subject after a previous dose of CAR-T. In some embodiments, the implementation of the subject can be assessed for the absence of any significant safety problems after any course of CAR-T (first or subsequent courses). In some embodiments, a subject can be assessed for suitability for subsequent lymphodepletion associated with CAR-T dosing. In some embodiments, the absence of donor-specific anti-HLA, anti-scFv CAR, and/or anti-TALEN antibodies following dosing of CAR-T cells indicates suitability of the subject for a subsequent dose of CAR-T cells.

[00150] In some embodiments, the absence of persistence of CAR-T cells after a day from the first response assessment after administration of a dose of CAR-T cells indicates the suitability of the subject for a subsequent dose of CAR-T cells. In some embodiments, the subject may be assessed for lack of persistence of CAR-T cells after a day from the first response assessment following dosing of CAR-T cells. The assessment can be made depending on the 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 dosing , regardless of the graded answer. Parameters for evaluating kinetics include, for example, the AUCO time point of response evaluation, Cmax (peak increase in CAR-T transgene, time of last quantifiable transgene, and level of increase in CAR-T cells in any other tissues or fluids. Suboptimal response after the first administration of CAR-T, regardless of persistence of CAR-T, may include one or more of the following: (a) complete response (CR) or complete response with incomplete recovery of the number of formed elements (CRi) with detectable minimal residual disease (for example, in patients with leukemia); (b) no cytogenetic response; (c) complete bone marrow response; (d) partial response and/or (e) stable response.

[00151] In some embodiments, the subject can be assessed for the presence of an adverse event after a day from the first response assessment after a dose of CAR-T cells. Evaluation can 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 dosing. In some embodiments, the evaluation can be performed, for example, during the first four weeks (up to 28 days) after dosing CAR-T. Illustrative adverse events are shown in Table 4.

[00152] In some embodiments, disease relapse may refer to relapse after optimal CR is achieved following the first dose of CAR-T. In some embodiments, disease relapse may refer to relapse after one or more CRs are achieved with CAR-T administration, regardless of when the relapse occurs.

[00153] The next dose of CAR-T may be from the same or a different donor (donor of healthy cells used to obtain CAR-T). A subsequent dose may use the same or a different (higher or lower) dose of CAR-T cells.

[00154] CAR-T cells can be administered as a single dose or as a divided dose. For example, the dose may be divided into two or more doses over a period such as, for example, a seven day period. In some embodiments, the implementation of a fractional dose provides for the first and second administration during the period. In some embodiments, an equal number of cells are administered with each of the first and second injections. In some embodiments, fewer cells are injected during the first injection than during the second injection. In some embodiments, more cells are administered during the first injection than during the second injection. In some embodiments, the implementation of a fractional dose provides for the first, second administration and third administration during the period. In some embodiments, an equal number of cells are administered with each of the first, second, and third injections. In some embodiments, fewer cells are injected during the first injection than during the second and third injections. In some embodiments, more cells are administered during the first administration than during the second and third administrations. In some embodiments, fewer cells are injected during the second injection than during the first and third injections. In some embodiments, more cells are administered during the second administration than during the first and third administrations. In some embodiments, fewer cells are injected during the third injection than during the first and second injections. In some embodiments, more cells are administered during the third injection than during the first and second injections.

[00155] In some embodiments, the divided dose is administered during the first day and the second day. In some embodiments, half the dose is administered on the first day and half 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, the implementation of each partial dose of a divided dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after each other. In specific embodiments, each partial dose of the split dose is administered within 7 days of each other. In some embodiments, each partial dose is administered on a subsequent day.

[00156] In some embodiments, the implementation of a fractional dose is administered during the first day, the second day and the third day. 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, the implementation of each partial dose of a divided dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after each other. In specific embodiments, each partial dose of the split dose is administered within 7 days of each other. In some embodiments, each partial dose is administered on a subsequent day.

[00157] In some embodiments, the divided dose is administered over at least three days, such as at least four days, at least five days, at least six days, or at least seven days.

Dosing regimens

[00158] In some embodiments, allogeneic CAR-T cells are administered using a fixed dose. In other embodiments, allogeneic CAR-T cells are administered using dose-range binding. For example, linking doses to ranges can be used to avoid the risk of a wide range of CAR-T cell exposures. In some embodiments, weight binding may be used. For example, without limitation, subjects <66 kg may be dosed X, and subjects >66 kg may be dosed approximately 1.33X. In some embodiments, subjects >50 kg may be given one dose and subjects <50 kg may be given another dose.

[00159] Exemplary dose levels for the first dose of UCART19 are shown in Table 5a for use in adult patients with relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[00160] Exemplary dose levels for the first dose of UCART19 are shown in Table 5b for use in pediatric patients at high risk for relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia.

[00161] Exemplary dose forms for the first dose of UCART19 are shown in Table 5c for use in pediatric patients at high risk for relapsed/refractory CD19+ B cell acute lymphoblastic leukemia.

[00162] Exemplary doses for the first dose of UCART19 are shown in Table 5d for use in adult patients with non-Hodgkin's lymphoma, such as relapsed/refractory large B-cell and/or follicular lymphoma.

[00163] In some embodiments, an allogeneic CAR-T cell dosing regimen provides for administering a first dose of CAR-T cells from about 1 x 10 ⁵ to 5 x 10 ⁸ cells, or from about 1 x 10 ⁴ cells/kg to approximately 3×10 ⁶ cells/kg. In some embodiments, the allogeneic CAR-T cell dosing regimen provides for the administration of a first dose of CAR-T cells of approximately 6×10 ⁶ cells (fixed dose). In some embodiments, the allogeneic CAR-T cell dosing regimen comprises administering a first dose of about 1x10 ⁴ , about 2x10 ⁴ , about 3x10 ⁴ , about 4x10 ⁴ , about 5x10 ⁴ , about 6×10 ⁴ , approximately 7×10 ⁴ , approximately 8×10 ⁴ , approximately 9×10 ⁴ , approximately 10×10 ⁴ , approximately 1×10 ⁵ , approximately 2×10 ⁵ , approximately 3×10 ⁵ , approximately 4× 10 ⁵ , about 5x10 ⁵ , about 6x10 ⁵ , about 7x10 ⁵ , about 8x10 ⁵ , about 9x10 ⁵ , about 1x10 ⁶ , about 2x10 ⁶ , about 3x10 ⁶ , approximately 4×10 ⁶ , approximately 5×10 ⁶ , approximately 6×10 ⁶ , approximately 7×10 ⁶ , approximately 8×10 ⁶ , approximately 9×10 ⁶ , approximately 1×10 ⁷ , approximately 2×10 ⁷ 3×10 ₇ , approximately 4×10 ⁷ , approximately 5×10 ⁷ , approximately 6×10 ⁷ , approximately 7×10 ⁷ , approximately 8×10 ⁷ , approx. 9×10 ⁷ approx. 1×10 ⁸ approx. 2×10 ⁸ approx. 3× ^{10 8} approx. 4× ^{10 8} approx ^. ×10 ⁸ or approximately 9×10 ⁸ CAR-T cells. In some embodiments, the dosing schedule provides for the administration of a first dose of about 1x10 ⁴ cells, about 6x10 ⁶ cells, about 7x10 ⁶ cells (weight-bound), or about 1.8x10 ⁸ cells (weight-bound). weight). In some embodiments, the dosing schedule provides for a first dose of about 20x10 ⁶ cells/dose, about 40x10 ⁶ cells/dose, about 80x10 ⁶ cells/dose, about 120x10 ⁶ cells/dose, 240 ×10 ⁶ cells/dose or approximately 360×10 ⁶ cells/dose (binding by weight). In some embodiments, the dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 80x10 ⁶ cells/dose, and about 240x10 ⁶ cells/dose if the subject has a weight of 50 kg or less. . In some embodiments, the dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 40x10 ⁶ cells/dose, about 120x10 ⁶ cells/dose, and about 360x10 ⁶ cells/dose, wherein the subject has weight more than 50 kg.

[00164] In some embodiments, the dosage regimen provides for the administration of a subsequent dose of about 1x10 ⁴ , about 2x10 ⁴ , about 3x10 ⁴ , about 4x10 ⁴ , about 5x10 ⁴ , about 6x10 ⁴ , approximately 7×10 ⁴ , approximately 8×10 ⁴ , approximately 9×10 ₄ , approximately 10×10 ⁴ , approximately 1×10 ⁵ , approximately 2×10 ⁵ , approximately 3×10 ⁵ , approximately 4×10 ⁵ , approximately 5×10 ⁵ , approximately 6×10 ⁵ , approximately 7×10 ⁵ , approximately 8×10 ⁵ , approximately 9×10 ⁵ , approximately 1×10 ⁶ , approximately 2×10 ⁶ , approximately 3×10 ⁶ , approximately 4 ×10 ₆ , approx. 5× 10 ⁶ , approx. ⁶ ×10 ⁶ , approx. ⁷ × ^{10 6} , approx. 8×10 ⁶ , approx. ⁷ approx 4×10 ⁷ approx 5×10 ⁷ approx 6×10 ⁷ approx 7×10 ⁷ approx 8×10 ⁷ approx 9×10 ⁷ , approximately 1×10 ⁸ , approximately 2×10 ⁸ , approximately 3×10 ⁸ , approximately 4×10 ⁸ , approximately 5×10 ⁸ , approximately 6×10 ⁸ , approximately 7×10 ⁸ , approximately 8× 10 ⁸ or approximately 9×10 ⁸ CAR-T cells. In some embodiments, the dosing schedule provides for the administration of a subsequent dose of about 1x10 ⁴ cells, about 6x10 ⁶ cells, about 7x10 ₆ cells, or about 1.8x10 ⁸ cells. In some embodiments, the allogeneic CAR-T cell dosing regimen provides for the administration of a subsequent dose of CAR-T cells of approximately 6×10 ⁶ cells (fixed dose). In some embodiments, the dosing schedule provides for the administration of a subsequent dose of about 20x10 ⁶ cells/dose, about 40x10 ⁶ cells/dose, about 80x10 ⁶ cells/dose, about 120x10 ⁶ cells/dose, 240 ×10 ⁶ cells/dose or approximately 360×10 ⁶ cells/dose (binding by weight). In some embodiments, the dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 80x10 ⁶ cells/dose, and about 240x10 ⁶ cells/dose if the subject has a weight of 50 kg or less. . In some embodiments, the dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 40x10 ⁶ cells/dose, about 120x10 ⁶ cells/dose, and about 360x10 ⁶ cells/dose, wherein the subject has weight more than 50 kg.

[00165] In some embodiments, the allogeneic CAR-T cell dosing regimen includes a first dose of about 6x10 ⁶ CAR-T cells and a subsequent dose of about 6x10 ⁶ CAR-T cells. In some embodiments, the subsequent dose of CAR-T cells contains approximately the same number of cells as the previous dose of CAR-T cells. In some embodiments, the subsequent dose of CAR-T cells contains more cells than the previous dose of CAR-T cells. In some embodiments, the subsequent dose of CAR-T cells contains fewer cells than the previous dose of CAR-T cells.

[00166] In some embodiments, a subsequent dose of allogeneic CAR-T cells can be administered within a specific time window relative to the original or previous doses of allogeneic CAR-T cells. In some embodiments, a subsequent dose of allogeneic CAR-T cells is administered at approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 weeks after initial or previous doses of allogeneic CAR-T cells. In some embodiments, the subsequent dose of allogeneic CAR-T cells is administered in the range of 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 even 24 months after initial or previous doses of allogeneic CAR-T cells. In some embodiments, a subsequent dose of allogeneic CAR-T cells is administered at approximately 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, or 110 days after initial or previous doses of allogeneic CAR-T cells.

[00167] In some embodiments, the time between the first dose (initial dose), for example, the start of the first or previous dose, and the start of the next dose (repeated dose, for example, the start of the next dose) is more than about 4 weeks, for example, more than about 5, 6, 7, 8 or 9 weeks, for example, more than about 20 weeks, for example, from about 9 to about 35 weeks, from about 14 to about 28 weeks, from 15 to 27 weeks, or from 16 weeks to approximately 18 weeks; and/or is or is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 weeks. In some embodiments, the next dose (eg, start of administration) is administered more than about 5 weeks after the first or previous dose (eg, start of administration) and less than about 24 weeks thereafter. In some embodiments, the next dose is started 17 weeks after the start of the first dose. In some embodiments, the time between administration of the first and subsequent dose (e.g., onset of administration) or the previous and subsequent subsequent dose is from more than about 5 weeks to less than about 24 weeks, such as from 10 to 24 weeks, such as about 17 weeks. In some embodiments, the time between the first and subsequent doses (eg, start of administration) is approximately 17 weeks.

[00168] In some embodiments, the subject receives a repeat dose 28 days to 9 months after the first CAR-T infusion. The same regimen for lymphodepletion can be used as for the first CAR-T dosing, or the regimen can be adapted to the condition of the patient and the degree of recovery of the host's immune system.

[00169] In some embodiments, if the subject has a relapse of the disease after administration of a subsequent dose of allogeneic CAR-T cells, an additional subsequent dose of allogeneic CAR-T cells can be administered. In some embodiments, if the subject has a relapse of the disease after administration of a subsequent dose of allogeneic CAR-T cells and there is no persistence of CAR-T, an additional subsequent dose of allogeneic CAR-T cells can be administered. In some embodiments, two or more subsequent doses of allogeneic CAR-T cells may be administered after the first dose. Various post-dose schedules can be used to increase the duration of allogeneic CAR-T response. For example, subsequent doses of CAR-T can be administered, for example, one month later (after the first dose of CAR-T) or every 3 months. You can enter any number of subsequent infusions. The timing of dosing may be adapted to the health of the patient. An exemplary dosing schedule is shown in FIG. one.

[00170] In some embodiments, the dosing regimen provides for the administration of a first dose of approximately 6×10 ⁶ CAR-T cells, followed by a subsequent dose of approximately 6×10 ⁶ CAR-T cells administered approximately 99 days later after the first dose. In other embodiments, the dosing regimen is to administer a first dose of approximately 6x10 ⁶ CAR-T cells followed by a maintenance dose of approximately 6x10 ⁶ CAR-T cells every 4 weeks until molecular remission is achieved. In other embodiments, the dosing schedule provides for a first dose of approximately 6x10 ⁶ CAR-T cells followed by a maintenance dose of approximately 6x10 ⁶ CAR-T cells every 8 weeks until molecular remission is achieved.

[00171] In other embodiments, the dosing schedule provides for a bimonthly dose of approximately 6×10 ⁶ CAR-T cells.

In other embodiments, the implementation of the scheme of dosing provides for the introduction of a dose of approximately 6×10 ⁶ CAR-T cells.

[00172] However, other dosage regimens may be applicable, depending on the type of pharmacokinetic degradation that the practitioner wishes to achieve. The progress of such therapy is easily monitored using traditional methods and assays. In preferred embodiments, the first dose and the first subsequent and additional subsequent doses are separated in time from each other by at least four weeks. The dosage regimen can be changed over time.

[00173] Typically, for the introduction of allogeneic CAR-T cells, the initial potential dose can be from about 1.1 to about 2.3×10 ⁵ cells/kg of CAR-T cells. For the purpose of the present invention, a typical fixed dose of CAR-T cells could range from about 1x10 ⁴ to 1x10 ⁵ to 1x10 ⁶ to 1x10 ⁷ to 1x10 ⁷ to 1x 10 ⁸ cells or more, depending on the factors mentioned above. For example, a dose of approximately 0.3×10 ⁴ cells, approximately 0.5×10 ⁴ cells, approximately 1×10 ⁴ cells, approximately 1.5×10 ⁴ cells, approximately 2×10 ⁴ cells, approximately 2 .5×10 ⁴ cells, approximately 3×10 ⁴ cells, approximately 3.5×10 ⁴ cells, approximately 4×10 ⁴ cells, approximately 4.5×10 ⁴ cells, approximately 5×10 ⁴ cells, approximately 5.5 ×10 ⁴ cells, approximately 6×10 ⁴ cells, approximately 6.5×10 ⁴ cells, approximately 7×10 ⁴ cells, approximately 7.5×10 ⁴ cells, approximately 8×10 ⁴ cells, approximately 8.5×10 ⁴ cells, approximately 9×10 ⁴ cells, approximately 9.5×10 ⁴ cells, approximately 1×10 ⁵ cells, approximately 1.5×10 ⁵ cells, 2×10 ⁵ cells, approximately 2.5×10 ⁵ cells, approximately 3×10 ⁵ cells, approximately 3.5×10 ⁵ cells, approximately 4×10 ⁵ cells, approximately 4.5×10 ⁵ cells, approximately 5×10 ⁵ cells, approximately 5.5×10 ⁵ cells, approximately 6×10 ₅ cells, approx. 6.5×10 ⁵ cells, approximately 7×10 ⁵ cells, approximately 7.5×10 ⁵ cells, approximately 8×10 ⁵ cells, approximately 8.5×10 ⁵ cells, approximately 9×10 ₅ cells, approximately 9 .5×10 ⁵ cells, approximately 1×10 ⁶ cells, approximately 1.5×10 ⁶ cells, 2×10 ⁶ cells, approximately 2.5×10 ⁶ cells, approximately 3×10 ⁶ cells, approximately 3.5× ¹⁰⁶ cells, approximately 4× ¹⁰⁶ cells, approximately 4.5× ¹⁰⁶ cells, approximately 5× ¹⁰⁶ cells, approximately 5.5×106 cells, approximately ⁶ × ¹⁰⁶ cells, approximately ^6.5 ×106 cells cells, approximately 7×10 ⁶ cells, approximately 7.5×10 ⁶ cells, approximately 8×10 ⁶ cells, approximately 8.5×10 ⁶ cells, approximately 9×10 ⁶ cells, approximately 9.5×10 ⁶ cells, approximately 1×10 ⁷ cells, approximately 1.5×10 ⁷ cells, 2×10 ⁷ cells, approximately 2.5×10 ⁷ cells, approximately 3×10 ⁷ cells, approximately 3.5×10 ⁷ cells, approximately 4× 10 ⁷ cells, approx. 4.5×10 ⁷ cells, approx. o 5×10 ⁷ cells, approximately 5.5×10 ⁷ cells, approximately 6×10 ⁷ cells, approximately 6.5×10 ⁷ cells, approximately 7×10 ⁷ cells, approximately 7.5×10 ⁷ cells, approximately 8 ×10 ⁷ cells, approximately 8.5×10 ⁷ cells, approximately 9×10 ⁷ cells, approximately 9.5×10 ⁷ cells, approximately 1×10 ⁸ cells, approximately 1.5×10 ⁸ cells, 2×10 ⁸ cells, approximately 2.5×10 ⁸ cells, approximately 3×10 ⁸ cells, approximately 3.5×10 ⁸ cells, approximately 4×10 ⁸ cells, approximately 4.5×10 ⁸ cells, approximately 5×10 ⁸ cells, approximately 5.5×10 ⁸ cells, approximately 6×10 ⁸ cells, approximately 6.5×10 ⁸ cells, approximately 7×10 ⁸ cells, approximately 7.5×10 ⁸ cells, approximately 8×10 ⁸ cells, approximately 8 .5×10 ⁸ cells, approximately 9×10 ⁸ cells, approximately 9.5×10 ⁸ cells, or approximately 1×10 ⁹ cells or more. For subsequent administrations of several weeks or longer, depending on the disease, treatment is continued until the desired suppression of symptoms occurs or until sufficient therapeutic levels, such as molecular remission, are achieved.

[00174] Exemplary dosing regimens include administering a first dose of approximately 0.3×10 ⁴ cells, approximately 0.5×10 ⁴ cells, approximately 1×10 ⁴ cells, approximately 1.5×10 ⁴ cells, approximately 2× 10 ⁴ cells, approximately 2.5×10 ⁴ cells, approximately 3×10 ⁴ cells, approximately 3.5×10 ⁴ cells, approximately 4×10 ⁴ cells, approximately 4.5×10 ⁴ cells, approximately 5×10 ⁴ cells, approximately 5.5×10 ⁴ cells, approximately 6×10 ⁴ cells, approximately 6.5×10 ⁴ cells, approximately 7×10 ⁴ cells, approximately 7.5×10 ⁴ cells, approximately 8×10 ⁴ cells, about 8.5x10 ⁴ cells, about 9x10 ⁴ cells, about 9.5x10 ⁴ cells, about 1x10 ⁵ cells, about 1.5x10 ⁵ cells, 2x10 ⁵ cells, about 2, 5×10 ⁵ cells, approximately 3×10 ⁵ cells, approximately 3.5×10 ⁵ cells, approximately 4×10 ⁵ cells, approximately 4.5×10 ⁵ cells, approximately 5×10 ⁵ cells, approximately 5.5× ten ⁵ cells, approximately 6×10 ⁵ cells, approximately 6.5×10 ⁵ cells, approximately 7×10 ⁵ cells, approximately 7.5×10 ⁵ cells, approximately 8×10 ⁵ cells, approximately 8.5×10 ⁵ cells , approximately 9×10 ⁵ cells, approximately 9.5×10 ⁵ cells, approximately 1×10 ⁶ cells, approximately 1.5×10 ⁶ cells, 2×10 ⁶ cells, approximately 2.5×10 ⁶ cells, approximately 3 ×10 ⁶ cells, approximately 3.5×10 ⁶ cells, approximately 4×10 ⁶ cells, approximately 4.5×10 ⁶ cells, approximately 5×10 ⁶ cells, approximately 5.5×10 ⁶ cells, approximately 6×10 ⁶ cells, approximately 6.5×10 ⁶ cells, approximately 7×10 ⁶ cells, approximately 7.5×10 ⁶ cells, approximately 8×10 ⁶ cells, approximately 8.5×10 ⁶ cells, approximately 9×10 ⁶ cells , approximately 9.5×10 ⁶ cells, approximately 1×10 ⁷ cells, approximately 1.5×10 ⁷ cells, 2×10 ⁷ cells, approximately 2.5×10 ⁷ cells, approximately 3×10 ⁷ cells, approximately 3 .5×10 ⁷ cells, approximately 4×10 ⁷ cells , approximately 4.5×10 ⁷ cells, approximately 5×10 ⁷ cells, approximately 5.5×10 ⁷ cells, approximately 6×10 ⁷ cells, approximately 6.5×10 ⁷ cells, approximately 7×10 ⁷ cells, approximately 7.5×10 ⁷ cells, approximately 8×10 ⁷ cells, approximately 8.5×10 ⁷ cells, approximately 9×10 ₇ cells, approximately 9.5×10 ⁷ cells, approximately 1×10 ⁸ cells, approximately 1, 5×10 ⁸ cells, approximately 2×10 ₈ cells, approximately 2.5×10 ⁸ cells, approximately 3×10 ⁸ cells, approximately 3.5×10 ₈ cells, approximately 4×10 ⁸ cells, approximately 4.5× 10 ⁸ cells, approximately 5×10 ₈ cells, approximately 5.5×10 ⁸ cells, approximately 6×10 ⁸ cells, approximately 6.5×10 ⁸ cells, approximately 7×10 ⁸ cells, approximately 7.5×10 ⁸ cells, approximately 8×10 ⁸ cells, approximately 8.5×10 ⁸ cells, approximately 9×10 ⁸ cells, approximately 9.5×10 ⁸ cells, or approximately 1×10 ⁹ cells of allogeneic CAR-T cells. In some embodiments, the subsequent dose is administered monthly. In some embodiments, the subsequent dose is administered one month later. In some embodiments, a subsequent dose is administered approximately every three months. In some embodiments, a subsequent dose is administered approximately every four months. In some embodiments, a subsequent dose is administered approximately every five months. In some embodiments, a subsequent dose is administered approximately every six months. In some embodiments, a subsequent dose is administered approximately every seven months. In some embodiments, a subsequent dose is administered approximately every eight months. In preferred embodiments, the first dose and the first subsequent and additional subsequent doses are separated in time from each other by at least about four weeks. In some embodiments, the subsequent dose is administered one month later.

[00175] In some embodiments, the dosing regimen provides for administering to the subject a first dose of approximately 6x10 ⁶ CAR T cells per D. 0 and a subsequent dose of approximately 6x10 ⁶ CAR T cells from approximately D. 30 to about D. 110. In some embodiments, the dosing schedule provides for administering to the subject a first dose of about 1 x 10 ⁶ CAR-T cells at D. 0 and a subsequent dose of about 6 x 10 ⁶ CAR-T -cells from about D. 30 to about D. 110. In some embodiments, the implementation of the dosing regimen provides for the introduction to the subject of a first dose of approximately 2x10 ⁶ CAR-T cells in D. 0, and a subsequent dose of approximately 6x 10 ⁶ CAR-T cells from about D. 30 to about D. 110. In some embodiments, the implementation of the dosing regimen provides the subject with a first dose of approximately 3x10 ⁶ CAR-T cells in D. 0, and thereafter a progressive dose of about ^{6x10 6} CAR T cells from about D.30 to about D.110. D. 0, followed by a dose of approximately 6×10 ⁶ CAR T cells from approximately D. 30 to approximately D. 110. In some embodiments, the dosing regimen provides for administering to the subject a first dose of approximately 5×10 ⁶ CAR -T cells at D.0, and a subsequent dose of approximately 6×10 ⁶ CAR-T cells from approximately D.30 to approximately D.110. 7×10 ⁶ CAR-T cells in D.0, followed by a dose of about 6×10 ⁶ CAR-T cells from about D.30 to about D.110. considers administering to the subject a first dose of approximately 8×10 ⁶ CAR-T cells at D.0 and a subsequent dose of approximately 6×10 ⁶ CAR-T cells from approximately D.30 to approximately D.110. In some embodiments, the dosing schedule provides for administering to the subject a first dose of about 1 x 10 ⁶ CAR T cells in D. 0 and a subsequent dose of about 9 x 10 ⁶ CAR T cells from about D. 30 to about D 110. In some embodiments, the dosing regimen provides for administering to the subject a first dose of approximately 1x10 ⁷ CAR T cells per D. 0 and a subsequent dose of approximately 6x10 ⁶ CAR T cells from approximately D. .30 to approximately D.110.

[00176] In some embodiments, the dosing schedule provides for administering to the subject a regimen for lymphodepletion at D. -7 to D. -2, followed by a first dose of approximately 6×10 ⁶ CAR-T cells per D. 0 followed by an optional regimen for lymphodepletion from D. 60 to D. 100, followed by a subsequent dose of 6×10 ⁶ CAR-T cells from D. 63 to D. 104. In some embodiments, implementation the dosing schedule is to administer to the subject a regimen for lymphodepletion at D.-7 to D.-2, followed by a first dose of approximately 6×10 ⁶ CAR-T cells at D. 0, followed by an optional regimen for lymphodepletion from D. 60 to D. 90, followed by a subsequent dose of 6x10 ⁶ CAR-T cells from D. 63 to D. 104. for lymphodepletion (LD) at D. -7 followed by the first dose, consisting approximately 6×10 ⁶ CAR-T cells at D. 0, followed by a reduced-intensity LD (compared to previous LD) at D. 70, followed by a subsequent dose of 6×10 ⁶ CAR-T cells at D. 99. In some embodiments, the dosing regimen comprises administering to the subject a regimen for lymphodepletion at D. -15, followed by a first dose of approximately 6×10 ⁶ CAR-T cells at D. 0, followed by the regimen for lymphodepletion at D. 60, followed by a subsequent dose of 6×10 ⁶ CAR-T cells at D. 75. In some embodiments, the dosing regimen provides for the administration of the regimen for lymphodepletion to the subject from D. -7 to D. .-2 followed by a first dose of approximately 6×10 ⁶ CAR-T cells at D.0 followed by an optional regimen for lymphodepletion between D.60 and D.90 followed by a subsequent dose, component 6×10 ⁶ CAR-T cells in the period from D. 63 to D. 104. In some embodiments, The dosing regimen involves administering to the subject a regimen for lymphodepletion at D.-7 to D.-2, followed by a first dose of approximately 6×10 ⁶ CAR-T cells at D. 0, followed by an optional regimen for lymphodepletion from D. 60 to D. 90, followed by a subsequent dose of 6×10 ⁶ CAR-T cells from D. 63 to D. 104.

[00177] In some embodiments, the dosing regimen comprises administering a regimen for lymphodepletion between D. -5 and D. -3, followed by a first dose of about 20 x 10 ⁶ cells, about 40 x 10 ⁶ cells, about 80×10 ⁶ cells, approximately 120×10 ⁶ cells, approximately 240×10 ⁶ cells, or approximately 360×10 ⁶ cells, followed by a subsequent dose of CAR-T cells. In some embodiments, the lymphodepletion regimen comprises fludarabine administered at a dose of approximately 30 mg/m ² ; cyclophosphamide administered at a dose of approximately 300 mg/m ² ; and optionally an anti-CD52 antibody (eg, 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.

[00178] However, other dosage regimens may be applicable, depending on the type of pharmacokinetic degradation that the practitioner wishes to achieve. The progress of such therapy is easily monitored using traditional methods and assays. In some embodiments, the first dose and the first subsequent and additional subsequent doses are separated in time from each other by at least four weeks. The dosage regimen can be changed over time.

Allogeneic CAR-T cells and supplements

[00179] The following is a description of exemplary CAR-T cells used in accordance with the present invention.

[00180] The methods include administering CAR-T cells that specifically bind to an antigen associated with a disease and result in a response, such as an immune response against such molecules when bound to such an antigen. Any allogeneic CAR-T cell can be used in the methods described herein unless a particular method is specifically associated with a particular type of allogeneic CAR-T cell (eg, UCART19 cells). In some embodiments, the methods and compositions of the present invention are practiced using an allogeneic CAR-T cell specific for a tumor-specific antigen.

[00181] In some embodiments, the methods and compositions of the present invention are practiced using an allogeneic CAR-T cell specific for CD 19. In some embodiments, the CAR-T cell expresses a CAR containing a CD 19 binding domain. in some embodiments, the CD 19 binding domain contains a single chain variable fragment (scFv). In some embodiments, the scFv is derived from an anti-CD 19 antibody (eg, 4G7 antibody). In some embodiments, the scFv contains VH and VL isolated or derived from an anti-CD 19 antibody (eg, 4G7 antibody). In some embodiments, the scFv comprises HCDR1, HCDR2, HCDR3, LCDR1, LDCR2, and LCDR3 isolated or derived from an anti-CD 19 antibody (eg, 4G7 antibody).

[00182] In some embodiments, the CAR T cell expresses a CAR containing a CD 19 binding domain, a 4-1 BB domain, and/or a CD3 zeta domain.

[00183] In some embodiments, the CAR-T cell additionally expresses a "safety switch". The security switches may contain an epitope that allows selection of transduced cells and an epitope that allows detection and/or removal of cells expressing the polypeptide. In some embodiments, the security switch comprises a mimotope. Exemplary security switches are disclosed, for example, in WO 2013/153391, which is incorporated herein by reference in its entirety. In some embodiments, the safety switch is associated with rituximab. In some embodiments, the security switch is expressed in trans with the CAR (eg, as a separate polypeptide in the same cell). In some embodiments, the security switch is RQR8. In some embodiments, the RQR8 containing the security switch is expressed in trans. Full length and component sequences for RQR8 are shown in Table 6 below.

[00184] In some embodiments, the security switch is R2.

[00185] In some embodiments, CAR-T cells do not contain a security switch.

[00186] In some embodiments, the security switch encoding sequence is provided in the same vector containing the CAR encoding sequence. In some embodiments, the security switch encoding sequence is provided in a vector other than the vector that contains the CAR encoding sequence.

[00187] In some embodiments, the sequence encoding the security switch is provided in the same polypeptide that contains the CAR (i.e., provided in the cis position). In some embodiments, the security switch is provided in a polypeptide other than CAR (ie, provided in trans).

[00188] Accordingly, in some embodiments, the security switch is expressed in trans with a CAR (eg, as a separate polypeptide in the same cell). In some embodiments, the security switch is RQR8. In some embodiments, the RQR8 containing the security switch is expressed in trans.

[00189] In some embodiments, the security switch is expressed in cis relative to CAR and contains an antibody specific mimotope as described in WO 2013/153391.

[00190] In some embodiments, the CAR-T cell further comprises an inactivated CD52 gene. In some embodiments, the CAR-T cell is CD52 deficient. In some embodiments, the CAR-T cells to be administered to a patient contain a mixture of CD52-deficient and CD52-positive cells.

[00191] In some embodiments, the composition of CAR-T cells for administration to a patient includes a mixture of cells with different genotypes. In some embodiments, the composition comprises CAR-T cells, where 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, where 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 cells have an inactivated TRAC gene. In some embodiments, the composition comprises CAR-T cells, where 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, and additionally, where 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 cells have an inactivated TRAC gene.

[00192] In some embodiments, the CAR-T cells are UCART19 CAR-T cells. UCART19 is an allogeneic engineered human T cell drug being developed for the treatment of CD19-expressing B-cell leukemias, including acute lymphoblastic leukemia in youth and adolescents, adults and children (B-ALL). Published information related to UCART19 includes: 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 patent US Publication No. 14/891296 published May 2, 2016 as US 20160145337, each of which is incorporated herein by reference in their entirety. UCART19 mediates the expression of the CAR directed against human CD 19, together with the RQR8 security switch, combining CD34 and CD20 antigen epitopes. UCART19 contains the CAR 4-1BB/CD3-zeta for human CD19, consisting of one or more of the peptides shown in Table 7 and/or as described in US Patent Application Publication No. 2016/0145337, which is incorporated herein by reference in its full extent.

[00193] UCART19 was further subjected to simultaneous destruction of the TRAC and CD52 genes, followed by depletion of the remaining TCRαβ+ cells. UCART19 contains a single chain variable fragment (scFv) derived from the mouse anti-human CD 19 hybridoma 4G7, a CD8 hinge and a transmembrane domain, a BB 4-1 costimulatory domain, and a CD3 zeta signaling domain. Drug substance (DS) UCART19 is defined as allogeneic genetically modified CD19CAR/RQR8+_TCRαβ-_T cells: (i) containing an integrated self-inactivating (SIN) recombinant lentiviral delivery vector that expresses the CAR for CD 19 to redirect T cells to tumor cells CD19+ and their elimination, (ii) expressing the RQR8 safety switch conferring rituximab susceptibility, (iii) which are TCRαβ negative due to disruption of the TRAC gene using mRNA-based TALEN® gene editing followed by depletion of remaining TCRαβ+ cells during production process and (iv) which are a mixture of CD52-knockout and CD52-positive cells due to disruption of the CD52 gene using mRNA-based TALEN® gene editing to allow administration of UCART19 to patients previously treated or treated with an anti-CD52 antibody (for example, an antibody containing the sequence under SEQ ID NO: 8 and/or SEQ ID NO: 10).

[00194] Exemplary procedures for obtaining UCART19 are shown in FIG. 4A and FIG. 4 V.

[00195] As used herein, the term UCART19 (CD19CAR/RQR8+_TCRαβ-_T cells) refers to UCART19 cells expressing CAR CD 19 and the RQR8 security switch. The cells also contain an inactivated TRAC gene. Optionally, the cells contain an inactivated CD52 gene.

[00196] As used herein, the term UCART19 (CD19CAR/R2+_TCRαβ-_T cells) refers to UCART19 cells expressing CAR CD 19 and the R2 security switch. The cells also contain an inactivated TRAC gene. Optionally, the cells contain an inactivated CD52 gene.

[00197] As used herein, the term UCART19 (CD19CAR/TCRαβ T cells) refers to UCART19 cells expressing CAR CD 19. The cells also contain an inactivated TRAC gene. Optionally, the cells contain an inactivated CD52 gene.

[00198] In some embodiments, the CAR-T cells are allogeneic CAR-T cells expressing the amino acid sequence shown under SEQ ID NO: 1.

[00199] For all methods described herein, reference to CAR-T cell compositions also includes compositions containing one or more additional agents. These compositions may additionally contain suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art. The compositions and methods disclosed herein may be used alone or in combination with other conventional therapies.

[00200] CAR-T cells can be administered to a subject via any suitable route. One of skill in the art should appreciate that the examples described herein are not meant to be limiting, but to illustrate available techniques. Accordingly, in some embodiments, CAR-T cells are administered to a subject according to known methods, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intracerebrospinal, transdermal subcutaneous, intraarticular , sublingual, intrasynovial, via insufflation, intrathecal, oral, inhalation or topical routes of administration. Administration may be systemic, eg intravenous, or localized. Commercially available nebulizers for liquid formulations are suitable for administration, including jet nebulizers and ultrasonic nebulizers. Liquid formulations can be sprayed directly, and lyophilized powder can be sprayed after reconstitution.

[00201] Various formulations of CAR-T cells can be used for administration. In some embodiments, the CAR-T cells and the pharmaceutically acceptable excipient may be in different formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of a pharmacologically effective substance. For example, an excipient may impart shape or texture or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.

[00202] These agents can be combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, and the like. The specific dosing regimen, ie. the dose, time of administration and repetition will depend on the particular individual and his medical history.

[00203] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations employed, and may include buffers such as those based on phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechin; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; 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 (eg Zn-protein complexes) and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutical kits and products

[00204] Pharmaceutical kits are also provided for the treatment of patients suffering from a disease such as cancer. In some embodiments, the pharmaceutical kit contains CAR-T cells and an anti-CD52 antibody. In some embodiments, the pharmaceutical kit contains UCART19 cells and an antibody. In some embodiments, the pharmaceutical kit comprises a first container containing CAR-T cells (e.g., UCART19 cells) and a second container containing an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO :10) In some embodiments, CAR-T cells (eg, UCART 19 cells) are CD52-deficient. In some embodiments, the kit contains multiple containers containing CAR-T cells, and the number of CAR-T cells in each container is the same or different. In some embodiments, the first and/or second containers are flexible cell infusion bags. In some embodiments, the first and/or second containers are vials or vials (eg, glass or plastic vials or vials). The pharmaceutical kit may further comprise a label or package insert containing instructions for administering the CAR-T cells and the anti-CD52 antibody to the subject.

[00205] Also provided are articles containing a number of resealable containers, each of which individually contains a unit dose of allogeneic chimeric antigen receptor (CAR) T cells for administration to a subject, said unit dose being from about 1×10 ⁶ to about 5×10 ⁸ cells; packaging material and a label or package insert containing instructions for administering to a subject a number of said unit doses by making a first administration and a subsequent administration, said first administration comprising delivering to the subject one of said unit doses, and said subsequent administration comprising administering to the subject one or more the number of specified standard doses. In some embodiments, the articles indicate that said subsequent administration is to be from about 30 to 150 days, optionally about 30 days, about 60 days, about 90 days, or about 99 days, after said first administration. In some embodiments, the containers are or contain flexible bags for cell infusion. In some embodiments, the containers are or contain vials or vials (eg, glass or plastic vials or vials).

NUMBERED EMBODIMENTS OF THE PRESENT INVENTION

[00206] The present invention and the inventions described herein may be described with reference to the following numbered illustrative embodiments.

[00207] 1. A method of treating a subject who has refractory and/or recurrent non-Hodgkin's lymphoma, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) containing CAR 4 -1 BB/CD3-zeta for CD 19 human, where at least one dose is from about 20×10 ⁶ cells/dose to about 360×10 ⁶ cells/dose.

[00208] 2. The method of Embodiment 1 wherein the non-Hodgkin's lymphoma is large B cell lymphoma.

[00209] 3. The method of Embodiment 1, wherein the non-Hodgkin's lymphoma is follicular lymphoma.

[00210] 4. The method according to any one of embodiments 1-3, wherein at least one dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 40x10 ⁶ cells/dose, about 80x10 ⁶ cells/dose, approximately 120×10 ⁶ cells/dose, approximately 240×10 ⁶ cells/dose and approximately 360×10 ⁶ cells/dose.

[00211] 5. The method according to any one of embodiments 1-4, wherein at least one dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 80x10 ⁶ cells/dose, and about 240x10 ⁶ cells/dose if the subject has a weight of 50 kg or less.

[00212] 6. The method according to any one of embodiments 1-4, wherein at least one dose is selected from the group consisting of about 20x10 ⁶ cells/dose, about 40x10 ⁶ cells/dose, about 120x10 ⁶ cells/dose and approximately 360×10 ⁶ cells/dose, where the subject has a weight of more than 50 kg.

[00213] 7. The method according to any one of embodiments 1-6, wherein the CAR-T cells are CD52-deficient.

[00214] 8. The method according to any one of embodiments 1-6, wherein the CAR-T cells comprise a mixture of CD52-deficient and CD-52-positive cells.

[00215] 9. The method according to any one of embodiments 1-8, wherein the CAR-T cells contain the CAR of SEQ ID NO: 1.

[00216] 10. The method according to any one of embodiments 1-9, wherein the CAR-T cells comprise UCART19(CD19)CAR/RQR8+_TCRαβ-_T cells.

[00217] 11. The method according to any one of embodiments 1-9, wherein the CAR-T cells do not express a security switch.

[00218] 12. The method according to any one of embodiments 1-11, wherein CAR expression is detectable in the subject for a period of at least 14 days following administration of CAR-T cells.

[00219] 13. The method according to any one of embodiments 1-12, wherein CAR expression is detectable in the subject for a period of at least 28 days following administration of CAR-T cells.

[00220] 14. The method according to any one of embodiments 1-13, wherein the subject has a CR or Cri state for at least 1 month following CAR-T administration.

[00221] 15. The method according to any one of embodiments 1-14, wherein the subject has a CR or Cri state for at least 2 months after administration of CAR-T.

[00222] 16. The method according to any one of embodiments 1-15, wherein the subject has a CR or Cri state for at least 6 months after CAR-T administration.

[00223] 17. The method according to any one of embodiments 1-16, wherein the subject has a CR or Cri state for at least 12 months after administration of CAR-T.

[00224] 18. The method according to any one of embodiments 1-17, wherein the subject receives the first regimen for lymphodepletion prior to at least one dose.

[00225] 19. The method of Embodiment 18, wherein the first regimen for lymphodepletion comprises administering fludarabine and cyclophosphamide.

[00226] 20. The method of Embodiment 18, wherein the first regimen for lymphodepletion comprises administering fludarabine, cyclophosphamide, and an anti-CD52 antibody.

[00227] 21. The method of embodiment 19 or 20 wherein the first regimen for lymphodepletion further comprises administering Mensa (sodium 2-mercaptoethanesulfonate).

[00228] 22. The method according to any one of embodiments 19-21, wherein the first regimen for lymphodepletion further comprises administering at least one corticosteroid.

[00229] 23. The method according to embodiment 22, wherein the corticosteroid is administered immediately prior to administration of the anti-CD52 antibody.

[00230] 24. The method according to embodiment 22 or 23, wherein the corticosteroid is methylprednisolone.

[00231] 25. The method according to embodiment 24 wherein methylprednisolone is administered at a dose of 2 mg/kg.

[00232] 26. The method according to any one of embodiments 1-25, wherein the patient is premedicated for an infusion-related reaction prior to the regimen for lymphodepletion.

[00233] 27. The method of Embodiment 26, wherein the premedication comprises at least one antihistamine.

[00234] 28. The method according to embodiment 26 or 27, wherein the premedication comprises administering acetaminophen.

[00235] 29. The method according to any one of embodiments 19-28, wherein fludarabine is administered at a dose of approximately 30 mg/m ² /day; cyclophosphamide is administered at a dose of about 300 mg/m ² /day, or an anti-CD52 antibody is administered at a dose of about 10 to about 13 mg/day.

[00236] 30. The method according to any one of embodiments 20-28, wherein fludarabine is administered at a dose of approximately 30 mg/m ² /day; cyclophosphamide is administered at a dose of about 300 mg/m ² /day, and anti-CD52 antibody is administered at a dose of about 10 to about 13 mg/day.

[00237] 31. The method of any one of embodiments 18-30, wherein the first regimen for lymphodepletion is started about 1-15 days prior to at least one dose.

[00238] 32. The method according to any one of embodiments 18-31, wherein the first regimen for lymphodepletion is administered over a course of 1, 2, 3, 4, or 5 days.

[00239] 33. The method according to any one of embodiments 1-32, wherein the subject receives a subsequent dose of CAR-T cells.

[00240] 34. The method of embodiment 33, wherein the subject has a suboptimal response at the time of the next dose.

[00241] 35. The method of embodiment 34, wherein the suboptimal response comprises:

(a) complete response (CR), complete response with incomplete recovery of blood cells (CRi) with detectable minimal residual disease (patients with leukemia) and no cytogenetic response;

(b) complete bone marrow response;

(c) a partial response, or

(d) stable response.

[00242] 36. The method according to any one of embodiments 33-35, wherein the subsequent dose contains approximately the same number of cells as the number of cells in the first dose.

[00243] 37. The method according to any one of embodiments 33-35, wherein the subsequent dose contains an increased number of cells compared to the first dose.

[00244] 38. The method according to any one of embodiments 33-35, wherein the subsequent dose contains a reduced number of cells compared to the first dose.

[00245] 39. The method according to 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.

[00246] 40. A pharmaceutical kit for treating a patient suffering from cancer, the kit comprising:

(a) T cells with CAR for CD 19 and

(b) anti-CD52 antibody.

[00247] 41. Pharmaceutical kit according to embodiment 40, wherein the CAR-T cells are UCART19 cells.

[00248] 42. Pharmaceutical kit according to embodiment 40 or 41, wherein the CAR-T cells express the CAR of SEQ ID NO: 1.

[00249] 43. A pharmaceutical kit according to any one of embodiments 40-42, wherein the anti-CD52 antibody comprises the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10.

[00250] 44. A pharmaceutical kit according to any one of embodiments 40-43, wherein the kit comprises a first container containing CAR-T cells and a second container containing an anti-CD52 antibody.

[00251] 45. The pharmaceutical kit of Embodiment 44, wherein at least one of the first and second containers is a flexible cell infusion bag.

[00252] 46. A pharmaceutical kit according to any one of embodiments 40-45, wherein the kit further comprises a label or package insert containing instructions for administering CAR-T cells and an anti-CD52 antibody to a subject.

[00253] 47. A method of treatment, comprising:

(a) administering to the subject having the disease a first dose of chimeric antigen receptor (CAR) T cells (CAR-T cells), and

(b) administering to the subject a subsequent dose of CAR-T cells at a time point that is at least or more than about 28 days after the start of administration in (a) to less than about 200 days thereafter.

[00254] 48. The method of embodiment 47 wherein the CAR-T cells are allogeneic.

[00255] 49. The method according to embodiment 47 or 48, wherein the first dose contains from about 1x10 ⁴ to about 5x10 ⁸ total cells.

[00256] 50. The method of Embodiment 49, wherein the first dose contains about 6x10 ⁵ total cells, about 6x10 ⁶ total cells, about 6x10 ⁷ total cells, about 8x10 ⁷ total cells , approximately 1.8×10 ⁸ total cells, approximately 2.4×10 ⁸ total cells, or approximately 5×10 ⁸ total cells.

[00257] 51. The method according to embodiment 47 or 48, wherein the first dose contains 1×10 ⁴ to 2×10 ⁷ cells per kilogram of the subject's body weight.

[00258] 52. The method according to embodiment 51, wherein the first dose contains about 1x10 ⁴ , about 1x10 ⁵ , about 1x10 ⁶ , about 3x10 ⁶ , or about 9x10 ⁶ cells per kilogram of body weight of the subject .

[00259] 53. The method according to any one of embodiments 47-52, wherein the next dose of CAR-T cells is administered at about 28 days, about 60 days, or about 99 days after the start of said administration according to (a).

[00260] 54. The method according to embodiment 53, wherein the subsequent dose of CAR-T cells is administered about 99 days after the start of said administration according to (a).

[00261] 55. The method according to any one of embodiments 47-54, wherein after step (a) and before step (b), the subject is administered an intermediate regimen for lymphodepletion.

[00262] 56. The method according to any one of embodiments 47-55, wherein the intermediate regimen for lymphodepletion comprises administering to the subject fludarabine, cyclophosphamide, and an anti-CD52 antibody for about 0 to 14 days prior to step (b).

[00263] 57. The method of Embodiment 56 wherein fludarabine is administered at a dose of about 90 to 150 mg/m ² ; cyclophosphamide is administered at a dose of about 1000 to 4000 mg/m ² , and anti-CD52 antibody is administered at a dose of about 0.3 to 1 mg/kg.

[00264] 58. The method according to any one of embodiments 47-57, wherein the subject is administered the first regimen for lymphodepletion.

[00265] 59. The method of embodiment 47, wherein the first regimen for lymphodepletion comprises administering fludarabine, cyclophosphamide, and an anti-CD52 antibody to the subject.

[00266] 60. The method of Embodiment 59 wherein fludarabine is administered at a dose of about 90 to 150 mg/m ² ; cyclophosphamide is administered at a dose of about 1000 to 4000 mg/m ² , and anti-CD52 antibody is administered at a dose of about 0.3 to 1 mg/kg.

[00267] 61. The method according to any one of embodiments 58-60, wherein the first regimen for lymphodepletion is started approximately 15-5 days prior to step (a).

[00268] 62. The method according to any one of embodiments 58-60, wherein the first regimen for lymphodepletion is completed approximately 2-10 days prior to step (a).

[00269] 63. The method according to any one of embodiments 47-62, wherein the response is evaluated between step (a) and step (b).

[00270] 64. The method according to embodiment 63, wherein the response is assessed prior to intermediate lymphodepletion.

[00271] 65. The method according to any one of embodiments 47-64, wherein the subsequent dose contains from about 1x10 ⁵ to about 5x10 ⁸ total cells.

[00272] 66. The method according to any one of embodiments 47-65, wherein the subsequent dose contains approximately 6×10 ⁶ total cells.

[00273] 67. The method according to any one of embodiments 47-66, wherein administration according to (a) results in alleviation of the disease in the subject, as indicated by the reduction of one or more symptoms of the disease after said in (a) administration.

[00274] 68. The method of Embodiment 67, wherein the subject has relapsed at the time of administration according to (b).

[00275] 69. The method according to any one of embodiments 47-68, wherein the subsequent dose of cells contains cells in an amount sufficient to alleviate the disease in the subject.

[00276] 70. The method according to any one of embodiments 47-69, wherein administration according to (b) results in further alleviation of the disease in the subject.

[00277] 71. The method according to any one of embodiments 47-70, wherein administration of the subsequent dose results in improvement of the disease in the subject as compared to the state immediately prior to the commencement of the subsequent dose.

[00278] 72. The method according to any one of embodiments 47-71, wherein the method results in relief of the disease to a greater extent and/or for a longer period of time compared to the method comprising an alternative dosing schedule, where the subject is administered cells according to ( a) and cells according to (b) in a single dose.

[00279] 73. The method of any one of embodiments 47-72, wherein the disease persists after the first dose is administered and/or the first dose is insufficient to eliminate the disease in the subject.

[00280] 74. The method of any one of embodiments 47-73, wherein the subject has no persistence of CAR-T cells at the time of administration according to (b).

[00281] 75. The method according to any one of embodiments 47-74, wherein the subject has one or more symptoms of the disease at the time of administration according to (b).

[00282] 76. The method according to any one of embodiments 47-75, wherein the subject has a suboptimal response at the time of administration according to (b).

[00283] 77. The method of embodiment 76, wherein the suboptimal response comprises:

(a) complete response (CR), complete response with incomplete recovery of blood cells (CRi) with detectable minimal residual disease (patients with leukemia) and no cytogenetic response;

(b) complete bone marrow response;

(c) a partial response, or

(d) stable response.

[00284] 78. The method according to any one of embodiments 47-77, wherein the cytokine release syndrome (CRS) outcome in the subject on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 after administration according to (b) is not detectable or decreases by a value from about 20% to about 99% compared with a method involving an alternative dosing regimen, where cells according to (b) are administered to the subject without performing the administration of the first doses.

[00285] 79. The method according to any one of embodiments 47-78, wherein the subsequent dose contains approximately the same number of cells as the number of cells in the first dose.

[00286] 80. The method according to any one of embodiments 47-78, wherein the subsequent dose contains an increased number of cells compared to the first dose.

[00287] 81. The method of embodiment 80, wherein the subsequent dose contains at least about 5% more cells than the number of cells in the first dose.

[00288] 82. The method according to any one of embodiments 47-78, wherein the subsequent dose contains a reduced number of cells compared to the first dose.

[00289] 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.

[00290] 84. The method according to any one of embodiments 47-83, wherein the disease is a tumor or cancer.

[00291] 85. The method of Embodiment 84 wherein the cancer is leukemia or lymphoma.

[00292] 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, slow growing B-cell lymphoma, B-cell malignancies, forms of colon, lung, liver, breast, prostate, ovary, skin, melanoma, bone and head cancer brain, ovarian cancer, forms of epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, or mesothelioma.

[00293] 87. The method of embodiment 86 wherein ALL is relapsed or refractory ALL.

[00294] 88. A method of treatment comprising administering a subsequent dose of allogeneic chimeric antigen receptor (CAR) T cells to a subject who has previously received a first dose of allogeneic CAR-T cells, wherein the subsequent dose of cells is administered at a time point that is from at least or more than about 5 weeks after the start of the first dose to less than about 24 weeks thereafter.

[00295] 89. 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 CAR-T cells in an amount sufficient to demonstrate a clinical effect in the subject; and at the time of administration, the subject does not show a detectable adaptive immune response of the body, specific for CAR-T cells; and/or the time between said previous and subsequent doses is greater than about 5 weeks to less than about 24 weeks.

[00296] 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.

[00297] 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.

[00298] 92. The 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 in a time point from about 5 to about 24 weeks after the previous treatment; and/or the composition is formulated to administer a subsequent dose in an amount sufficient to alleviate the disease in a subject previously treated with CAR-T cells.

[00299] 93. A composition comprising allogeneic chimeric antigen receptor (CAR) T cells for use in the treatment of a disease in a subject previously treated with CAR-T cells, wherein the cells are to be used at a time point from about 5 up to 24 weeks after previous treatment; and wherein the cells are formulated to administer a subsequent dose in an amount sufficient to alleviate the disease in a subject previously treated with CAR-T cells.

[00300] 94. Use according to Embodiment 92 or composition for use according to Embodiment 93, wherein the dose of CAR-T cells in the previous treatment provided relief of one or more symptoms of the disease in the subject prior to administration of the next dose.

[00301] 95. Use of allogeneic chimeric antigen receptor (CAR) T cells in the manufacture of a medicament for use in a method of treating a disease, said method comprising administering to a subject having the disease a first dose of CAR-T cells, wherein said first dose contains from about 1x10 ⁶ to about 5x10 ⁸ total cells; administering to the subject a subsequent dose of CAR-T cells at a time point that is at least or greater than about 4 weeks after and less than about 24 weeks after the start of said administration according to (a).

[00302] 96. The use according to embodiments 92, 94, or 95, or the composition for use according to embodiment 93, wherein the disease is leukemia or lymphoma.

[00303] 97. The method according to any one of embodiments 47-83, the use according to any one of embodiments 92 or 94-95, or the composition for use according to embodiment 93, wherein the CAR T cells are CAR T cells specific for the tumor antigen.

[00304] 98. The method according to any one of embodiments 47-83, the use according to any one of embodiments 92 or 94-95, or the composition for use according to embodiment 93, wherein the CAR-T cells are UCART19 cells.

[00305] 99. An article comprising:

a number of sealed containers, each of which individually contains a unit dose of allogeneic chimeric antigen receptor (CAR) T cells for administration to a subject, said unit dose containing from about 1x10 ⁶ to about 5x10 ⁸ cells; packaging material and a label or package insert containing instructions for administering to a subject a number of said unit doses by making a first administration and a subsequent administration, said first administration comprising delivering to the subject one of said unit doses, and said subsequent administration comprising administering to the subject one or more the number of specified standard doses.

[00306] 100. The article of manufacture of Embodiment 99, wherein the instructions indicate that said follow-up administration is to take place between about 30 and 150 days, optionally on about day 30, about day 60, about day 90, or about day 99, after said first injection.

[00307] 101. The article of manufacture according to embodiment 99 or 100, wherein the containers are or contain flexible cell infusion bags.

[00308] 102. A method of treating an adult subject who has refractory and/or recurrent acute lymphoblastic CD 19+ B cell leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR- T cells) containing CAR 4-1BB/CD3-zeta for human CD19, wherein at least one dose is selected from the group consisting of approximately 6×10 ⁵ cells/dose, 6×10 ⁶ cells/dose, approximately 6- 8×10 ⁷ cells/dose and approximately 1.8-2.4×10 ⁸ cells/dose.

[00309] 103. A method of treating a pediatric subject who has refractory and/or recurrent acute lymphoblastic CD 19+ B cell leukemia, comprising administering to the subject at least one dose of allogeneic T cells with a chimeric antigen receptor (CAR) (CAR- T-cells) containing CAR 4-1BB/CD3-zeta for human CD19, where at least one dose is approximately 2-8×10 ⁷ cells/dose.

[00310] 104. The method of embodiment 102 or 103, wherein the CAR-T cells are CD52 deficient.

[00311] 105. The method according to embodiment 102 or 103, wherein the CAR-T cells comprise a mixture of CD52-deficient and CD-52-positive cells.

[00312] 106. The method according to any one of embodiments 102-105, wherein the CAR-T cells express a CAR with the sequence of SEQ ID NO: 1.

[00313] 107. The method according to any one of embodiments 102-106, wherein the CAR-T cells comprise UCART19 (CD19CAR/RQR8+_TCRαβ-_T cells).

[00314] 108. The method of any one of embodiments 102-107, wherein CAR expression is detectable in a subject for a period of at least 42 days following administration of CAR-T cells.

[00315] 109. The method of any one of embodiments 102-108, wherein CAR expression is detectable in a subject for a period of at least 56 days following administration of CAR-T cells.

[00316] 110. The method of any one of embodiments 102-109, wherein the subject has a CR or Cri state for at least 1.3 months following administration of UCART19.

[00317] 111. The method of any one of embodiments 102-110, wherein the subject has a CR or Cri state for at least 1.8 months following administration of UCART19.

[00318] 112. The method of any one of embodiments 102-111, wherein the subject has a CR or Cri state for at least 3.6 months following administration of UCART19.

[00319] 113. The method according to any one of embodiments 102-112, wherein the subject has a CR or Cri state for at least 12.4 months after administration of UCART19.

[00320] 114. The method according to any one of embodiments 102-113, wherein the subject receives the first regimen for lymphodepletion prior to at least one dose.

[00321] 115. The method of embodiment 114 wherein the first regimen for lymphodepletion comprises administering fludarabine and cyclophosphamide.

[00322] 116. The method of embodiment 114, wherein the first regimen for lymphodepletion comprises administering fludarabine, cyclophosphamide, and an anti-CD52 antibody.

[00323] 117. The method of embodiment 115 or 116 wherein fludarabine is administered at a dose of about 30 to 150 mg/m ² ; cyclophosphamide is administered at a dose of about 300 to 4000 mg/m ² , and anti-CD52 antibody is administered at a dose of about 0.3 to 1 mg/kg.

[00324] 118. The method of any one of embodiments 114-117, wherein the first regimen for lymphodepletion is started about 1-15 days prior to at least one dose.

[00325] 119. The method according to any one of embodiments 114-118, wherein the subject receives a subsequent dose of CAR-T cells.

[00326] 120. The method of embodiment 119, wherein the subject has a suboptimal response at the time of the next dose.

[00327] 121. The method of embodiment 120, wherein the suboptimal response comprises:

(a) complete response (CR), complete response with incomplete recovery of blood cells (CRi) with detectable minimal residual disease (patients with leukemia) and no cytogenetic response;

(b) complete bone marrow response;

(c) a partial response, or

(d) stable response.

[00328] 122. The method of embodiment 119, wherein the subsequent dose contains approximately the same number of cells as the number of cells in the first dose.

[00329] 123. The method of embodiment 119, wherein the subsequent dose contains an increased number of cells compared to the first dose.

[00330] 124. The method of embodiment 119, wherein the subsequent dose contains a reduced number of cells compared to the first dose.

[00331] 125. A method of obtaining a population of allogeneic chimeric antigen receptor (CAR) T cells (CAR-T cells) directed to a target of interest, comprising:

- isolation of peripheral blood mononuclear cells (PBMC) from a healthy donor;

- activation of T-cells in RVMS;

- transducing activated T cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing the CAR of interest;

- violation of the expression of genes TCRαβ and CD-52 in a subpopulation of T cells;

- an increase in the population of T-cells and

- enrichment of the T-cell population for TCRαβ-negative cells

- thereby obtaining a population of allogeneic T cells with a chimeric antigen receptor (CAR) (CAR-T cells).

[00332] 126. The method of Embodiment 125, wherein step (c) is performed approximately 3-4 days after step (b).

[00333] 127. The method according to embodiment 125 or 126 wherein step (d) is performed about 2 days after step (c).

[00334] 128. The method according to any one of embodiments 125-127, wherein step (f) is performed approximately 10-12 days after step (d).

[00335] 129. The method of any one of embodiments 125-128, wherein the CAR-T cells are CD52-deficient.

[00336] 130. The method according to any one of embodiments 125-129, wherein the CAR-T cells comprise a mixture of CD52-deficient and CD-52-positive cells.

[00337] 131. The method according to any one of embodiments 125-130, wherein the CAR-T cells express a CAR with the sequence of SEQ ID NO: 1.

[00338] 132. The method according to any one of embodiments 125-131, wherein the CAR-T cells comprise UCART19 (CD19CAR/RQR8+_TCRαβ-_T cells).

[00339] 133. The method according to any one of embodiments 125-132, wherein the lentiviral vector from step (c) further expresses a security switch.

[00340] 134. The method of Embodiment 133, wherein the security switch is RQR8.

EXAMPLES

[00341] The following examples are intended to illustrate the methods and materials of the present invention. Suitable modifications and adaptations of the described diseases and parameters commonly encountered in the art, which are obvious to those skilled in the art, are within the spirit and scope of the present invention.

Example 1 Allogeneic CAR-T Dosing Schedule

[00342] This example illustrates treatment with multiple doses of allogeneic CAR-T cells.

[00343] Prior to administration of CAR-T cells, diseased subjects were administered a schedule for lymphodepletion. Such a regimen may include, for example, treatment with fludarabine (total dose in the range of about 30 to 150 mg/m ² ) and with cyclophosphamide (total dose in the range of about 300 to 4000 mg/m ² ), drug anti-CD52 agents (eg, with an anti-CD52 antibody, such as an antibody containing the sequence under SEQ ID NO: 8 and SEQ ID NO: 10) (total dose in the range of about 0.3 to 1 mg/kg).

[00344] Subjects received a first dose of CAR-T cells in an amount of from about 1 x 10 ⁴ to about 5 x 10 ⁸ cells per D.0.

[00345] Approximately between D. 24 and D. 62, the response to the first dose of CAR-T cells was assessed. The number of CAR-T cells present in the peripheral blood of treated subjects was determined by performing flow cytometry on a cell sample for surface expression of a CAR-specific marker, CD4 and/or CD8. A positive MRD and/or loss of persistence suggested subsequent doses of CAR-T cells.

[00346] Prior to subsequent dosing of CAR-T cells, subjects with MRD were administered a regimen for lymphodepletion. Such a regimen may include, for example, treatment with fludarabine (total dose in the range of about 30 to 150 mg/m ² ) and cyclophosphamide (total dose in the range of about 300 to 4000 mg/m ² ), with an anti-CD52 antibody, or without it (total dose in the range of about 0.3 to 1 mg/kg). Between 2 and 14 days post lymphodepletion, subjects were dosed with about 1x10 ⁴ to about 1x10 ⁸ CAR-T cells. The response to a dose of CAR-T cells was assessed and the number of CAR-T cells present in the peripheral blood of treated subjects was determined. A positive MRD and/or loss of persistence suggested subsequent doses of CAR-T cells. Multiple doses of allogeneic CAR-T could be administered until MRD-negativity was achieved.

[00347] Example 2 Use of an allogeneic T cell product with CAR for CD19 (UCART19) in adult patients with relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia

[00348] This study was conducted in relation to the treatment of B-ALL. B-ALL is incurable in ~60% of adult patients. With recurrence, the prognosis is very poor (OS <10%). Standard therapy includes a combination of chemotherapy ± allogeneic SCT.

[00349] Subjects were administered a universal adoptive therapy based on allogeneic T cells directed at CD19+ malignancies.

[00350] Expression of the trans gene was achieved using lentiviral transduction. UCART19 contains scFv for CD19 and an intracellular domain containing CD3ζ+4-1BB. The CAR-T cell additionally contains RQR8 (CD20 mimotope), a security switch expressed in trans. CAR-T cells were generated by additional knockout using TALEN-based technologies. 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 anti-CD52 antibodies in lymphodepletion. An exemplary UCART19 cell used herein (CD19CAR/RQR8+_TCRαβ-_T cells) is shown in FIG. 5.

[00351] This study had at least 3 objectives, and schematic representations of the study design are presented in FIG. 6A and FIG. 6B (BMA = bone marrow aspiration).

[00352] Goal #1: Evaluate the safety and tolerability of UCART19 at different doses and determine the maximum tolerated dose (MTD). (FIG. 3B).

[00353] Goal #2: Evaluation of antileukemic activity.

[00354] Objective #3: Evaluate the increase in the number and persistence of UCART19.

[00355] FIG. 3B shows a study design having the following features:

- An open label non-comparative dose escalation study to evaluate up to 4 dose levels (DL) of UCART19 and to determine the maximum tolerated dose (MTD) in adult patients with R/R (relapsed/refractory) B-ALL.

- Dose escalation was followed by a safety extension part, patients received the MTD-dose or the recommended dose (RD).

- Schedule for lymphodepletion (LD) started at D.-7 previous UCART19 infusion and combined cyclophosphamide 1500 mg/m ² and fludarabine 90 mg/m ² without anti-CD52 antibody (FC) or with anti-CD52 antibody 1 mg/kg (FCA) ).

- During the expansion part, the role of anti-CD52 antibody could be investigated in 2 groups of patients (LD with FC or FCA).

- In D. 0, UCART19 was administered as a single, non-split dose via slow IV infusion (5 minutes).

- A dose-limiting toxicity assessment was performed 28 days after infusion (D. 28).

- Bone marrow aspiration/biopsy was performed before LD at D. -1, D. 28 and D. 84.

- Minimal residual disease (MRD) was determined by <10-4 blasts in the bone marrow, detected by flow cytometry (FLC) and/or qPCR.

- At the completion of the study (D. 84 post-infusion), the patient was transferred to a long-term follow-up study (LTFU) over a 15-year period.

[00356] Key eligibility criteria for this study included:

- Age ≥16 years, no more than <70 (male or female).

- Patient with CD19 ⁺ relapsed or refractory (relapsed/refractory; R/R) B-ALL as recommended by the National Comprehensive Cancer Network (NCCN, 2017).

- Morphological or MRD ⁺ (≥1×10 ^-3 determined using flow cytometry and/or qPCR).

- Who have exhausted the available treatment options.

- No previous treatment with investigational gene or cell therapy products.

- No extramedullary involvement was clinically suspected.

- Adequate function of the kidneys, liver, lungs and heart.

- No active infection.

- No active CNS leukemia.

[00357] The data presented herein refer to 12 patients who received treatment, with six patients at the first dose level of 6×10 ⁶ cells (about 1×10 ⁵ cells per kilogram) and six patients at the second dose level of 6-8×10 ⁷ total cells (about 1×10 ⁶ cells per kilogram). No patient has yet received treatment at third and final dose levels ranging from 1.8 to 2.4×10 ⁸ total cells. The majority of patients received three or more previous treatment lines, three of which were previously treated with blinatumomab and seven were previously treated with allo-SCT (allogeneic stem cell transplantation), reflecting clinical practice in Europe where 10 out of 12 patients were included into research.

[00358] Patient characteristics are shown in Table 8 below.

Safety

[00359] All 12 included patients received UCART19 at a targeted cell dose following lymphodeplement chemotherapy consisting of cyclophosphamide and fludarabine, with 10 patients receiving anti-CD52 antibody (FCA regimen) and two patients receiving no anti-CD52 antibody (FC regimen). Table 6 below summarizes adverse events by grade according to UCART19 infusion, as well as those associated with the regimen for lymphodepletion. Grade 1 represents mild toxicity, Grade 2 represents moderate toxicity, Grade 3 represents severe toxicity, and Grade 4 represents life-threatening toxicity. Toxicity grade 5 represents toxicity leading to death.

[00360] The most common adverse event associated with UCART19 was CRS, reported in 11 patients (two patients suffering from severe cases of CRS, one grade 3 and one grade 4). Tocilizumab was administered to 6/11 patients. CRS correlated with an increase in serum cytokines (IL-6, IL-10 and IFN-gamma) and an increase in the amount of UCART19 in the blood in all patients.

[00361] Viral reactivation (CMV and/or adenovirus) occurred in 4 patients (G1 to G3).

[00362] Three patients developed persistent cytopenia, defined as persistent cytopenia, 42 days after UCART19 infusion.

[00363] Three patients experienced mild or Grade 1 neurotoxic events.

[00364] One patient experienced grade 1 GvHD adverse events on the skin, which were resolved with topical steroids.

[00365] Two cases of dose-limiting toxicity have been reported. The first event occurred at the first dose level and was grade 4 CRS associated with UCART19 and associated with grade 5 neutropenic sepsis associated with lymphodepletion and UCART19. Death occurred on day 15 after UCART19 infusion. The second case, grade 4 prolonged cytopenia, occurred at the second dose level and was described as being associated with lymphodepletion and UCART19. This patient underwent allo-SCT and had grade 5 unrelated pulmonary hemorrhage under conditions of infection on day 19 after allo-SCT or day 82 due to UCART19 infusion. Grade 5 adverse events have been reported in other trials of autologous T-cell-based therapy with CAR for CD19 due in part to advanced disease and comorbidities.

[00366] Two additional deaths were also reported, which were not attributed to the consequences of the use of UCART19. One patient died from progressive disease and one patient from complications associated with allo-SCT. Transplant-related mortality occurred in approximately 20-30% of patients due to allo-SCT.

Efficiency

[00367] FIG. 7A shows the status of the study. FIG. 7B shows the response, duration of remission, and retries of UCART19 in this trial (antileukemic activity).

[00368] MRD-CR occurs when a patient reaches CR and there is no evidence of ALL cells in the bone marrow when sensitive tests such as polymerase chain reaction or flow cytometry are used. The CR or CRi rate is a typical normative standard, but studies in both children and adults with ALL have demonstrated a strong correlation between minimal residual disease (MRD+) and relapse risk.

[00369] Of the 12 patients who were administered UCART19, two could not be assessed (one died on day 15, as noted above, and one did not reach the assessment on day 28 at the time of data collection). Eight of the 10 patients evaluated achieved a CR, defined as the absence of any signs or symptoms of cancer, or a CR with incomplete recovery of the number of formed blood cells (CRi). Seven patients achieved MRD-CR.

[00370] No increase in UCART19 was observed in these 2 patients with refractory disease.

[00371] Six patients were treated with allo-SCT, including four patients after the first dose of UCART19 and two patients after the second dose. According to the data collected in this study, four patients maintained MRD-CR at 12.4, 3.6, 1.8, and 1.3 months after UCART19 infusion (molecular remission).

[00372] An increase in the number of CAR-T cells was detected in the blood from day 7 after infusion of UCART19 with a peak in reproduction between day 10 and day 17. One patient at the second dose level had the highest peak associated with long-term persistence up to day 42 still remaining at the time data collection was stopped. At the second dose level, the longest persistence observed at the time data collection was stopped was at day 56.

[00373] Two patients on the FC regimen did not show any increase in CAR-T cells. A similar lack of increase in CAR-T cells was observed in two out of 10 patients on the FCA regimen.

[00374] FIG. 8A and FIG. 8B depicts the PK profile of flow petometry and UCART19 kinetics in adult patients with relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia. In particular, with reference to FIG. 8A and FIG. 8B Preliminary flow cytometric data at DL1 and DL2 show that UCART19 was detectable in blood from D.3 to D.14, with a proliferation peak between D.10 and D.17. One patient at DL2 showed the highest peak associated with the longest persistence. Among these patients with an increase in the number of cells in DL1: 1 patient demonstrated UCART19 persistence up to D. 42; at DL2: 2 patients had persistence up to D. 42 and ongoing persistence at D. 56. Preliminary data indicate that the rate of increase in UCART19 did not correlate with response at D. 28; instead, MRD-CR at D. 28 was observed even at low levels of UCART19 increase. After the first dose of UCART19, no increase was observed in 2 of 10 patients treated with LD with FCA and 2 of 2 patients treated with FC.

[00375] The role of the anti-CD52 antibody in increasing the amount of UCART19 was also evaluated.

Example 3 Re-dosing of UCART19 in a patient with relapsed/refractory CD19-positive B-ALL

[00376] This example illustrates treatment with UCART19, where the treatment involves a subsequent dose of UCART19 administered after the first dose of UCART19 as administered in Example 2.

[00377] Two patients from the above study in Example 2 received a second dose of UCART19 and both achieved MRD-CR.

Patient #4

[00378] A 23-year-old male patient with relapsed/refractory CD19-positive B-ALL received a single dose of UCART19 (6×10 ⁶ total cells, DL1 dose) on day 0 (D. 0) of the study. The regimen for lymphodepletion (fludarabine, 122 mg/m ² ; cyclophosphamide, 1230 mg/m ² and anti-CD52 antibody, 1 mg/kg) was administered for six days prior to UCART19 infusion. No significant toxicity was observed. This patient had a relapse of CD19+ disease in D. 61 after the 1st dose (LD with FCA); The 2nd dose (LD with FC) provided this patient with an MRD- at D. 28. The patient then received a regimen for low-intensity lymphodepletion over a period of six days (fludarabine and cyclophosphamide without anti-CD52 antibody). 99 days after the initial UCART19 infusion, the patient was re-administered the same dose (6×10 ⁶ total cells) of UCART19.

[00379] The data is plotted in FIG. 2A. At the time immediately preceding the next dose of UCART19, UCART19 was not detectable in the patient (FIG. 2A).

[00380] The patient achieved MRD-negativity at D. 28 after infusion of the second dose of UCART19. Thus, administration of UCART19 doses after relapse after molecular remission after treatment with the first dose of UCART19 was effective in achieving MRD-negativity. The patient was transferred to the stage of transplantation of allogeneic stem cells 6 weeks after the second dose.

Patient #13

[00381] Male patient, 31 years old, diagnosed with refractory B-ALL. The patient received the 1st infusion of UCART19; the resulting dose was 8×10 ⁷ total cells (dose level 2) after lymphodepletion combining fludarabine (total dose 90 mg/m ² ) and cyclophosphamide (total dose 1500 mg/m ² ) (FC). After 1st dosing: UCART19 was not detectable (<1 cells/µl by flow cytometry). On day 28 (28 days after UCART19 infusion), no antileukemic activity was observed (89% of blasts in the bone marrow). During the 28 days following UCART19 infusion, no toxicity-related mechanism of action was reported. The patient was excluded from the study and received a second infusion of UCART19 (total dose of 8×10 ⁷ total cells) (48 days after the first dose) on humanitarian grounds ("Specials" in the UK). The regimen for lymphodepletion combined fludarabine (total dose 90 mg/m ² ), cyclophosphamide (total dose 1500 mg/m ² ) and anti-CD52 antibody (total dose 1 mg/kg) (FCA). The patient was characterized by a low level increase in the number of UCART19 (10 cells/μl) on day 10, after D. 14, no UCART19 was detected. A low degree of CRS has been reported. Anti-leukemic activity was observed at day 14 and D. 28 with negative minimal residual disease (MRD-negative) as determined by flow cytometry and qPCR. The patient underwent allotransplantation (63 days after the second dose). He eventually relapsed on August 15, 2018 (112 days post-transplant) with a low MRD (molecular relapse) detected. The patient is still alive 9.3 months after the first dose of UCART19 and 7.8 months after the second dose of UCART19. The data is provided in FIG. 2B.f

[00382] Both patients were subsequently transferred to allo-SCT.

Patient #18

[00383] Male patient, 22 years old, diagnosed with B-ALL. The patient received the 1st infusion of 1.8×10 ⁸ cells UCART19 (dose level 3) after lymphodepletion combining fludarabine (total dose 90 mg/m ² ), cyclophosphamide (total dose 1500 mg/m ² ) and anti-CD52 antibody ( total dose 40 mg) (FCA). No UCART19 was detectable and no toxicity-related mechanism of action was reported. On day 28 (28 days after UCART19 infusion), no antileukemic activity was observed (medullary biopsy showed 15% blasts). The patient was refractory and received a second infusion of UCART19 (75 days after the first dose) with an enhanced regimen for lymphodepletion with fludarabine (total dose 120 mg/m ² ), cyclophosphamide (total dose 1500 mg/m ² ) and anti-CD52 antibody (total dose 1 mg/kg=65 mg) (FCA). The patient continued to participate in the study as repeat doses were allowed under the modified protocol. A peak increase in UCART19 was observed on day 7, with UCART19 being detectable on day 10 (10 cells/µl) and on day 14 UCART19 was not detectable. On day 28 after the second dose, the patient showed no response and progress. Patient #18's response to repeated dosing is shown in FIG. fourteen.

[00384] This result demonstrates that repeated doses of allogeneic CAR-T are effective in achieving MRD-negativity after relapse.

[00385] Example 4 Use of an allogeneic T cell product with CAR for CD19 (UCART19) in pediatric patients at high risk for relapsed/refractory CD19+ B-cell acute lymphoblastic leukemia

[00386] B-cell acute lymphoblastic leukemia is the most common malignant disease in children and accounts for 80% of childhood leukemias. The most common age is 2–10 years (peaks at 3–4 years), with ~90% survival. Relapse can occur in 10% of children, with about 40-50% surviving, unless there is a high risk, in which case approximately 10-30% survive.

[00387] This study used the same levels of UCART19 as shown in Example 2.

[00388] This study had at least the following objectives:

- Assessing the safety and tolerability of UCART19 at a fixed dose.

- Evaluation of the ability of UCART19 to induce molecular remission at D. 28, D. 56, D. 84 and/or before starting the allo-SCT regimen.

- Assessment of remission rate, duration of remission, time to remission, disease-specific survival and progression-free survival.

- Estimated proportion of patients undergoing allo-SCT.

- Evaluation of the phenotype, migration, increase in the number and persistence of UCART19.

[00389] Key eligibility criteria for this study included:

- Ages 6 months to <18 years (male and female).

- Patient with CD19+R/R B-ALL, morphological or MRD+ (≥1×10 ^-3 determined by flow cytometry and/or qPCR)

- Who have exhausted the available treatment options.

- Meets inclusion parameters for allo-SCT with a matching donor.

- No previous treatment with investigational gene or cell therapy products.

- No active infection.

- No active CNS leukemia.

[00390] FIG. 9 shows the study plan.

[00391] The regimen for lymphodepletion was started with D.-7 (within the week of the previous UCART19 infusion) and combined: cyclophosphamide (C) (60 mg/kg/day for 2 days), fludarabine (F) (30 mg/m ² /day for 5 days) and without antibody to CD52 (A) (0.2 mg/kg/day for 5 days). In D. 0, a fixed dose of UCART19 (2x10 ⁷ total cells, equivalent to 1.1-2.3x10 ⁶ cells/kg) was administered as a single, non-split dose via slow IV infusion over 5 minutes.

[00392] The safety assessment was performed at D. 28 after administration of UCART19. VMA was performed at baseline, D. -1, D. 14 (optional), D. 28, D. 56, D. 84 or before the start of the allo-SCT conditioning regimen or at the early termination visit (at the discretion of the investigator). During the 12-month follow-up period, MMAs will be performed at M. 1, M. 2, M. 3, M. 6, and M. 12 after allotransplantation to assess disease. For refractory patients, VMA will be optional, at the discretion of the investigator.

results

[00393] Six patients were included in the study. Six patients were treated at a weight-bound cell dose equivalent to 1.1-2.3×10 ⁶ cells/kg. Five patients received three or more previous treatment lines, and three received four or more previous treatment lines. Two patients were previously treated with allo-SCT. Patient characteristics are presented in Table 10 below.

Safety

[00394] All six enrolled patients received UCART19 at a target cell dose following lymphodeplement chemotherapy consisting of cyclophosphamide and fludarabine. Five patients also received anti-CD52 antibodies. Table 11 below summarizes the adverse events by severity by UCART19 infusion, as well as those associated with the regimen for lymphodepletion and/or UCART19. [Grade 1 represents mild toxicity, Grade 2 represents moderate toxicity, Grade 3 represents severe toxicity, and Grade 4 represents life-threatening toxicity. Toxicity grade 5 represents toxicity leading to death.]

[00395] The most common adverse events associated with UCART19 were CRS in all treated patients, with one patient suffering from grade 3 CRS. Mild to moderate neurotoxic events occurred in three of the six treated patients. Three patients had prolonged cytopenia, which was reported to be associated with lymphodepletion and, in some cases, possibly associated with UCART19. Viral reactivation by cytomegalovirus (CMV), adenovirus, BK virus, and metapneumovirus has been attributed to the consequences of lymphodepletion. One patient experienced transient GvHD on grade 1 skin. Two patients died of disease recurrence due to allo-SCT and one patient died from complications of allo-SCT. There were no treatment-related deaths. The data are presented in table 12.

Efficiency

[00396] FIG. 10 shows the status of the study. FIG. 11 shows the response and duration of remission (anti-leukemic activity).

[00397] All patients completed the 28-day evaluation period and were evaluated for antileukemic activity. Five of the six patients achieved MRD-CR and all five underwent allo-SCT. Two patients were in remission more than 13 months after UCART19 infusion at the time data collection was stopped, and three patients died due to allo-SCT, two due to disease recurrence and one due to graft-related complications. One patient left the study due to lack of response and received follow-up treatment with blinatumomab outside the study. This is the only patient who received the FC regimen.

[00398] Cellular kinetics were evaluated and shown in FIG. 12. The copy number of the UCART19 vector (VCN) was measured in blood and bone marrow by qPCR. Preliminary data showed that for 5 out of 6 patients, UCART19 was detected in the blood at D. 7, with a peak of proliferation observed around D. 14. UCART19 was not detected in one patient who subsequently experienced a relapse. In 3 of 5 patients, UCART19 persisted in the blood until D. 28. In 2 of 5 patients, UCART19 remained detectable in the blood at D. 42. Persistence after D. 42 was not measured because UCART 19 was ruled out by an allo-SCT conditioning regimen. .

[00399] The kinetics of cytokines was assessed. 2 out of 6 patients had elevated levels of IL-6 and IFNγ. An increase in cytokines was not observed in 4 of 6 patients. All 6 patients suffered from CRS. CRS G3 was observed in patient 1 and CRS G2 in patient 8. The time to onset of the first symptoms of CRS ranged from D. 5 to D. 9.

[00400] T cell donor chimerism was assessed (FIG. 13A, FIG. 13B, FIG. 13C). UCART19 was detected in blood from D. 7 to at least D. 42 in all but one patient, based on molecular signatures of T cell donor chimerism.

[00401] Example 5 Use of an allogeneic T cell product with CAR for CD19 (UCART19) in patients with relapsed/refractory large B-cell and/or follicular lymphoma

[00402] Diffuse large B-cell lymphoma (DLBCL), which accounts for 20% to 30% of cases of B-cell non-Hodgkin's lymphoma (B-NHL), is fatal if left untreated. Primary mediastinal large B cell lymphoma (PMBCL) and transformed follicular lymphoma (FL) are usually treated according to the DLBCL paradigm. DLBCL includes a group of molecularly diverse aggressive lymphomas that differ not only in chromosomal changes, but also in their activation signaling pathway and clinical outcome. The disease itself may be de novo or may be the result of transformation of indolent B lymphomas such as transformed follicular lymphoma. In the US, the annual incidence rate of DLBCL is 6.9 per 100,000 and is the most common form of B-NHL.

[00403] Approximately half of all patients with aggressive B-NHL have relapsed or refractory disease, with an estimated 10% to 15% of patients with DLBCL having primary refractory disease and an additional 20% to 30% relapse after baseline objective response (Chaganti et al, 2016). High-grade B-cell lymphomas with aberrations in MYC, BCL2, and/or BCL6, including double-hit and triple-hit lymphomas, are associated with poor prognosis, even in early-diagnosed settings (Rosenthal and Younes, 2017) .

[00404] This study is a phase 1/2 non-comparative, open-label, multicentre, phase 1/2 study evaluating the safety, efficacy, cellular kinetics, and pharmacodynamics of UCART19 in adult patients with relapsed/refractory large B-cell lymphoma or follicular lymphoma. The study is divided into dose escalation (phase 1) and dose expansion (phase 2) phases. Phase 1 of the study will use CAR T cells containing the RQR8 security switch (CD19CAR/RQR8+_TCRαβ T cells). During the Phase 2 study, one or more patients may receive CAR-T cells that do not contain the RQR8 safety switch (CD19CAR/TCRαβ-_T cells or CD19CAR/R2+_TCRαβ-_T cells).

[00405] Criteria for inclusion in a study may include one or more of the following.

1. Histologically and cytologically confirmed diagnosis of large B-cell lymphoma according to the 2018 WHO revision of the classification of lymphomas: diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS) (terminal center [GCB] and non-GCB B cells ), DLBCL coexisting with any grade of follicular lymphoma, intravascular large B-cell lymphoma, DLBCL associated with chronic inflammation, anaplastic lymphomakinase positive (ALK+) DLBCL Epstein-Barr virus (EBV+) positive DLBCL-NOS, T-cell /Histiocyte-rich large B-cell lymphoma, DLBCL with IRF4/MUM1 rearrangement, High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 translocation (double/triple hit), primary cutaneous DLBCL-leg type, transformation of follicular lymphoma into DLBCL, primary mediastinal B-cell lymphoma (PMBCL), or follicular lymphoma.

2. Recurrent or refractory aggressive large B-cell lymphoma or follicular lymphoma, as defined by the following:

Первичная рефрактерность, определяемая как лучший ответ при прогрессирующем заболевании или стабильном заболевании после по меньшей мере 4 циклов терапии первой линии (например, 4 цикла RCHOP) со стабильной продолжительностью заболевания, не превышающей 6 месяцев после последней дозы терапии.

Primary refractoriness, defined as the best response in progressive disease or stable disease after at least 4 cycles of first-line therapy (eg, 4 cycles of RCHOP) with a stable disease duration not exceeding 6 months after the last dose of therapy.

Рефрактерность (определяемая как лучший ответ при стабильном заболевании или прогрессирующем заболевании) в отношении второй или большей линией терапии.

Refractory (defined as the best response in stable disease or progressive disease) to a second or greater line of therapy.

Рецидив через ≤1 год после трансплантации аутологичных стволовых клеток (SCT).

Relapse ≤1 year after autologous stem cell transplant (SCT).

3. Patients must have received at least 2 lines of previous therapy, including an anthracycline and an anti-CD20 monoclonal antibody.

4. Male or female patients ≥18 years of age.

5. General condition status according to the Eastern United Cancer Group (ECOG) was 0 or 1.

6. Estimated elimination period ≥12 weeks after ALLO-501 infusion (according to the investigator).

7. Absence of donor-specific antibodies to HLA.

8. Adequate hematological function, including:

Абсолютное число нейтрофилов (ANC)≥1500/мм³ или >1,5×10⁹/л.

Absolute neutrophil count (ANC)≥1500/mm ³ or >1.5×10 ⁹ /l.

Абсолютное число лимфоцитов (ALC)≥200/мм³ или ≥0,2×10⁹/л.

Absolute lymphocyte count (ALC)≥200/mm ³ or ≥0.2×10 ⁹ /l.

Число тромбоцитов (ALC)≥50000/мм³ или ≥50×10⁹/л.

Platelet count (ALC) ≥50000/mm ³ or ≥50×10 ⁹ /l.

Гемоглобин ≥9 г/дл.

Hemoglobin ≥9 g/dl.

9. Adequate renal function, including:

Расчетный клиренс креатинина ≥60 мл/мин, рассчитанный с использованием стандартного способа для учреждения и не зависящий от диализа. В сомнительных случаях для более точной оценки клиренса креатинина можно использовать 24-часовой тест сбора мочи.

Estimated creatinine clearance ≥60 mL/min calculated using the standard facility method and independent of dialysis. In doubtful cases, a 24-hour urine collection test can be used to more accurately assess creatinine clearance.

10. Adequate liver function, including:

Общий билирубин ≤2,0 мг/дл, за исключением пациентов с синдромом Гилберта, у которых общий билирубин должен составлять менее чем 3,0 мг/дл.

Total bilirubin ≤2.0 mg/dL, except in patients with Gilbert's syndrome, in which total bilirubin should be less than 3.0 mg/dL.

Аспартат- и аланинаминотрансфераза (AST и ALT)≤3×ULN, ≤5,0×ULN, если печень поражена опухолью.

Aspartate and alanine aminotransferase (AST and ALT) ≤3×ULN, ≤5.0×ULN if the liver is affected by a tumor.

Щелочная фосфатаза ≤2,5×ULN (≤5×ULN при метастазах в костях).

Alkaline phosphatase ≤2.5xULN (≤5xULN for bone metastases).

11. Normal levels of blood oxygen saturation (SpO2) ≥91% in room air.

12. Left ventricular ejection fraction (LVEF) ≥45% and no hemodynamically significant pericardial effusion at screening.

13. Eliminated acute effects of any previous therapy to baseline severity or CTCAE ≤1, with the exception of adverse events (AE) that do not pose a risk to safety in the opinion of the investigator.

14. Seronegative result for hepatitis B virus antigen; positive tests for hepatitis B can be further assessed with confirmatory tests, and if the confirmatory tests are negative, the patient can be enrolled in the study.

15. Sero-negative for hepatitis C antibody but negative for antigen. If the hepatitis C antibody test is positive, then patients should be tested for antigen by RT-PCR and be negative for HCV RNA.

16. Negative serum test for pregnancy (for women of childbearing potential) at screening.

17. Female patients without childbearing potential must meet at least 1 of the following criteria:

Достигли постменопаузального статуса, определяемого следующим образом: прекращение регулярных менструаций в течение не менее 12 последовательных месяцев без альтернативной патологической или физиологической причины; статус может быть подтвержден уровнем фолликулостимулирующего гормона в сыворотке (FSH), подтверждающим состояние постменопаузы.

Have achieved postmenopausal status, defined as: cessation of regular menses for at least 12 consecutive months without an alternative pathological or physiological cause; status can be confirmed by serum follicle-stimulating hormone (FSH) levels, confirming postmenopausal status.

Подверглись документально подтвержденной гистерэктомии и/или двусторонней овариэктомии.

Have undergone a documented hysterectomy and/or bilateral oophorectomy.

Характеризуются медицински подтвержденной недостаточностью яичников.

They are characterized by medically confirmed ovarian failure.

All other female patients (including female patients with tubal ligation) are considered to be of childbearing potential.

18. Confirmation of a personally signed and dated informed consent document indicating that the patient was informed of all relevant aspects of this study.

19. Willingness and ability to comply with scheduled visits, treatment plan, laboratory tests and other procedures.

[00406] Phase 1 and Phase 2 studies will be divided into different periods, including screening, lymphodeficiency, treatment, and follow-up. One cycle is defined as the combination of one period of lymphodepletion and one period of treatment.

[00407] Screening will begin upon signing the informed consent form. The patient will be subject to eligibility screening. The screening period will be no more than 28 days. Patients who meet all of the eligibility criteria move to the lymphodepletion period at the end of the screening period.

[00408] Lymph depletion will begin on day -5 and end with an infusion of UCART19 on day 0. Prior to treatment with UCART19, all patients will receive an intravenous regimen for lymph depletion with fludarabine (30 mg/m ² /day), cyclophosphamide (300 mg/m ² /day) and anti-CD52 antibodies (13 mg/day) on an outpatient basis on day -5, day -4 and day -3. The anti-CD52 antibody will be administered over about 4 hours, and patients should be closely monitored for no more than 2 hours after completion of the infusion. Premedication, including high doses of corticosteroids, is required prior to administration of anti-CD52 antibody. If dose-dependent toxic effects (DLT) associated with anti-CD52 antibodies are observed at the initial dose level, then a lower dose of 30 mg total cells (10 mg/day for 3 days) will be evaluated. At the end of the period of lymphodepletion, the eligibility criteria for the use of UCART19 should be assessed to ensure patient safety.

[00409] The treatment period starting on day 0 will end on day 56 after UCART19 infusion. Patients will receive UCART19 as an intravenous infusion on day 0 for approximately 5 minutes. To closely monitor UCART19-associated toxicity, patients should be hospitalized for a minimum of 5 days from day 0 or until UCART19-associated non-hematological toxicity returns to grade ≤1, or receive UCART19 on an outpatient basis at study sites where direct treatment is possible. patient admission.

[00410] A UCART19 dosing strategy will be implemented using two different weight bindings as shown in Table 13 below.

[00411] Follow-up will last from day 56 to month 9. Patients will be followed for the follow-up period until the EOS visit at month 9 after the first infusion of UCART19 or until their early termination of participation in the study. Patients will then be immediately referred for a 15-year long-term follow-up according to a separate long-term follow-up (LTFU) protocol.

[00412] Following evaluation of cellular kinetics and/or disease, patients may be given one optional retreatment with UCART19. Retreatment with UCART19 may be administered at the highest dose level considered safe in Phase 1 or at RP2D in Phase 2. A different batch and/or donor may be used for retreatment than those at the original dose. Each re-treatment with UCART19 should be at least 4 weeks apart from baseline after routine tumor evaluation at Day 56, Month 4, and Month 6 and should be done after lymphodepletion with Flu/Cy or Flu/Cy and anti-CD52 antibody. Patients with undetectable whole blood UCART19 at D. 14 may be re-treated followed by lymphodepletion starting after the end of the DLT observation window in phase 1 or on day 28 in phase 2. Patients who are eligible for re-treatment will be re-cycled, starting with lymphodepletion (day -5), up to month 2 post-dose (day 56) and will follow the same schedule of visits and assessments as described for the initial UCART19 infusion. At the end of the treatment period (Day 56) after the last UCART19 infusion, the patient will continue the observation period as planned in the study plan. Retreatment cannot be performed after month 6 (6 months after baseline day 0).

[00413] Although the disclosed ideas have been described with reference to various uses, methods, and compositions, it should be understood that various changes and modifications can be made without departing from the teachings of this document and the claims below. The above examples are provided to better illustrate the ideas disclosed and are not intended to limit the scope of the ideas presented herein. Although the present ideas have been described in the definitions of these illustrative embodiments, one skilled in the art will readily appreciate that numerous variations and modifications to these illustrative embodiments are possible without undue experimentation. All such variations and modifications are within the scope of current ideas.

[00414] All references cited in this document, including patents, patent applications, articles, textbooks, and the like, as well as references cited in this document, to the extent that they are no longer such, incorporated herein by reference in their entirety. In the event that one or more of the references to the literature and similar materials differ or conflict with this application, including, without limitation, certain terms, terms regarding use, described techniques, and the like, this application will take precedence.

[00415] In the foregoing description and examples, specific embodiments of the present invention are defined and the best mode of implementation contemplated by the present inventors is described. However, it will be understood that no matter how detailed the above may be in the text, the present invention may be practiced in many ways and the present invention is to be construed in accordance with the appended claims and any equivalents thereof.

--->

SEQUENCE LIST

<110> ALLOGENE THERAPEUTICS, INC.

LES LABORATOIRES SERVIER

<120> METHODS AND COMPOSITIONS FOR DOSING ALLOGENOUS T-CELLS

WITH CHIMERIC ANTIGEN RECEPTOR

<130> ALGN-012/03WO 333466-2218

<150> US 62/750,215

<151> 2018-10-24

<150> US 62/716,898

<151> 2018-08-09

<150> US 62/579,426

<151> 2017-10-31

<160> 35

<170>PatentIn version 3.5

<210> 1

<211> 495

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CAR full-length amino acid sequence (version 2)

<400> 1

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile

20 25 30

Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly

50 55 60

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr

65 70 75 80

Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

145 150 155 160

Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val Ser

165 170 175

Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn Thr

180 185 190

Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu

195 200 205

Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu

225 230 235 240

Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro

245 250 255

Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp Pro

260 265 270

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

275 280 285

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

290 295 300

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

305 310 315 320

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

325 330 335

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

340 345 350

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

355 360 365

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 2

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HCDR1 antibodies CD52

<400> 2

Asp Phe Tyr Met Asn

fifteen

<210> 3

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HCDR2 antibodies CD52

<400> 3

Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro Ser

1 5 10 15

Val Lys Gly

<210> 4

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HCDR3 antibodies CD52

<400> 4

Glu Gly His Thr Ala Ala Pro Phe Asp Tyr

1 5 10

<210> 5

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR1 antibody CD52

<400> 5

Lys Ala Ser Gln Asn Ile Asp Lys Tyr Leu Asn

1 5 10

<210> 6

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR2 antibody CD52

<400> 6

Asn Thr Asn Asn Leu Gln Thr

fifteen

<210> 7

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR3 antibody CD52

<400> 7

Leu Gln His Ile Ser Arg Pro Arg Thr

fifteen

<210> 8

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH antibody CD52

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro

50 55 60

Ser Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln

65 70 75 80

Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 9

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<223> VH antibody CD52

<400> 9

caagtgcagc ttcaagaatc cggccctggt ctggtccgcc cctcccaaac cctctccctg 60

acatgcaccg tgtcgggatt cacctttacc gatttctaca tgaactgggt ccggcagccg 120

ccgggaagag gtctggagtg gatcggcttc attcgggaca aagccaaggg gtacaccacc 180

gagtacaacc cgtccgtgaa gggacgcgtg actatgctcg tggacacgtc caagaaccag 240

ttcagcttga ggctgagcag cgtgactgcc gcggataccg cagtgtacta ctgtgcccgg 300

gaagggcaca ctgccgctcc attcgactat tggggccagg gatcactggt cactgtgtcg 360

tccgcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540

tcaggactct actccctcag cagcgtagtg accgtgccct ccagcagctt gggcacccag 600

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggac 1080

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acgcagaaga gcctctccct gtctccggga aaa 1353

<210> 10

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL antibody CD52

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asp Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln His Ile Ser Arg Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 11

<211> 699

<212> DNA

<213> Artificial sequence

<220>

<223> VL antibody CD52

<400> 11

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggcgt gcactccgac 60

atccaaatga cccaatcccc atcctcactt tccgcctccg tgggcgaccg cgtgactatt 120

acctgtaaag cgtcacagaa tatcgacaag tacctgaact ggtaccagca gaagcctgga 180

aaggccccca agctcctgat ctacaacacc aacaacttgc agactggagt gccgagcaga 240

ttttccggct ccggctcggg gactgatttc accttcacca tctcgagcct gcagccggag 300

gatattgcta cctattactg cctgcaacac attagccggc ccaggacgtt cggacagggt 360

accaaggtcg aaatcaagcg tacggtggct gcaccatctg tcttcatctt cccgccatct 420

gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480

agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540

agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600

agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660

agctcgcccg tcacaaagag cttcaacagg ggagagtgt 699

<210> 12

<211> 177

<212> PROTEIN

<213> Artificial sequence

<220>

<223> RQR8 Full Size Safety Switch

<400> 12

Met Leu Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly

20 25 30

Gly Gly Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser

35 40 45

Thr Asn Val Ser Pro Ala Lys Pro Thr Thr Thr Thr Ala Cys Pro Tyr Ser

50 55 60

Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro

65 70 75 80

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

85 90 95

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

100 105 110

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

115 120 125

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg

130 135 140

Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Arg Ala Glu

145 150 155 160

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

165 170 175

Pro

<210> 13

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human TCR-beta leader sequence (with G replaced with L)

<400> 13

Met Leu Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala

twenty

<210> 14

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Rituximab epitope

<400> 14

Cys Pro Tyr Ser Asn Pro Ser Leu Cys

fifteen

<210> 15

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> glycine-serine linker

<400> 15

Ser Gly Gly Gly Gly Ser

fifteen

<210> 16

<211> 16

<212> PROTEIN

<213> Homo sapiens

<400> 16

Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser

1 5 10 15

<210> 17

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 17

Pro Ala Lys Pro Thr Thr Thr

fifteen

<210> 18

<211> 42

<212> PROTEIN

<213> Homo sapiens

<400> 18

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

1 5 10 15

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

20 25 30

His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40

<210> 19

<211> 21

<212> PROTEIN

<213> Homo sapiens

<400> 19

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr

twenty

<210> 20

<211> 19

<212> PROTEIN

<213> Homo sapiens

<400> 20

Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg

1 5 10 15

Pro Val Val

<210> 21

<211> 19

<212> PROTEIN

<213> Thosea asigna virus

<400> 21

Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu

1 5 10 15

Asn Pro Gly

<210> 22

<211> 495

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CAR full-length amino acid sequence (version 1)

<400> 22

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

20 25 30

Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln

50 55 60

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys

65 70 75 80

Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser

85 90 95

Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val

165 170 175

Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn

180 185 190

Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu

195 200 205

Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe

210 215 220

Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val

225 230 235 240

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr

245 250 255

Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp

260 265 270

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

275 280 285

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

290 295 300

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

305 310 315 320

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

325 330 335

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

340 345 350

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

355 360 365

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 23

<211> 672

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Full-length amino acid sequence of CAR UCART19

with safety switch RQR8

<400> 23

Met Leu Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly

20 25 30

Gly Gly Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser

35 40 45

Thr Asn Val Ser Pro Ala Lys Pro Thr Thr Thr Thr Ala Cys Pro Tyr Ser

50 55 60

Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro

65 70 75 80

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

85 90 95

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

100 105 110

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

115 120 125

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg

130 135 140

Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Arg Ala Glu

145 150 155 160

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

165 170 175

Pro Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val

180 185 190

Pro Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

195 200 205

Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

210 215 220

Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln

225 230 235 240

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys

245 250 255

Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser

260 265 270

Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser

275 280 285

Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val

290 295 300

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

325 330 335

Met Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val

340 345 350

Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn

355 360 365

Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu

370 375 380

Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe

385 390 395 400

Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val

405 410 415

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr

420 425 430

Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp

435 440 445

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

450 455 460

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

465 470 475 480

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

485 490 495

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

500 505 510

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

515 520 525

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

530 535 540

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

545 550 555 560

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

565 570 575

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

580 585 590

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

595 600 605

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

610 615 620

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

625 630 635 640

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

645 650 655

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 24

<211> 20

<212> PROTEIN

<213> Mus musculus

<400> 24

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

twenty

<210> 25

<211> 121

<212> PROTEIN

<213> Mus musculus

<400> 25

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 26

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Glycine-serine linker

<400> 26

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 27

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mouse kappa (4G7) light chain for human CD19 (with A to S substitution) (CDR)

<400> 27

Asp Ile Val Met Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Arg Ser Asp

115

<210> 28

<211> 252

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extracellular binding domain (version 2)

<400> 28

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe Asp Tyr Trp Gly

100 105 110

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ala Ala

130 135 140

Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg

145 150 155 160

Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn Thr Tyr Leu Tyr Trp

165 170 175

Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met

180 185 190

Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val

210 215 220

Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly

225 230 235 240

Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp Pro

245 250

<210> 29

<211> 251

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extracellular binding domain (version 1)

<400> 29

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe Asp Tyr Trp Gly

100 105 110

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ala Ala

130 135 140

Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg

145 150 155 160

Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn Thr Tyr Leu Tyr Trp

165 170 175

Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met

180 185 190

Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val

210 215 220

Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly

225 230 235 240

Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp

245 250

<210> 30

<211> 46

<212> PROTEIN

<213> Homo sapiens

<400> 30

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

1 5 10 15

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

20 25 30

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 31

<211> 21

<212> PROTEIN

<213> Homo sapiens

<400> 31

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr

twenty

<210> 32

<211> 69

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fragment of CD8 alpha isoform 1 precursor

T cell surface glycoprotein (residues 138-206)

<400> 32

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 33

<211> 3

<212> PROTEIN

<213> Homo sapiens

<400> 33

Leu Tyr Cys

one

<210> 34

<211> 42

<212> PROTEIN

<213> Homo sapiens

<400> 34

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 35

<211> 112

<212> PROTEIN

<213> Homo sapiens

<400> 35

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<---

Claims (44)

1. A method of treating a subject who has refractory and/or recurrent non-Hodgkin's lymphoma, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR) T cells (CAR-Τ cells) containing CAR 4-1BB/ CD3-zeta for human CD19, where at least one dose is from about 20x10 ⁶ cells/dose to about 360x10 ⁶ cells/dose, and CAR-T cells contain CAR under SEQ ID NO: 1. 2. The method of claim 1 wherein the non-Hodgkin's lymphoma is large B cell lymphoma. 3. The method of claim 1 wherein the non-Hodgkin's lymphoma is follicular lymphoma. 4. The method according to any one of paragraphs. 1-3, wherein at least one dose is selected from the group consisting of approximately 20×10 ⁶ cells/dose, approximately 40×10 ⁶ cells/dose, approximately 80×10 ⁶ cells/dose, approximately 120×10 ⁶ cells/dose. dose, approximately 240×10 ⁶ cells/dose and approximately 360×10 ⁶ cells/dose. 5. The method according to any one of paragraphs. 1-4, wherein at least one dose is selected from the group consisting of about 20 x 10 ⁶ cells/dose, about 80 x 10 ⁶ cells/dose, and about 240 x 10 ⁶ cells/dose if the subject is overweight, equal to 50 kg or less. 6. The method according to any one of paragraphs. 1-4, wherein at least one dose is selected from the group consisting of approximately 20×10 ⁶ cells/dose, approximately 40×10 ⁶ cells/dose, approximately 120×10 ⁶ cells/dose, and approximately 360×10 ⁶ cells/dose. dose where the subject has a weight of more than 50 kg. 7. The method according to any one of paragraphs. 1-6, where CAR-T cells are CD52-deficient. 8. The method according to any one of paragraphs. 1-6, where CAR-T cells contain a mixture of CD52-deficient and CD-52-positive cells. 9. The method according to any one of paragraphs. 1-8, where CAR-T cells include UCART19(CD 19)CAR/RQR8+_TCRαβ-_T cells. 10. The method according to any one of paragraphs. 1-8 where CAR-T cells do not express a security switch. 11. The method according to any one of paragraphs. 1-10, wherein CAR expression is detectable in the subject for a period of at least 14 days following administration of CAR-T cells. 12. The method according to any one of paragraphs. 1-11, wherein CAR expression is detectable in the subject for a period of at least 28 days following administration of CAR-T cells. 13. The method according to any one of paragraphs. 1-12, wherein the subject has a CR or Cri state for at least 1 month following CAR-T administration. 14. The method according to any one of paragraphs. 1-13, where the subject has a CR or Cri state for at least 2 months after administration of CAR-T. 15. The method according to any one of paragraphs. 1-14, wherein the subject has a CR or Cri state for at least 6 months following CAR-T administration. 16. The method according to any one of paragraphs. 1-15, wherein the subject has a CR or Cri state for at least 12 months following CAR-T administration. 17. The method according to any one of paragraphs. 1-16 wherein the subject receives a first regimen for lymphodepletion prior to at least one dose. 18. The method of claim 17 wherein the first regimen for lymphodepletion comprises the administration of fludarabine and cyclophosphamide. 19. The method of claim 17, wherein the first regimen for lymphodepletion comprises the administration of fludarabine, cyclophosphamide, and an anti-CD52 antibody. 20. The method of claim 18 or 19, wherein the first regimen for lymphodepletion further comprises administering Mensa (sodium 2-mercaptoethanesulfonate). 21. The method according to any one of paragraphs. 18-20, wherein the first regimen for lymphodepletion further comprises administering at least one corticosteroid. 22. The method of claim 21, wherein the corticosteroid is administered immediately prior to administration of the anti-CD52 antibody. 23. The method of claim 21 or 22 wherein the corticosteroid is methylprednisolone. 24. The method according to p. 23, where methylprednisolone is administered at a dose of 2 mg/kg. 25. The method according to any one of paragraphs. 1-24 wherein the patient is premedicated for an infusion-related reaction prior to the regimen for lymphodepletion. 26. The method of claim 25, wherein the premedication comprises at least one antihistamine. 27. The method of claim 25 or 26, wherein the premedication comprises the administration of acetaminophen. 28. The method according to any one of paragraphs. 18-27 wherein fludarabine is administered at a dose of approximately 30 mg/m ² /day; cyclophosphamide is administered at a dose of about 300 mg/m ² /day, or an anti-CD52 antibody is administered at a dose of about 10 to about 13 mg/day. 29. The method according to any one of paragraphs. 19-27 wherein fludarabine is administered at a dose of approximately 30 mg/m ² /day; cyclophosphamide is administered at a dose of about 300 mg/m ² /day, and anti-CD52 antibody is administered at a dose of about 10 to about 13 mg/day. 30. The method according to any one of paragraphs. 17-29, where the first lymphodepletion regimen is started approximately 1-15 days prior to at least one dose. 31. The method according to any one of paragraphs. 17-30 wherein the first regimen for lymphodepletion is administered over a course of 1, 2, 3, 4, or 5 days. 32. The method according to any one of paragraphs. 1-31, where the subject receives a subsequent dose of CAR-T cells. 33. The method of claim 32, wherein the subject has a suboptimal response at the time of the next dose. 34. The method of claim 33, wherein the suboptimal response includes: (a) complete response (CR), complete response with incomplete recovery of blood cells (CRi) with detectable minimal residual disease (patients with leukemia) and no cytogenetic response; (b) complete bone marrow response; (c) a partial response, or (d) stable response. 35. The method according to any one of paragraphs. 32-34, where the subsequent dose contains approximately the same number of cells as the number of cells in the first dose. 36. The method according to any one of paragraphs. 32-34, where the subsequent dose contains an increased number of cells compared to the first dose. 37. The method according to any one of paragraphs. 32-34, where the subsequent dose contains a reduced number of cells compared to the first dose. 38. The method according to any one of paragraphs. 32-37, wherein the subsequent dose is administered at least 14, at least 28, at least 42, or at least 56 days after the first dose. 39. The method of claim 32, wherein the first dose of CAR-T cells is approximately 120×10 ⁶ cells and the second dose of CAR-T cells is approximately 120×10 ⁶ cells.

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