US20190125799A1 - Methods and compositions for dosing of allogeneic chimeric antigen receptor t cells - Google Patents

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

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US20190125799A1
US20190125799A1 US16/175,663 US201816175663A US2019125799A1 US 20190125799 A1 US20190125799 A1 US 20190125799A1 US 201816175663 A US201816175663 A US 201816175663A US 2019125799 A1 US2019125799 A1 US 2019125799A1
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cells
dose
car
subject
administered
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Cyril Alkis KONTO
Amina ZINAI
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Laboratoires Servier SAS
Allogene Therapeutics Inc
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Laboratoires Servier SAS
Allogene Therapeutics Inc
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Priority to US16/175,663 priority Critical patent/US20190125799A1/en
Assigned to LES LABORATOIRES SERVIER reassignment LES LABORATOIRES SERVIER ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ZINAI, AMINA
Assigned to ALLOGENE THERAPEUTICS, INC. reassignment ALLOGENE THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KONTO, CYRIL ALKIS
Publication of US20190125799A1 publication Critical patent/US20190125799A1/en
Priority to US18/484,244 priority patent/US20240041930A1/en
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Definitions

  • the present disclosure concerns therapeutic regimens for treatment of disorders, including proliferative disorders and immune disorders.
  • the subject therapeutic regimens involve administration of one or multiple doses of immune cells comprising allogeneic chimeric antigen receptor T cells.
  • the subject therapeutic regimens can be used in the prevention and/or treatment of disorders, including cancer.
  • CAR-T cells chimeric antigen receptor (CAR)-T cells.
  • CAR-T cells chimeric antigen receptor-T cells
  • the doses of allogeneic CAR-T cells required to be effective to treat disease and achieve remission in human patients are not known.
  • Provided herein are methods, compositions, and articles of manufacture for dosing and re-dosing patients with allogeneic CAR-T cells.
  • the disclosed methods reduce the risk of toxicity and/or increase efficacy, for example, by increasing exposure of the subject to the administered cells (e.g., by improving expansion and/or persistence of the administered cells).
  • This disclosure relates to therapeutic regimens for treatment of a disorder, such as cancer. More specifically, this disclosure relates to therapeutic regimens for treatment of a disorder, such as cancer, by administration of allogeneic CAR-T-cells.
  • the methods generally involve administering single or multiple doses of such cells, and/or administering a subsequent dose to a subject having been previously treated with a prior (e.g., first) dose of such cells. Also provided are cells, compositions, and articles of manufacture for use in such methods.
  • the methods involve treating an adult subject who has refractory and/or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR (e.g. UCART19 (CD19CAR/RQR8+_TCR ⁇ _T-cells), wherein the at least one dose is selected from the group consisting of about 6 ⁇ 10 5 cell/dose, about 6 ⁇ 10 6 cells/dose, about 6-8 ⁇ 10 7 cells/dose, and about 1.8-2.4 ⁇ 10 8 cells/dose.
  • CAR allogeneic chimeric antigen receptor
  • the methods involve treating a pediatric subject who has refractory and/or relapsed CD19+ B-cell acute lymphoblastic leukemia, comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR (e.g. UCART19 (CD19CAR/RQR8+_TCR ⁇ _T-cells) or UCART19 (CD19CAR/R2+_TCR ⁇ _T-cells) or UCART19 (CD19CAR/TCR ⁇ _T-cells), wherein the at least one dose is about 2-8 ⁇ 10 7 cells/dose.
  • CAR allogeneic chimeric antigen receptor
  • the methods involve treating a subject who has refractory and/or relapsed 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 allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR (CD19CAR/RQR8+_TCR ⁇ _T-cells), wherein the at least one dose is about 20 ⁇ 10 6 cells/dose to about 360 ⁇ 10 6 cells/dose, for example about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 80 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, 240 ⁇ 10 6 cells/dose, or about 360 ⁇ 10 6 cells/dose.
  • CAR allogeneic chimeric antigen receptor
  • CAR-T cells comprising an anti-human CD19 4-1BB/CD3zet
  • the methods involve (a) administering to a subject with a disease a first dose of allogeneic CAR-T cells; and (b) administering to the subject a subsequent dose of allogeneic CAR-T cells. In other embodiments, the methods are carried out by administering to the subject the subsequent dose or doses as in (b), to a subject that has been previously administered the first dose as in (a).
  • the CAR-T cells are tumor antigen-specific CAR-T cells.
  • the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19 (CD19CAR/RQR8+_TCR ⁇ _T-cells)).
  • the disease is a tumor.
  • it is a cancer, malignancy, neoplasm, or other proliferative disease or disorder, such as leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL (e.g., relapsed/refractory ALL)), non-Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung (e.g., small and non-small cell lung cancer), liver, breast, prostate, ovarian, skin, melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorec
  • CLL
  • 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 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.
  • NHL non-Hodgkin lymphoma
  • the disease is acute lymphoblastic leukemia (ALL). In some embodiments the disease is pediatric 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, e.g. adult refractory B-ALL. In some embodiments, the disease is relapsed B-ALL, e.g. adult relapsed B-ALL. In some embodiments, the disease is extramedullary disease associated, for example, with one or more of the cancers described herein.
  • the subject exhibits detectable molecular disease and/or minimum residual disease at the time of the administration of the subsequent dose. In some embodiments, the subject exhibits one or more symptoms of the disease at the time of the administration of the subsequent dose. In some embodiments, the disease is a cancer and the subject exhibits relapse at the time of initiation of the administration of the subsequent dose. In some embodiments, the disease is a leukemia or lymphoma and the subject exhibits greater than 5% blast cells in the bone marrow at the time of the administration of the subsequent dose.
  • the disease is a cancer and the subject exhibits morphologic disease.
  • the disease is a leukemia or lymphoma and the subject exhibits extramedullary disease (i.e., presence of blasts outside bone marrow) at the time of the administration of the subsequent dose.
  • the disease is a leukemia or lymphoma and the subject exhibits greater than 5% blast cells in the bone marrow at the time of the administration of the subsequent dose.
  • the disease is a cancer and the subject does not exhibit morphologic disease at the time of initiation of the administration of the subsequent dose.
  • the disease is a leukemia or lymphoma and the subject does not exhibit greater than 5% blast cells in the bone marrow at the time of the administration of the subsequent dose.
  • the methods involve (a) administering to a subject having a disease a first dose of allogeneic CAR-T cells.
  • the first dose contains about 1 ⁇ 10 4 cells, about 5 ⁇ 10 4 cells, about 1 ⁇ 10 5 cells, about 5 ⁇ 10 5 cells, about 1 ⁇ 10 6 cells, about 5 ⁇ 10 6 cells, about 6 ⁇ 10 6 cells, about 1 ⁇ 10 7 cells, about 6 ⁇ 10 7 cells, about 1 ⁇ 10 8 , about 1.8 ⁇ 10 8 cells, or about 4.8 ⁇ 10 8 cells.
  • the first dose contains about 20 ⁇ 10 6 cells to about 360 ⁇ 10 6 cells, for example about 20 ⁇ 10 6 cells, about 40 ⁇ 10 6 cells, about 80 ⁇ 10 6 cells, about 120 ⁇ 10 6 cells, 240 ⁇ 10 6 cells, or about 360 ⁇ 10 6 cells.
  • the methods further involve (b) administering to the subject a subsequent dose of CAR-T cells at a time point that is at least or more than about 5 weeks after and less than about 24 weeks after initiation of the administration in (a).
  • a subject with relapsed/refractory ALL is administered a first and subsequent dose of allogeneic CAR-T cells each containing about 6 ⁇ 10 6 cells, and the subsequent dose of CAR-T cells in (b) is administered about 99 days after initiation of the administration in (a).
  • the methods further involve the administration of additional subsequent or subsequent doses, such that a first and multiple subsequent doses are administered, e.g., in accordance with the dosing amounts and timing schedules as specified for the first and subsequent doses.
  • the first of one or more subsequent doses is administered at a time that is at least or greater than 5 weeks after the initiation of the administration of the subsequent dose.
  • the administration of the first, second, and subsequent doses includes administering at least three of the doses within about 5 weeks.
  • the subsequent dose is administered at about 16 weeks following the initiation of administration of the first dose, and an additional subsequent or subsequent dose is administered at week 17 following the initiation of administration of the first dose.
  • additional subsequent doses are administered at week 17 and/or week 34 following the initiation of administration of the first dose.
  • the time of administering the subsequent dose(s) is further one at which the subject does not exhibit an immune response, e.g., does not exhibit a detectable adaptive host immune response specific for the CAR-T after said first (or prior) dose.
  • the time between the administration of the first dose (initial dose), e.g., the initiation of the administration of the first or prior dose, and the initiation of the administration of the subsequent dose (e.g., the initiation of the administration of the subsequent dose) is greater than about 4 weeks, e.g., greater than about 5, 6, 7, 8, or 9 weeks, e.g., greater than about 20 weeks, e.g., between about 9 and about 35 weeks, between about 14 and about 28 weeks, between 15 and 27 weeks, or between 16 weeks and about 18 weeks; and/or at or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 weeks.
  • administration of the subsequent dose is more than about 5 weeks after and less than about 24 weeks after administration of the first or prior dose (e.g., initiation thereof). In some embodiments, the administration of the subsequent dose is initiated 17 weeks following the initiation of the first dose. In some embodiments, the time between administration of the first and the subsequent dose (e.g., initiation thereof) or prior and next subsequent dose is greater than about 5 weeks and less than about 24 weeks, such as between 10 and 24 weeks, such as about 17 weeks. In some embodiments, the time between administration of the first and the subsequent dose (e.g., initiation thereof) is about 17 weeks.
  • administration of the subsequent dose is more than about 7 days after and less than about 365 days after administration of the first or prior dose (e.g., initiation thereof). In some embodiments, administration of the subsequent dose (e.g., initiation thereof) is more than about 30 days after and less than about 110 days after administration of the first or prior dose (e.g., initiation thereof).
  • the subject exhibits an absence of persistence of CAR-T cells at the time of the administration of the subsequent dose.
  • the subject exhibits a suboptimal response at the time of the administration of the subsequent dose.
  • a suboptimal response may comprise any one or more of the following: (i) complete response (CR), complete response with incomplete recovery of blood count (CRi), with detectable minimal residual disease (leukemic patients) and absence of cytogenetic response; (ii) marrow complete response; (iii) partial response; or (iv) stable response.
  • administration of the first dose leads to amelioration of one or more symptoms of the disease in the subject following administration of the first dose.
  • the subject at the time of the administration of the subsequent dose, the subject has relapsed and/or one or more symptoms of the disease have increased following an initial amelioration (e.g., remission) experienced after the first dose.
  • the subsequent dose of cells contains CAR-T cells in an amount sufficient for amelioration of the disease in the subject.
  • the administration of the subsequent dose leads to a further amelioration of the disease in the subject.
  • administration of the subsequent dose leads to amelioration of the disease in the subject as compared with immediately prior to initiation of the administration of the subsequent dose.
  • administration of the subsequent dose leads to MRD negativity.
  • the method ameliorates the disease to a greater degree and/or for a greater period of time as compared to a method with an alternative dosing regimen wherein the subject is administered the cells in the first dose and the cells in the subsequent dose in a single dose.
  • the amelioration may comprise a reduction in total number of cells, e.g., tumor cells, of the disease in the subject, in an organ of the subject, in a tissue of the subject, or in a bodily fluid of the subject.
  • the amelioration may comprise a reduction in molecular detection by flow cytometry or quantitative PCR, mass or volume of a tumor, and/or a reduction in number and/or extent of metastases.
  • the amelioration comprises improvement in survival of the subject, e.g., increased time of survival or incident-free, progression-free, or relapse-free survival.
  • the disease persists following the administration of the first dose and/or the administration of the first dose is not sufficient to eradicate the disease in the subject.
  • the administration of said subsequent dose leads to amelioration of the disease in the subject as compared with immediately prior to initiation of the administration of the subsequent dose.
  • the administration of the first dose does not induce severe cytokine release syndrome (CRS) in the subject.
  • CRS severe cytokine release syndrome
  • the severity of cytokine release syndrome (CRS) may be assessed according to the modified grading described by Lee D W, et al., Blood 2014; 124(2):188-195, which is incorporated by reference herein in its entirety.
  • administration of the first dose does not induce CRS in the subject.
  • administration of the first dose does not induce severe CRS in a majority of subjects.
  • the administration of the first dose does not induce CRS encompassing a combination of (1) persistent fever (fever of at least 38 degrees Celsius for at least three days) and (2) a serum level of C reactive protein (CRP) of at least at or about 20 mg/dL, and/or does not induce CRS encompassing hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation.
  • CRP C reactive protein
  • administration of the first dose does not induce grade 3 or higher neurotoxicity in the subject. In some embodiments, based on clinical data, administration of the first dose does not induce grade 3 or higher neurotoxicity in a majority of subjects.
  • symptoms associated with a clinical risk of neurotoxicity and/or grade 3 or higher neurotoxicity include confusion, delirium, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (optionally as confirmed by electroencephalogram [EEG]), elevated levels of beta amyloid (A ⁇ ), elevated levels of glutamate, and elevated levels of oxygen radicals.
  • the subject has been treated with a therapeutic agent targeting the tumor prior to the administration of the first dose.
  • the subject is refractory or non-responsive to said therapeutic agent at the time of the administration of the first dose and/or the subsequent dose.
  • the methods subsequent to administration of the first dose and before said administration of the subsequent dose, or prior to administration of the first dose, the methods further include assessing a serum level of a factor indicative of CRS, a symptom of the disease, and/or an indicator of a host anti-recombinant receptor (e.g., anti-CAR) immune response in the subject, such as a humoral or cell-mediated immune response.
  • a host anti-recombinant receptor e.g., anti-CAR
  • the symptom of the disease detected is or comprises a total number of cells of the disease in the subject, in an organ of the subject, in a tissue of the subject, or in a bodily fluid of the subject, molecular detection by flow cytometry or quantitative PCR, mass or volume of a solid tumor, or number or extent of metastases.
  • the methods include assessing a symptom of the disease prior to administration of the subsequent dose, and based on the result of the assessment, determining the subsequent dose of cells to be administered to the subject. In some embodiments, if the assessment determines that the subject has morphologic disease, the subject is administered a subsequent dose containing less than, greater than, or about the same number of allogeneic CAR-T cells as the number of allogeneic CAR-T cells in the first dose. In some embodiments, if the assessment determines that the subject has minimal residual disease, the subject is administered a subsequent dose containing an increased number of allogeneic CAR-T cells as compared to the first dose.
  • the subsequent dose comprises about the same number of allogeneic CAR-T cells as the number 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 is the same or about the same as the number of allogeneic CAR-T cells per kilogram administered in the first dose. In other embodiments, the number of allogeneic CAR-T cells administered in the subsequent dose is greater than the number of allogeneic CAR-T cells administered in the first dose. In some embodiments, the subsequent dose comprises an increased number of such cells as compared to the first dose, such as at least 2-fold, 5-fold, or 10-fold greater than the number in the first dose. In some embodiments, the number of CAR-T cells per kilogram administered in the subsequent dose is at least at or about 2 times or at or about 3 times greater than the number of receptor-expressing (e.g., CAR-expressing) cells per kilogram administered in the first dose.
  • the number of CAR-T cells per kilogram administered in the subsequent dose is at least at or
  • the population of allogeneic CAR-T cells in the first dose expands in the subject following administration of the first dose and/or following the administration of the subsequent dose.
  • the expansion is evidenced by an increase in serum C-reactive protein (CRP) level following the administration of the first dose and/or subsequent dose as compared to just prior to the administration.
  • CCP serum C-reactive protein
  • the expansion is evidenced by PK as assessed by, e.g., without limitation, flow cytometry.
  • the expansion is evidenced by an increase in a level of CAR-encoding nucleic acid in the serum, as measured by qPCR, following the administration of the first dose and/or subsequent dose as compared to just prior to the administration.
  • the increase is at least 1, 2, or 3-fold.
  • the cells of the first dose and the cells of the second or subsequent dose are derived from the same donor. In some embodiments, the cells of the first dose and the cells of the second or subsequent dose are derived from different donors.
  • the first and/or subsequent dose is not a split dose.
  • the cells of the first dose are administered in a single pharmaceutical composition comprising the cells of the first dose and/or the cells of the subsequent dose are administered in a single pharmaceutical composition comprising the cells of the subsequent dose.
  • the first and/or subsequent dose is a split dose, for example, where the cells of the first dose are administered in a plurality of compositions, collectively comprising the cells of the first dose, over a period of no more than three days; and/or the subsequent dose is a split dose, where the cells of the subsequent dose are administered in a plurality of compositions, collectively comprising the cells of the subsequent dose, over a period of no more than three days.
  • the methods include administering a subsequent dose of allogeneic CAR-T cells to a subject previously administered a first dose of allogeneic CAR-T cells.
  • the subsequent dose of cells is administered at a time point that is at least or more than about 5 weeks after and less than about 24 weeks after initiation of the first dose.
  • the number of allogeneic CAR-T cells administered in the subsequent dose is the same as the first dose.
  • the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).
  • the number of cells administered in the first dose is between about 0.5 ⁇ 10 6 cells and 5 ⁇ 10 8 cells, between about 0.75 ⁇ 10 6 cells and 8 ⁇ 10 7 cells or between about 5 ⁇ 10 6 cells and 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 between about 0.5 ⁇ 10 6 cells and 1 ⁇ 10 9 cells, between about 1 ⁇ 10 5 cells and 3 ⁇ 10 8 cells or between about 6 ⁇ 10 5 cells and 2.4 ⁇ 10 8 cells, each inclusive.
  • the number of cells administered in the first dose of UCART19 cells is about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells. In some embodiments, the number of cells administered in the first dose of UCART19 cells is about 6 ⁇ 10 6 cells.
  • the number of cells administered in the subsequent dose of CD19-specific CAR-T cells is between about 0.5 ⁇ 10 6 cells and 1 ⁇ 10 9 cells, between about 1 ⁇ 10 5 cells and 3 ⁇ 10 8 cells or between about 6 ⁇ 10 5 cells and 2.4 ⁇ 10 8 cells, each inclusive. In some embodiments, the number of cells administered in the subsequent dose of UCART19 cells is about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells. In some embodiments, the number of cells administered in the subsequent dose of UCART19 cells is about 6 ⁇ 10 6 cells. In some embodiments, the number of cells administered in the first and subsequent dose of UCART19 cells is about 6 ⁇ 10 6 cells.
  • the methods further include administering a lymphodepletion regimen prior to the first dose and/or prior to the administration of the subsequent dose.
  • the lymphodepletion regimen comprises cyclophosphamide, fludarabine, and/or a combination thereof.
  • the lymphodepletion regimen comprises one or more of cyclophosphamide, fludarabine, a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10), and/or a combination thereof.
  • the administration of the lymphodepletion regimen includes administration of cyclophosphamide and fludarabine prior to the administration of the first dose, and a CD-52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) after administration of the first dose.
  • the administration of the lymphodepletion regimen includes administration of a lymphodepletion regimen prior to the administration of the first dose and optionally not prior to the administration of the subsequent dose.
  • the lymphodepletion regimen is administered between 2 and 10 days prior to the administration of the first dose.
  • the lymphodepletion regimen is administered between 2 and 10 days prior to the administration of the second dose.
  • the lymphodepletion regimen is administered between 2 and 10 days prior to the administration first dose and between 2 and 14 days prior to the administration of the second dose. In some embodiments, the lymphodepletion regimen is administered over the course of 1, 2, 3, 4, or 5 days.
  • a lymphodepletion regimen comprises administering about 90-120 mg/m 2 fludarabine and about 1500 mg/m 2 cyclophosphamide. In some embodiments, a lymphodepletion regimen comprises administering about 150 mg/m 2 fludarabine and about 120 mg/m 2 cyclophosphamide.
  • a lymphodepletion regimen comprises administering fludarabine at a dosage of about 30 to 150 mg/m 2 ; cyclophosphamide at a dosage of about 300 to 4000 mg/m 2 ; or a CD52 antibody (e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) at a dosage of about 0.3 to 1 mg/kg.
  • fludarabine is administered at a dosage of about 30 to 150 mg/m 2 ; cyclophosphamide is administered at a dosage of about 300 to 4000 mg/m 2 ; and a CD52 antibody (e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) is administered at a dosage of about 10 to about 13 mg.
  • a CD52 antibody e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10.
  • fludarabine is administered at a dosage of about 30 mg/m2/day; cyclophosphamide is administered at a dosage of about 300 mg/m 2 /day; or a CD52 antibody (e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) is administered at a dosage of about 10 to about 13 mg/day.
  • a CD52 antibody e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10
  • fludarabine is administered at a dosage of about 30 mg/m 2 /day; cyclophosphamide is administered at a dosage of about 300 mg/m 2 /day; and a CD52 antibody (e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) is administered at a dosage of about 10 to about 13 mg/day.
  • a CD52 antibody e.g., a CD52 antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10.
  • a lymphodepletion regimen comprises administering fludarabine and cyclophosphamide. In some embodiments, a lymphodepletion regimen comprises administering fludarabine, cyclophosphamide, and an anti-CD52 drug (e.g., a CD52 antibody having the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10). In some embodiments, a lymphodepletion regimen further comprises administering Mensa (sodium-2-mercaptoethanesolfonate). In some embodiments, a lymphodepletion regimen further comprises administering at least one corticosteroid.
  • an anti-CD52 drug e.g., a CD52 antibody having the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10
  • a lymphodepletion regimen further comprises administering Mensa (sodium-2-mercaptoethanesolfonate). In some embodiments, a lymphodepletion regimen further comprises administering at least one corticosteroid.
  • the corticosteroid is administered immediately prior to administration of an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10).
  • the corticosteroid is methylprednisolone administered at a dose of 1-5 mg/kg (e.g., 2 mg/kg).
  • the corticosteroid is administered at least two days prior to administration of CAR-T cells. In some embodiments, no corticosteroid is administered for at least two days prior to the administration of CAR-T cells.
  • a patient receives a premedication for infusion-related reaction prior to the lymphodepletion regimen.
  • the premedication may comprise, for example, an antihistamine or acetaminophen.
  • the components of the lymphodepletion regimen of fludarabine/cyclophosphamide (FC) or fludarabine/cyclophosphamide/anti-CD52 antibody (FCA) are administered simultaneously; in other embodiments, the components are administered serially.
  • the subject receives a FC regimen prior to the first dose of the CAR-T cell therapy; and a FCA regimen prior to a redosing of the CAR-T cell therapy.
  • the subject receives a FCA regimen prior to the first dose of the CAR-T cell therapy; and a second FCA regimen prior to a redosing of the CAR-T cell therapy.
  • the subject receives a FC regimen prior to the first dose of the CAR-T cell therapy, and a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) after the first dose of the CAR-T cell therapy.
  • a CD52 antibody e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10.
  • the subject receives a FC regimen prior to the first dose of the CAR-T cell therapy, and a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) after the first dose of the CAR-T cell therapy; and the subject further receives a FC regimen prior to the second/subsequent dose of the CAR-T cell therapy, and a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) after the second/subsequent dose of the CAR-T cell therapy.
  • a CD52 antibody e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10
  • the methods further include administering an agent to debulk the disease prior to administration of the first dose and/or prior to the administration of the second/subsequent dose.
  • the methods include administering a chemotherapeutic agent prior to the first dose and/or prior to the administration of the second/subsequent dose.
  • the subject has been previously treated with a chemotherapeutic agent prior to the first dose.
  • the chemotherapeutic agent is or comprises a conditioning chemotherapy, which reduces burden of the disease in the subject prior to the first dose and/or subsequent dose.
  • the administration of the chemotherapeutic agent includes administration of a chemotherapeutic agent prior to the administration of the first dose and optionally not prior to the administration of the second/subsequent dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 10 days prior to the administration of the first dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 10 days prior to the administration of the second/subsequent dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 10 days prior to the administration first dose and between 2 and 14 days prior to the administration of the second/subsequent dose.
  • cells and compositions for use and uses of allogeneic CAR-T cells and compositions for treating a disease in a subject such as a tumor or cancer.
  • cells and compositions for use and uses of allogeneic CAR-T cells and compositions for the manufacture of a medicament for treatment of a disease in a subject previously treated with allogeneic CAR-T cells are for use 4 to 24 weeks after the previous treatment.
  • the compositions or cells for use are formulated for administration of a subsequent dose in an amount sufficient for reduction in burden of a disease in the subject having been previously treated with the CAR-T cells.
  • the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).
  • the compositions or cells are for use that includes administering to a subject having the disease a first dose of allogeneic CAR-T cells.
  • the first dose contains about 1 ⁇ 106 total cells, about 6 ⁇ 106 total cells, or about 1 ⁇ 107 total cells.
  • the compositions or cells are for use that includes administering to the subject a subsequent dose of allogeneic CAR-T cells at a time point that is at least or more than about 4 weeks after and less than about 24 weeks after initiation of the administration of the first dose.
  • allogeneic CAR-T cells are provided for use in methods of treating a disease in a subject previously treated with allogeneic CAR-T cells.
  • the cells are for use between about 4 and 24 weeks after the previous treatment.
  • the cells for use are formulated for administration of a subsequent dose in an amount sufficient for reduction in burden of a disease in the subject having been previously treated with the allogeneic CAR-T cells.
  • the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).
  • the subject does not exhibit morphologic disease and/or the subject does not exhibit greater than 5% blast cells in the bone marrow.
  • the compositions or cells are for use in a method including administering to a subject having the disease a first dose of allogeneic CAR-T cells.
  • the first dose about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells.
  • the cells are for use in a method that includes administering to the subject a subsequent dose of allogeneic CAR-T cells at a time point that is at least or more than about 5 weeks after and less than about 24 weeks after initiation of said administration of the first dose.
  • the treatment includes administering the cells to the subject in a first and a subsequent dose, where the first dose includes about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells.
  • the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).
  • the cells for use are formulated and/or packaged for administration to the subject in a first and a subsequent dose and/or the treatment includes administering the cells to the subject in a first and a subsequent dose.
  • the first dose contains about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells.
  • the use includes where the first and subsequent administrations include administering the cells in one or more unit dose, each unit dose comprising about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells.
  • the cells or use includes where the first administration comprises administering a single unit dose. In some embodiments, the cells or use includes where the subsequent administration comprises administration of two or more unit doses. In some embodiments, the cells or use includes where the subsequent administration comprises administration a single unit dose.
  • a use of the cells is provided for treating a disease, wherein the disease is a tumor or a cancer.
  • the disease is a lymphoid malignancy such as for example without limitation, acute and chronic leukemias, lymphomas, multiple myelomas, a myeloid malignancy (e.g., leukemias, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms), or a mixed lineage malignancy.
  • a composition or cells are provided for use in treating acute lymphoblastic leukemia (ALL).
  • the ALL is relapsed/refractory ALL.
  • a composition or cells are provided for use in treating Non-Hodgkin's lymphoma, such relapsed or refractory Non-Hodgkin's lymphoma. In some embodiments, a composition or cells are for use in treating relapsed or refractory large B-cell or follicular lymphoma.
  • the cells, composition, or use includes where the subsequent dose is formulated for administration of about the same number of allogeneic CAR-T cells as the allogeneic CD19-specific CAR-T cells in the previous dose. In some embodiments, the composition containing the cells of the subsequent dose is formulated for administration of an increased number of allogeneic CAR-T cells as compared to the first dose or previous dose. In some embodiments, the cells, composition, or use includes where the subsequent dose is formulated for administration of less than the number of allogeneic CAR-T cells as the allogeneic CD19-specific CAR-T cells in the previous dose.
  • kits for treating a patient suffering from a disease such as cancer comprising CAR-T cells and a CD52 antibody.
  • the CAR-T cells are CD19 specific CAR-T cells, such as UCART19 cells.
  • the CAR-T cells express the CAR of SEQ ID NO: 1.
  • the CD52 antibody comprises the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10.
  • the kit comprises a first container comprising the CAR-T cells, and a second container comprising the CD52 antibody.
  • at least one of the first and the second container is a flexible cell infusion bag.
  • the kit further comprises a label or package insert comprising instructions for administering the CAR-T cells and the CD52 antibody to the subject.
  • the article of manufacture includes a plurality of containers, e.g., sealable containers, each individually comprising a unit dose of allogeneic CAR-T cells, for administration to the subject, packaging material, and/or a label or package insert.
  • the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).
  • the unit dose comprises the amount of cells to be given in the lowest dose in the methods, such as the size of the first dose. In some embodiments, the unit dose includes about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells.
  • the label or package insert includes instructions for administering a plurality of the unit doses to the subject, for example, by administering a certain number of such unit doses, e.g., one unit dose, in administration of a first dose, and then administering a subsequent dose including one or a plurality of the unit doses.
  • the instructions specify carrying out a first administration, said first administration comprising delivering one of said unit doses to the subject, and carrying out a subsequent administration, said subsequent administration comprising administering one or a plurality of said unit doses to the subject.
  • the containers are or comprise flexible cell infusion bags.
  • the methods are for allogeneic administration.
  • the label and/or packaging material further includes an identifier specific to the subject, indicating that the cells were derived from the subject and/or should be administered to the subject specifically.
  • the CAR-T cells are CD19-specific CAR-T cells (e.g., UCART19).
  • CAR-T cells are also provided.
  • a method of producing a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) directed to a target of interest comprising: (a) isolating peripheral blood mononuclear cells (PBMCs) from a healthy donor; (b) activating the T-cells in the PBMCs; (c) transducing the activated T-cells with a lentiviral vector, wherein the lentiviral vector is a self-inactivating recombinant vector expressing a CAR of interest; (d) disrupting TCR ⁇ and CD-52 gene expression in a subset of the T-cells; (e) expanding the population of T-cells; and (f) enriching the population of T-cells for TCR ⁇ -negative cells; whereby generating a population of “off the shelf” allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells).
  • the CAR-T cells
  • CAR-T cells allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR, for use in a method of treating a subject who has refractory and/or relapsed Non-Hodgkin's Lymphoma, the method comprising administering to the subject at least one dose of the CAR-T cells, wherein the at least one dose is about 20 ⁇ 10 6 cells/dose to about 360 ⁇ 10 6 cells/dose.
  • CAR-T cells comprising administering to the subject at least one dose of the CAR-T cells, wherein the at least one dose is about 20 ⁇ 10 6 cells/dose to about 360 ⁇ 10 6 cells/dose.
  • CAR-T cells for use in a method of treatment comprising: (a) administering to a subject a first dose of the 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 and less than about 200 days after initiation of said administration in (a).
  • allogeneic chimeric antigen receptor (CAR)-T cells for use in a method of treatment comprising administering a subsequent dose of the allogeneic CAR-T cells to a subject previously administered a first dose of allogeneic CAR-T cells, wherein: the subsequent dose of cells is administered at a time point that is at least or more than about 5 weeks after and less than about 24 weeks after initiation of the first dose.
  • the subject does not exhibit a detectable adaptive host immune response specific for the CAR-T cells.
  • allogeneic chimeric antigen receptor (CAR)-T cells for use in a method of treatment comprising administering to a subject a subsequent dose of the allogeneic chimeric antigen receptor (CAR)-T cells, wherein: prior to said administration, the subject has received a previous dose of the CAR-T cells in an amount sufficient to demonstrate clinical benefit in the subject; and at the time of administration, the subject does not exhibit a detectable adaptive host immune response specific for the CAR-T cells; and/or the time between said previous and subsequent doses is greater than about 5 weeks and less than about 24 weeks.
  • CAR allogeneic chimeric antigen receptor
  • CAR-T cells comprising an anti-human CD19 4-1BB/CD3zeta CAR, for use in a method of treating an adult subject who has refractory and/or relapsed CD19+ B-cell acute lymphoblastic leukemia, wherein the method comprises administering to the subject at least one dose of the CAR-T cells, wherein the at least one dose is selected from the group consisting of about 6 ⁇ 10 5 cell/dose, 6 ⁇ 10 6 cells/dose, about 6-8 ⁇ 10 7 cells/dose, and about 1.8-2.4 ⁇ 10 8 cells/dose.
  • CAR-T cells comprising an anti-human CD19 4-1BB/CD3zeta CAR
  • allogeneic chimeric antigen receptor (CAR)-T cells comprising an anti-human CD19 4-1BB/CD3zeta CAR
  • administering comprising administering to the subject at least one dose of the CAR-T cells comprising an anti-human CD19 4-1BB/CD3zeta CAR, wherein the at least one dose is about 2-8 ⁇ 10 7 cells/dose.
  • CAR-T cells for use in treating a disease in a subject previously treated with the CAR-T cells, wherein: the cells are for use between about 5 and 24 weeks after the previous treatment; and the cells are formulated for administration of a subsequent dose in an amount sufficient for amelioration of a disease in the subject having been previously treated with the CAR-T cells.
  • the methods of treatment disclosed herein are applied to subjects who have been treated with an antibody for the depletion of 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 methods of treatment.
  • These methods of treatment may comprise the use of a CAR-T cell (e.g. UCART19) deficient in CD52.
  • CAR-T cells chimeric antigen receptor-T cells
  • FIG. 1 depicts an exemplary schematic showing possible steps for a multiple dosing regimen for allogeneic CAR-T cell treatment.
  • FIG. 2A and FIG. 2B depict kinetic data from two patients receiving a second UCART19 infusion (redosing study).
  • FIG. 3A depicts an exemplary schematic showing the steps of a dosing regimen for allogeneic CAR-T cell treatment.
  • FIG. 3B depicts an exemplary schematic showing the steps of a dosing regimen for allogeneic CAR-T cell treatment.
  • FIG. 4A and FIG. 4B depict exemplary flow diagrams for allogeneic CAR-T cell manufacturing.
  • FIG. 5 depicts an exemplary engineered allogeneic anti-CD19 CAR T-cell product (CD19CAR/RQR8+_TCR ⁇ _T-cells).
  • FIG. 6A and FIG. 6B depict a study design for the use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in adult patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • UCART19 allogeneic anti-CD19 CAR-T cell product
  • FIG. 7A depicts the study status, following the initiation of the use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in adult patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • UCART19 allogeneic anti-CD19 CAR-T cell product
  • FIG. 7B depicts response, duration of remission and re-dosing of UCART19 in adult patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • FIG. 8A and FIG. 8B depict the flow cytometry PK profile and UCART19 kinetics in adult patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • FIG. 9 depicts a study design for the use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in high-risk pediatric patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • UCART19 allogeneic anti-CD19 CAR-T cell product
  • FIG. 10 depicts the study status, following the initiation of the use of an allogeneic anti-CD19 CAR-T cell product (UCART19) in high-risk pediatric patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • UCART19 allogeneic anti-CD19 CAR-T cell product
  • FIG. 11 depicts response and duration of remission (anti-leukemic activity) of UCART19 in high-risk pediatric patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • FIG. 12 depicts cellular kinetics following use of UCART19 in high-risk pediatric patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • FIG. 13A , FIG. 13B , and FIG. 13C depict chimerism data in blood following use of UCART19 in high-risk pediatric patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • FIG. 14 shows the response of Patient #18 to redosing, as described in Example 3.
  • allogeneic means that cells or population of cells used for treating patients are not originating from said patient but from a donor, but not from a Human Leucocyte Antigen (HLA) compatible donor.
  • HLA Human Leucocyte Antigen
  • autologous means that cells, a cell line, or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor.
  • HLA Human Leucocyte Antigen
  • immune cell refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptive immune response.
  • chimeric antigen receptor or alternatively a “CAR” refers to a molecule which, when in an immune effector cell, provides the cell with specificity for a target and intracellular signal generation upon engagement with the target.
  • the term “dosing regimen” refers to the total course of treatment administered to a patient, e.g., treatment with CAR-T cells.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of tumor, remission of a disease (e.g., cancer), decreasing symptoms resulting from a disease (e.g., cancer), increasing the quality of life of those suffering from a disease (e.g., cancer), decreasing the dose of other medications required to treat a disease (e.g., cancer), delaying the progression of a disease (e.g., cancer), curing a disease (e.g., cancer), and/or prolonging survival of patients having a disease (e.g., cancer).
  • “Reducing incidence” means any of reducing severity (which can include reducing need for and/or amount of (e.g., exposure to) other drugs and/or therapies generally used for this disease.
  • “Ameliorating” or “amelioration of” means a lessening or improvement of one or more symptoms as compared to not administering a treatment. “Ameliorating” also includes shortening or reduction in duration of a symptom.
  • an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results.
  • beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • An effective dosage can be administered in one or more administrations.
  • an effective dosage of drug, CAR-T cell, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • mammals are a mammal, e.g. a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • Refractory refers to a disease (e.g., cancer) that does not respond to a treatment.
  • a refractory cancer can be resistant to a treatment before or at the beginning of the treatment. In other embodiments, the refractory cancer can become resistant during a treatment.
  • a refractory cancer is also referred to herein as a resistant cancer.
  • Relapsed refers to the return of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of improvement, e.g., after prior treatment of a therapy, e.g., cancer therapy.
  • a disease e.g., cancer
  • a therapy e.g., cancer therapy
  • MRD minimal residual disease
  • vector means a construct, which is capable of delivering, and, expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • expression control sequence means a nucleic acid sequence that directs transcription of a nucleic acid.
  • An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer.
  • the expression control sequence is operably linked to the nucleic acid sequence to be transcribed.
  • an “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • antibody encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen binding fragment (i.e., “antigen-binding portion”) or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site including, for example without limitation, scFv, single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1126-1136).
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • antigen binding portion of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., at tumor antigen). Antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term “antigen binding portion” of an antibody include Fab; Fab′; F(ab′)2; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., 1989, Nature 341:544-546), and an isolated complementarity determining region (CDR).
  • Fab fragment antigen binding portion
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and 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.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • a monoclonal antibody refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • a monoclonal antibody of the disclosure exists in a homogeneous or substantially homogeneous population.
  • Humanized antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary 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 having the desired specificity, affinity, and capacity.
  • CDR complementary determining region
  • human antibody means an antibody having an amino acid sequence corresponding to that of an antibody that can be produced by a human and/or which has been made using any of the techniques for making human antibodies known to those skilled in the art or disclosed herein.
  • This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide.
  • One such example is an antibody comprising murine light chain and human heavy chain polypeptides.
  • Human antibodies can be produced using various techniques known in the art.
  • the human antibody is selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, Proc.
  • Human antibodies can also be made by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of the endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
  • the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from a subject or may have been immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.
  • variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination.
  • variable regions of the heavy and light chain each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) also known as hypervariable regions, that contribute to the formation of the antigen binding site of antibodies.
  • FRs framework regions
  • CDRs complementarity determining regions
  • variants of a subject variable region are desired, particularly with substitution in amino acid residues outside of a CDR region (i.e., in the framework region), appropriate amino acid substitution, often, conservative amino acid substitution, can be identified by comparing the subject variable region to the variable regions of other antibodies which contain CDR1 and CDR2 sequences in the same canonincal class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4): 901-917, 1987).
  • FR to flank subject CDRs e.g., when humanizing or optimizing an antibody, FRs from antibodies which contain CDR1 and CDR2 sequences in the same canonical class are preferred.
  • a “CDR” of a variable domain are amino acid residues within the variable region that are identified in accordance with the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, and/or conformational definitions or any method of CDR determination well known in the art.
  • “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as 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 comprising such carriers are formulated by 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 21st Ed. Mack Publishing, 2005).
  • PBS phosphate buffered saline
  • Compositions comprising such carriers are formulated by well
  • references to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range.
  • the disclosure encompasses 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 the main group absent one or more of the group members.
  • the disclosure also envisages the explicit exclusion of one or more of any of the claimed group members.
  • ranges throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
  • CAR chimeric antigen receptor
  • the methods may be used for treatment of, e.g., hematological neoplasms (WHO classification, 2008) and solid tumors.
  • Hematological neoplasms include, e.g., lymphoid malignancies (acute and chronic leukemias, lymphomas, multiple myelomas), myeloid malignancies (leukemias, myelodysplastic syndromes, myelodysplastic or myeloproliferative neoplasms), and mixed lineage malignancies.
  • Solid tumors include, e.g., solid tumors expressing antigen(s) targeted by the CAR-T cells used in treatment.
  • the methods are suitable for treatment of adults and pediatric population, including all subsets of age, and can be used as any line of treatment, including first line or subsequent lines.
  • the disease is a tumor.
  • 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, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung (e.g., small and non-small cell lung cancer), liver, breast, prostate, ovarian, skin, melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma,
  • CLL chronic lympho
  • 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 lymphoma (NHL).
  • NHL non-Hodgkin lymphoma
  • the disease is acute lymphoblastic leukemia (ALL). In some embodiments the disease is pediatric acute lymphoblastic leukemia (ALL).
  • the disease is an advanced lymphoid malignancy such as B-cell acute lymphoblastic leukemia (B-ALL).
  • B-ALL B-cell acute lymphoblastic leukemia
  • the disease is refractory B-ALL, e.g. adult refractory B-ALL.
  • the disease is relapsed B-ALL, e.g. adult relapsed B-ALL.
  • the disease is Non-Hodgkin's lymphoma, such as large B-cell and/or follicular lymphoma.
  • the large B-cell lymphoma may be a large B-cell lymphoma as diagnosed using histological or cytological methods according to the 2018 WHO revision of lymphoma classification: diffuse large B-cell lymphoma (DLBCL)-not-otherwise specified (NOS) (germinal center B-cell [GCB], and non-GCB), DLBCL coexistent with follicular lymphoma of any grade, intravascular large B-cell lymphoma, DLBCL associated with chronic inflammation, anaplastic lymphoma kinase positive (ALK+) DLBCL, Epstein-Barr virus positive (EBV+) DLBCL-NOS, T cell/histiocyte-rich large B cell lymphoma, DLBCL with IRF4/MUM1 rearrangement, high-grade B cell lymphomas with translocation of MYC and BCL2
  • Methods of treatment may comprise several stages including lymphodepletion (optionally, with premedication), treatment, and optional retreatment.
  • a lymphodepletion (LD) regimen is administered to the subject prior to a first and/or subsequent dose of CAR-T cells. In some embodiments, the lymphodepletion regimen is administered to the subject concurrently with a first and/or subsequent dose of CAR-T cells. In some embodiments, the lymphodepletion regimen is administered before, during, and/or after a first and/or subsequent dose of CAR-T cells.
  • LD lymphodepletion
  • LD starts prior to, concurrently with, or after a CAR-T infusion. Doses and timing of LD administration may be adapted with regard to the first or subsequent dosings of CAR-T. In some embodiments, the duration of LD is about 3 to 5 days. In some embodiments, a time window between the end of LD and start of CAR-T administration is between about of 2 days to about 2 weeks. In some embodiments, LD is initiated about 15 to 7 days prior to administration of a dose of CAR-T cells. In some embodiments, LD is initiated about 19 to 5 days prior to administration of a dose of CAR-T cells.
  • LD is initiated about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days prior to administration of a dose of CAR-T cells.
  • duration of a LD regimen is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.
  • a dose of CAR-T cells is administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the end of LD.
  • a LD regimen comprises administration of one or more chemotherapeutic drugs.
  • a LD regimen comprises administration of anti-CD52 antibody, such as an antibody that recognizes the human cluster of differentiation (CD) 52 antigen, a cell surface glycoprotein expressed on most lymphoid cells.
  • a CD52 monoclonal antibody is one that is directed against the 21-28 kD cell surface glycoprotein CD52.
  • CD52 is an abundant molecule (approximately 5 ⁇ 10 5 antibody binding sites per cell) present on at least 95% of all human peripheral blood lymphocytes and monocytes/macrophages.
  • Exemplary CD52 antibodies for use in the methods and compositions described herein include, for example, alemtuzumab.
  • a CD52 antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as shown in Table 1 below.
  • HCDR1 DFYMN SEQ ID NO: 2
  • HCDR2 FIRDKAKGYTTEYNPSVKG SEQ ID NO: 3
  • HCDR3 EGHTAAPFDY SEQ ID NO: 4
  • LCDR1 KASQNIDKYLN SEQ ID NO: 5
  • LCDR2 NTNNLQT SEQ ID NO: 6
  • LCDR3 LQHISRPRT SEQ ID NO: 7
  • a CD52 antibody comprises a VH and/or a VL comprising the sequences shown in Table 2 below.
  • a CD52 antibody comprises a VH having the sequence of SEQ ID NO: 8, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8.
  • a CD52 antibody comprises a VL having the sequence of SEQ ID NO: 10, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10.
  • a CD52 antibody comprises a VH having the sequence of SEQ ID NO: 8 and a VL having the sequence of SEQ ID NO: 10. In some embodiments, a 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.
  • the anti-CD52 antibody is a recombinant humanized IgG1 kappa monoclonal antibody (mAb).
  • the anti-CD52 antibody is alemtuzumab.
  • Alemtuzumab is a recombinant DNA-derived humanized monoclonal antibody directed against the 21-28 kD cell surface glycoprotein, CD52. See, e.g., Saif et al., Pediatr Transplant 2015 March; 19(2):211-8.
  • the anti-CD52 antibody comprises one or more CDR sequences isolated or derived from the CDRs of alemtuzumab.
  • the anti-CD52 antibody comprises the sequence of SEQ ID NO: 8, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8.
  • the anti-CD52 antibody comprises the sequence of SEQ ID NO: 10, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10.
  • the anti-CD52 antibody comprises an HCDR1 comprising the sequence of SEQ ID NO: 2, a HCDR2 comprising the sequence of SEQ ID NO: 3, a HCDR3 comprising the sequence of SEQ ID NO: 4, a LCDR1 comprising the sequence of SEQ ID NO: 5, a LCDR1 comprising the sequence of SEQ ID NO: 6, and/or a LCDR3 comprising the sequence of SEQ ID NO: 7.
  • the anti-CD52 antibody comprises an HCDR1 comprising the sequence of SEQ ID NO: 2, a HCDR2 comprising the sequence of SEQ ID NO: 3, a HCDR3 comprising the sequence of SEQ ID NO: 4, a LCDR1 comprising the sequence of SEQ ID NO: 5, a LCDR1 comprising the sequence of SEQ ID NO: 6, and a LCDR3 comprising the sequence of SEQ ID NO: 7; wherein the anti-CD52 antibody comprises the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8 and/or SEQ ID NO: 10.
  • LD comprises administration of a combination of therapies.
  • the combination includes: fludarabine (range total dose about 90 to 150 mg/m 2 ) and cyclophosphamide (range total dose about 1000 to 4000 mg/m 2 ), with or without an anti-CD52 drug (e.g., an anti-CD52 antibody such as an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) (total dose from about 0.3 to about 1 mg/kg, or a flat dose of from about 30 mg to about 40 mg, from about 25 to about 60 mg, or from about 100 mg to about 120 mg).
  • an anti-CD52 drug e.g., an anti-CD52 antibody such as an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10
  • total dose from about 0.3 to about 1 mg/kg, or a flat dose of from about 30 mg to about 40 mg, from about 25 to about 60 mg, or from about 100 mg to about 120 mg.
  • the combination includes: fludarabine (about 30 mg/m 2 ) and cyclophosphamide (range total dose about 500 to 600 mg/m 2 ), with or without an anti-CD52 drug (e.g., CD52 antibody) (total dose from about 0.3 to about 1 mg/kg, or a flat dose of from about 30 mg to about 40 mg, from about 25 to about 60 mg, or from about 100 mg to about 120 mg).
  • fludarabine about 30 mg/m 2
  • cyclophosphamide range total dose about 500 to 600 mg/m 2
  • an anti-CD52 drug e.g., CD52 antibody
  • the combination includes: fludarabine (about 30 mg/m 2 ) and cyclophosphamide (about 300 mg/m 2 ), with or without an anti-CD52 drug (e.g., CD52 antibody) (total dose from about 0.3 to about 1 mg/kg, or a flat dose of from about 30 mg to about 40 mg, from about 25 mg to about 60 mg, or from about 100 mg to about 120 mg).
  • the combination includes: fludarabine (about 90 mg/m 2 ), cyclophosphamide (about 1500 mg/m 2 ) and with or without an anti-CD52 drug (e.g. anti-CD52 antibody, about 1 mg/kg).
  • the combination includes: fludarabine (about 150 g/m 2 ) and cyclophosphamide (about 130 mg/kg), with or without an anti-CD52 drug (e.g. anti-CD52 antibody, total dose from about 0.3 to about 1 mg/kg, or a flat dose of from about 30 mg to about 40 mg, from about 25 to about 60 mg, or from about 100 mg to about 120 mg).
  • an anti-CD52 drug e.g. anti-CD52 antibody, total dose from about 0.3 to about 1 mg/kg, or a flat dose of from about 30 mg to about 40 mg, from about 25 to about 60 mg, or from about 100 mg to about 120 mg.
  • the combination includes: fludarabine (about 150 g/m 2 ) and cyclophosphamide (about 120 mg/kg or about 130 mg/kg), with or without an anti-CD52 drug (e.g.
  • an anti-CD52 antibody total dose from about 0.3 to about 1 mg/kg, or a flat dose of from about 30 mg to about 40 mg, from about 25 to about 60 mg, or from about 100 mg to about 120 mg).
  • 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).
  • 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).
  • these above doses are administered during the course of one day. In some embodiments, these above doses are administered over multiple days.
  • fludarabine and cyclophosphamide are administered on a first day, and the anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) is administered on a second day.
  • fludarabine and cyclophosphamide are administered on a first day before administration of the 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 on a second day; wherein the second day is the same day that CAR-T cells are administered or the second day is after the CAR-T cells are administered.
  • fludarabine and cyclophosphamide are administered on a first day
  • CAR-T cells are administered on a second day
  • an anti-CD52 antibody e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10
  • an anti-CD52 antibody 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 the second day.
  • fludarabine and cyclophosphamide are administered before 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 administration of the CAR-T cells.
  • an anti-CD52 antibody e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10
  • a lymphodepletion regimen comprises administration of fludarabine and cyclophosphamide (FC). In some embodiments, a lymphodepletion regimen comprises administration of fludarabine and anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) (FA). In some embodiments, a lymphodepletion regimen comprises administration of cyclophosphamide and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) (CA).
  • FC fludarabine and cyclophosphamide
  • FA anti-CD52 antibody
  • CA anti-CD52 antibody
  • a lymphodepletion regimen comprises administration of fludarabine, cyclophosphamide, and an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) (FCA).
  • an anti-CD52 antibody e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10.
  • a second lymphodepletion regimen can be more or less intense compared to a first lymphodepletion regimen (for example, based on recovery of lymphocytes, neutrophils, and viral reactivation after a first dose). For example, at the time of redosing, if lymphocyte and neutrophil levels are high, a strong or aggressive lymphodepletion regimen may be used. Alternatively, at the time of redosing, if lymphocyte levels are low, a weaker or less aggressive lymphodepletion regimen may be used.
  • a strong or aggressive lymphodepletion regimen is used. In some embodiments, if the number of blasts at the time of redosing is low, a weaker or less aggressive lymphodepletion regimen is used.
  • an increased intensity of LD regimen may be applied at the time of redosing (with or without anti-CD52 drug).
  • a reduced intensity of LD regimen may be applied, for example, in case of grade 3-4 lymphopenia at time of re-dosing (with or without anti-CD52 drug).
  • the components of the lymphodepletion regimen of fludarabine/cyclophosphamide (FC) or fludarabine/cyclophosphamide/anti-CD52 antibody (FCA) are administered simultaneously; in other embodiments, the components are administered serially.
  • the subject receives a FC regimen prior to the first dose of the CAR-T cell therapy; and a FCA regimen prior to a redosing of the CAR-T cell therapy.
  • the subject receives a FCA regimen prior to the first dose of the CAR-T cell therapy; and a second FCA regimen prior to a redosing of the CAR-T cell therapy.
  • Exemplary LD regimens are provided in Tables 3A, 3B, 3C, 3D and 3E.
  • the timing indicated under Schedule is relative to the timing of administration of a dose of CAR-T cells (D0), in days. Negative numbers indicate days prior to administration of CAR-T cells (at D0).
  • the LD regimen may further comprise treatment with Mensa (sodium 2-mercaptoethanesulfonate).
  • the methods may comprise premedication for infusion-related reaction prior to the lymphodepletion regimen.
  • the methods may comprise a premedication administered before treatment with an anti-CD52 antibody.
  • the premedication may comprise treatment with high dose corticosteroids.
  • the premedication may comprise treatment with 2 mg/kg methylprednisolone.
  • the premedication is administered immediately before infusion with an anti-CD52 antibody.
  • the premedication is discontinued at least one or at least two days prior to any infusion of CAR-T cells (e.g., UCART19).
  • the premedication may comprise treatment with one or more of an antihistamine, cimedidine, ranitidine, and acetaminophen.
  • the antihistamine may be administered, for example, about 1 day, about 1 hour, or about 0.5 hour before administration of an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10).
  • the acetaminophen may be administered, for example, about 1 day, about 1 hour, or about 0.5 hour before administration of an anti-CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10).
  • CAR-T cells are administered by intravenous infusion.
  • the intravenous infusion is over 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.
  • a method of treating an adult subject who has refractory and/or relapsed CD19+ B-cell acute lymphoblastic leukemia comprises comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR, wherein the at least one dose is selected from the group consisting of about 6 ⁇ 10 5 cell/dose, about 6 ⁇ 10 6 cells/dose, about 6-8 ⁇ 10 7 cells/dose, and about 1.8-2.4 ⁇ 10 8 cells/dose.
  • the CAR-T cells express the CAR of SEQ ID NO: 1.
  • the CAR-T cells are UCART19(CD19)CAR/RQR8+_TCR ⁇ _T-cells. In some embodiments, the CAR-T cells are CD-52 deficient. In some embodiments, the CAR-T cells are a mixture of CD52-deficient and CD52-positive cells. In some embodiments, the CAR-T cells express a safety switch, such as RQR8.
  • a method of treating a pediatric subject who has refractory and/or relapsed CD19+ B-cell acute lymphoblastic leukemia comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR, wherein the at least one dose is about 2-8 ⁇ 10 7 cells/dose.
  • the CAR-T cells express the CAR of SEQ ID NO: 1.
  • the CAR-T cells are UCART19(CD19)CAR/RQR8+_TCR ⁇ _T-cells.
  • the CAR-T cells are CD-52 deficient.
  • the CAR-T cells are a mixture of CD52-deficient and CD52-positive cells.
  • the CAR-T cells express a safety switch, such as RQR8.
  • a method of treating a subject who has Non-Hodgkin's lymphoma comprises administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising (e.g., an anti-human CD19 4-1BB/CD3zeta CAR), wherein the at least one dose is about 20 ⁇ 10 6 cells/dose to about 360 ⁇ 10 6 cells/dose.
  • CAR allogeneic chimeric antigen receptor
  • the at least one dose is selected from the group consisting of about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 80 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, 240 ⁇ 10 6 cells/dose, and about 360 ⁇ 10 6 cells/dose.
  • the CAR-T cells express the CAR of SEQ ID NO: 1.
  • the CAR-T cells are UCART19(CD19)CAR/RQR8+_TCR ⁇ _T-cells.
  • the CAR-T cells are CD-52 deficient.
  • the CAR-T cells are a mixture of CD52-deficient and CD52-positive cells.
  • the CAR-T cells express a safety switch, such as the RQR8 safety switch described herein.
  • CAR expression is detectable in the subject for up to at least 14 days or at least 28 days after administration of the CAR.
  • the subject exhibits a CR (complete response) or Cri (complete response with incomplete recovery of blood count) for at least 1 month, at least 1.3 month, at least 1.4 month, at least 1.6 month, at least 1.8 month, at least 2 months, at least 2.3 months, at least 1.4 month, at least 1.6 month, at least 1.8 month, at least 3 months, at least 3.3 months, at least 3.4 months, at least 3.6 months, at least 3.8 months, at least 6 months, at least 12 months, or at least 36 months after CAR-T (e.g., UCART19) administration.
  • CAR-T e.g., UCART19
  • the methods involve administering one or more subsequent doses of cells to subjects having received a first dose, and/or administering the first and one or more subsequent doses.
  • the doses generally are administered in particular amounts and according to particular timing parameters.
  • the methods generally involve administering a first dose of cells, thereby reducing disease burden, followed by a subsequent dose of cells, administered during a particular time window with respect to the first dose, or the administration of the subsequent dose to a subject having received such a first dose.
  • additional subsequent doses then are administered, for example, within the same or a similar window of time with respect to the subsequent dose.
  • the number of cells administered and timing of the multiple doses are designed to improve one or more outcomes, such as to reduce the likelihood or degree of toxicity to the subject, improve exposure of the subject to and/or persistence of the administered cells, and/or improve therapeutic efficacy.
  • articles of manufacture containing the cells and designed for administration following such dosing regimens.
  • a subject to be treated with one or more subsequent doses of cells is in relapse.
  • the relapse may be, for example, a morphological relapse characterized by greater than or equal to 5% of blasts in bone marrow, the presence of extramedullary disease associated with the presence of blasts in bone marrow, or the presence of a minimum residual disease (MRD) defined as greater than or equal to 3-10 blasts in bone marrow.
  • MRD minimum residual disease
  • a subject to be treated with one or more subsequent doses of cells exhibited a suboptimal response after administration of the initial dose.
  • the subject may exhibit the presence of a minimum residual disease (MRD) at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days post administration of the initial dose.
  • MRD minimum residual disease
  • a subject to be treated with one or more subsequent doses of cells exhibits a non-persistence of CARs after administration of the initial dose.
  • a subject may present CARs detectable after the initial dose (D0, CAR expansion), but does not have detectable CAR after D0.
  • the lack of detectable CAR occurs even though the subject exhibits some signs of activity of the CAR-T cells (such as cytokine release syndrome).
  • a subject to be treated with one or more subsequent doses of cells exhibits no substantial expansion of CAR-T cells.
  • the subject has no detectable CAR expansion and no response at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days post administration of the initial dose.
  • the subject has no detectable CAR expansion and some signs of treatment response (e.g., MRD positive subject) at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days post administration of the initial dose.
  • the subject has no detectable car expansion and complete response (e.g., MRD negative subject) at about 7, about 14, about 28, about 60, about 90, about 120, or about 365 days post administration of the initial dose.
  • the provided methods involve a subsequent dose of cells administered at about the same number, and hence at a similar dose, as the first dose of cells.
  • administration of a subsequent dose of cells can be advantageous compared to a single dose.
  • administration of subsequent dose(s) of cells such as a dose of about 6 ⁇ 10 5 cells, about 6 ⁇ 10 6 cells, about 6 ⁇ 10 7 cells, about 8 ⁇ 10 7 cells, about 1.8 ⁇ 10 8 cells, or about 2.4 ⁇ 10 8 cells, are associated with an increased overall survival in subjects, particularly in subjects that exhibit morphological disease prior to treatment.
  • the methods include administering a first dose of cells that can expand in the presence of disease-associated antigens and reduce symptoms associated with disease but without the same degree of toxic outcomes that may be associated with a higher dose.
  • the first dose is generally also large enough to be effective in reducing disease burden. In some cases, the first dose is large enough to reduce disease burden if the cell dose is sufficient to expand in vivo and debulk disease. In some embodiments, the cells of the first dose thereby debulk or reduce disease burden, e.g., tumor size, without effecting severe unwanted outcomes. In some cases, the first dose is an amount of cells that is effective to reduce tumor burden, such as by reducing disease from a morphological setting to minimum 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 burden, the first dose may be higher.
  • MRD minimum residual disease
  • a risk-adapted dosing regimen may be used for determining the appropriate number or amount or relative number or amount of cells or CAR-T cells in the dose. For example, in some aspects, prior to infusion of CAR-T cells, the disease burden of the subject is determined and, based on the disease burden, a first dose of CAR-T cells (e.g.
  • low or high dose is selected that can minimize toxicity and maximize efficacy, for example, based on observations described above and elsewhere herein in which higher doses of recombinant receptor-expressing cells (e.g., CAR-expressing, such as CAR-expressing T cells) can be associated with toxic outcomes, such as severe neurotoxicity, in subjects having morphological disease burden, which are not necessarily observed, or are not observed to as great of an extent, in subjects with relatively lower disease burden.
  • CAR-expressing such as CAR-expressing T cells
  • toxic outcomes such as severe neurotoxicity
  • subjects having morphological disease burden which are not necessarily observed, or are not observed to as great of an extent, in subjects with relatively lower disease burden.
  • subjects with a high marrow tumor burden prior to treatment which, in some cases, can be associated with a greater CAR T-cell expansion following treatment, were observed to have a higher risk of developing severe neurotoxicity that may require ICU care.
  • a subject is assessed for response after a first dose of allogeneic CAR-T cells. Such assessment may be conducted at about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks after administration of the first or previous dose of allogeneic CAR-T cells. In some embodiments, a subject is assessed for response at between about one, about two, or about three months after administration of a first dose of allogeneic CAR-T cells.
  • expression of antigens targeted by the CAR-T may be confirmed before administering a subsequent dose of CAR-T cells.
  • expression of antigens may be assessed by flow cytometry in blood, in other matrices, or in solid tumors.
  • a subject may be assessed for the absence of anti-HLA antibodies developed by the subject against prior CAR-T administered.
  • a subject may be assessed for the absence of anti-scFv antibodies developed by the subject after a prior CAR-T dosing.
  • a subject may be assessed for the absence any major safety issue after any course of CAR-T (first or subsequent courses).
  • a subject may be assessed for suitability for a subsequent lymphodepletion associated with CAR-T dosing.
  • absence of donor specific anti-HLA, anti-CAR scFv, and/or anti-TALEN antibodies after a CAR-T cell dosing indicates suitability of the subject for administration of a subsequent dose of CAR-T cells.
  • absence of persistence of CAR-T cells beyond the day of the first evaluation of response after a CAR-T cell dosing indicates suitability of the subject for administration of a subsequent dose of CAR-T cells.
  • a subject may be assessed for absence of persistence of CAR-T cells beyond the day of the first evaluation of response after a CAR-T cell dosing. Assessment may be conducted according to the disease at, 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 response evaluated.
  • Parameters for kinetics evaluation include, e.g., AUC0-timepoint of response evaluation, Cmax (peak expansion of transgene CAR-T, time of last observed quantifiable transgene, and level of CAR-T cell expansion in any other tissues or fluids.
  • a suboptimal response after first CAR-T, irrespective of CAR-T persistence, dosing may include one or more of the following: (a) complete response (CR) or complete response with incomplete recovery of blood count (CRi), with detectable minimal residual disease (e.g., in leukemic patients); (b) absence of cytogenetic response; (c) marrow complete response; (d) partial response; and/or (e) stable response.
  • a subject may be assessed for an adverse event beyond the day of the first evaluation of response after administration of a dose of CAR-T cells. Assessment may be conducted at, 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. In some embodiments, assessment may be conducted within, e.g., the first four weeks (up to Day 28) after a CAR-T dosing. Exemplary adverse events are summarized in Table 4.
  • disease relapse may refer to relapse after having achieved an optimal CR after first CAR-T dosing. In some embodiments, disease relapse may refer to relapse after one or more CR is obtained with CAR-T, irrespective of when the relapse occurs.
  • a subsequent CAR-T dose may be issued from the same or another donor (donor of healthy cells used for CAR-T manufacturing).
  • the same or different (higher or lower) dose of CAR-T cells may be applied in a subsequent dose.
  • the CAR-T cells may be administered as a single dose or as a split dose.
  • a dose can be split in two or more doses within a period, such as e.g., a seven day period.
  • a split dose comprises first and second administrations within a period.
  • an equal number of cells is administered at each of the first and second administrations.
  • fewer cells are administered during the first administration than are administered during the second administration.
  • a greater number of cells is administered during the first administration than is administered during the second administration.
  • a split dose comprises first, a second administration, and a third administration within a period.
  • an equal number of cells is administered at each of the first, second, and third administrations. In some embodiments, fewer cells are administered during the first administration than are administered during the second and third administrations. In some embodiments, a greater number of cells is administered during the first administration than is administered during the second and third administrations. In some embodiments, fewer cells are administered during the second administration than are administered during the first and third administration. In some embodiments, a greater number of cells is administered during the second administration than is administered during the first and third administration. In some embodiments, fewer cells are administered during the third administration than are administered during the first and second administration. In some embodiments, a greater number of cells is administered during the third administration than is administered during the first and second administration.
  • a split dose is administered over a first day and a second day. In some embodiments, half of the dose is administered on the first day and half of the dose is administered on the second day. In some embodiments, one-third of the dose is administered on the first day, and two-thirds of the dose are administered on the second day. In some embodiments, two thirds of the dose are administered on the first day, and one third of the dose is administered on the second day. In some embodiments, each partial dose of the split dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of one another. In particular embodiments, each partial dose of the split dose is administered within 7 days of one another. In some embodiments, each partial dose is administered on a sequential day.
  • a split dose is administered over a first day, a second day, and a 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, each partial dose of the split dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of one another. In particular embodiments, each partial dose of the split dose is administered within 7 days of one another. In some embodiments, each partial dose is administered on a sequential day.
  • a split dose is administered over at least three days, for example at least four days, at least five days, at least six days, or at least seven days.
  • allogeneic CAR-T cells are administered using a flat dose.
  • allogeneic CAR-T cells are administered using dose-banding.
  • dose-banding may be used to avoid the risk of a wide range of CAR-T cell exposure.
  • a weight band may be used.
  • subjects ⁇ 66 kg may be administered X dose, and subjects >66 kg may be administered about 1.33 ⁇ dose.
  • subjects >50 kg may be administered one dose, and subjects ⁇ 50 kg may be administered a different dose.
  • Exemplary dose levels for a first dose of UCART19 are provided in Table 5a, for use in adult patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • UCART19 dose expressed in number of cells Estimated UCART19 Patient
  • UCART19 dose level weight ⁇ 66 kg weight ⁇ 66 kg dose/kg DL ⁇ 1 6 ⁇ 10 5 cells 1 ⁇ 10 4 cells/kg DL1 6 ⁇ 10 6 cells 1 ⁇ 10 5 cells/kg DL2 6 ⁇ 10 7 cells 8 ⁇ 10 7 cells 1 ⁇ 10 6 cells/kg DL3 1.8 ⁇ 10 8 cells 2.4 ⁇ 10 8 cells 3 ⁇ 10 6 cells/kg
  • Exemplary dose levels for a first dose of UCART19 are provided in Table 5b, for use in high-risk pediatric patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • Exemplary dosage forms for a first dose of UCART19 are provided in Table 5c, for use in high-risk pediatric patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • Exemplary dosages for a first dose of UCART19 are provided in Table 5d, for use in adult patients with Non-Hodgkin's lymphoma, such as relapsed/refractory large B-cell and/or follicular lymphoma.
  • an allogeneic CAR-T cell dosing regimen comprises administering a first dose of CAR T-cells ranging between about 1 ⁇ 10 5 and 5 ⁇ 10 8 cells, or between about 1 ⁇ 10 4 cells/kg to about 3 ⁇ 10 6 cells/kg. In some embodiments, an allogeneic CAR-T cell dosing regimen comprises administering a first dose of CAR T-cells of about 6 ⁇ 10 6 cells (flat dose).
  • an allogeneic CAR-T cell dosing regimen comprises administering a first dose of about 1 ⁇ 10 4 , about 2 ⁇ 10 4 , about 3 ⁇ 10 4 , about 4 ⁇ 10 4 , about 5 ⁇ 10 4 , about 6 ⁇ 10 4 , about 7 ⁇ 10 4 , about 8 ⁇ 10 4 , about 9 ⁇ 10 4 , about 10 ⁇ 10 4 , about 1 ⁇ 10 5 , about 2 ⁇ 10 5 , about 3 ⁇ 10 5 , about 4 ⁇ 10 5 , about 5 ⁇ 10 5 , about 6 ⁇ 10 5 , about 7 ⁇ 10 5 , about 8 ⁇ 10 5 , about 9 ⁇ 10 5 , about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , about 6 ⁇ 10 6 , about 7 ⁇ 10 6 , about 8 ⁇ 10 6 , about 9 ⁇ 10 6 , about 1 ⁇ 10 7 , about 2 ⁇ 10 7 , about 3 ⁇ 10 7 , about 4 ⁇ 10 7 , about 5 ⁇ 10 4 , about
  • a dosing regimen comprises administering a first dose of about 1 ⁇ 10 4 cells, about 6 ⁇ 10 6 cells, about 7 ⁇ 10 6 cells (weight banded), or about 1.8 ⁇ 10 8 cells (weight banded). In some embodiments, a dosing regimen comprises administering a first dose of about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 80 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, 240 ⁇ 10 6 cells/dose, or about 360 ⁇ 10 6 cells/dose (weight banded).
  • the dose is selected from the group consisting of about 20 ⁇ 10 6 cells/dose, about 80 ⁇ 10 6 cells/dose and about 240 ⁇ 10 6 cells/dose when the subject has a weight of less than or equal to 50 kg. In some embodiments, the dose is selected from the group consisting of about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, and about 360 ⁇ 10 6 cells/dose when the subject has a weight of greater than 50 kg.
  • a dosing regimen comprises administering a subsequent dose of about 1 ⁇ 10 4 , about 2 ⁇ 10 4 , about 3 ⁇ 10 4 , about 4 ⁇ 10 4 , about 5 ⁇ 10 4 , about 6 ⁇ 10 4 , about 7 ⁇ 10 4 , about 8 ⁇ 10 4 , about 9 ⁇ 10 4 , about 10 ⁇ 10 4 , about 1 ⁇ 10 5 , about 2 ⁇ 10 5 , about 3 ⁇ 10 5 , about 4 ⁇ 10 5 , about 5 ⁇ 10 5 , about 6 ⁇ 10 5 , about 7 ⁇ 10 5 , about 8 ⁇ 10 5 , about 9 ⁇ 10 5 , about 1 ⁇ 10 6 , about 2 ⁇ 10 6 , about 3 ⁇ 10 6 , about 4 ⁇ 10 6 , about 5 ⁇ 10 6 , about 6 ⁇ 10 6 , about 7 ⁇ 10 6 , about 8 ⁇ 10 6 , about 9 ⁇ 10 6 , about 1 ⁇ 10 7 , about 2 ⁇ 10 7 , about 3 ⁇ 10 7 , about 4 ⁇ 10 7 , about 5 ⁇ 10 7 , about 6 ⁇ 10 7 , about 6
  • a dosing regimen comprises administering a subsequent dose of about 1 ⁇ 10 4 cells, about 6 ⁇ 10 6 cells, about 7 ⁇ 10 6 cells, or about 1.8 ⁇ 10 8 cells.
  • an allogeneic CAR-T cell dosing regimen comprises administering a subsequent dose of CAR-T cells of about 6 ⁇ 10 6 cells (flat dose).
  • a dosing regimen comprises administering a subsequent dose of about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 80 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, 240 ⁇ 10 6 cells/dose, or about 360 ⁇ 10 6 cells/dose (weight banded).
  • the dose is selected from the group consisting of about 20 ⁇ 10 6 cells/dose, about 80 ⁇ 10 6 cells/dose and about 240 ⁇ 10 6 cells/dose when the subject has a weight of less than or equal to 50 kg. In some embodiments, the dose is selected from the group consisting of about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, and about 360 ⁇ 10 6 cells/dose when the subject has a weight of greater than 50 kg.
  • an allogeneic CAR-T cell dosing regimen comprises administering a first dose of CAR T-cells of about 6 ⁇ 10 6 cells and a subsequent dose of CAR T-cells of about 6 ⁇ 10 6 cells.
  • a subsequent dose of CAR-T cells has about the same number of cells as the previous dose of CAR-T cells.
  • a subsequent dose of CAR-T cells has more cells as the previous dose of CAR-T cells.
  • a subsequent dose of CAR-T cells has fewer cells as the previous dose of CAR-T cells.
  • a subsequent dose of allogeneic CAR-T cells may be administered during a particular time window with respect to an initial or previous dose of allogeneic CAR-T cells.
  • a subsequent dose of allogeneic CAR-T cells is administered about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks after administration of an initial or previous dose of allogeneic CAR-T cells.
  • a subsequent dose of allogeneic CAR-T cells is administered between 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 administration of an initial or previous dose of allogeneic CAR-T cells.
  • a subsequent dose of allogeneic CAR-T cells is administered about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 days after administration of an initial or previous dose of allogeneic CAR-T
  • the time between the administration of the first dose (initial dose), e.g., the initiation of the administration of the first or prior dose, and the initiation of the administration of the subsequent dose (redose, e.g., the initiation of the administration of the subsequent dose) is greater than about 4 weeks, e.g., greater than about 5, 6, 7, 8, or 9 weeks, e.g., greater than about 20 weeks, e.g., between about 9 and about 35 weeks, between about 14 and about 28 weeks, between 15 and 27 weeks, or between 16 weeks and about 18 weeks; and/or at or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 weeks.
  • administration of the subsequent dose is more than about 5 weeks after and less than about 24 weeks after administration of the first or prior dose (e.g., initiation thereof). In some embodiments, the administration of the subsequent dose is initiated 17 weeks following the initiation of the first dose. In some embodiments, the time between administration of the first and the subsequent dose (e.g., initiation thereof) or prior and next subsequent dose is greater than about 5 weeks and less than about 24 weeks, such as between 10 and 24 weeks, such as about 17 weeks. In some embodiments, the time between administration of the first and the subsequent dose (e.g., initiation thereof) is about 17 weeks.
  • the subject receives a redose from 28 days to 9 months after the first CAR-T infusion.
  • the same lymphodepletion regimen may be applied as for the first CAR-T dosing, or the regimen may be adapted to the patient's status and the degree of recovery of the host immune system.
  • a further subsequent dose of allogeneic CAR-T cells may be administered.
  • a further subsequent dose of allogeneic CAR-T cells may be administered.
  • two or more subsequent doses of allogeneic CAR-T cells may be administered after a first dose.
  • Various regimens for subsequent dosing can used to extend durability of allogeneic CAR-T response.
  • subsequent doses of CAR-T can be administered, e.g., every other month (post first dose of CAR-T), or every 3 months. Any number of subsequent infusions may be administered. Timing of re-dosing may be adapted to patient's health status.
  • An exemplary dosing regimen is depicted in FIG. 1 .
  • a dosing regimen comprises administering a first dose of about 6 ⁇ 10 6 CAR-T cells, followed by a subsequent dose of about 6 ⁇ 10 6 CAR-T cells administered at about 99 days after administration of the first dose.
  • a dosing regimen comprises administering a first dose of about 6 ⁇ 10 6 CAR-T cells, followed by a maintenance dose of about 6 ⁇ 10 6 CAR-T cells every 4 weeks until molecular remission is achieved.
  • a dosing regimen comprises administering a first dose of about 6 ⁇ 10 6 CAR-T cells, followed by a maintenance dose of about 6 ⁇ 10 6 CAR-T cells every 8 weeks until molecular remission is achieved.
  • a dosing regimen comprises administering a bimonthly dose of about 6 ⁇ 10 6 CAR-T cells. In other embodiments, a dosing regimen comprises administering a dose of about 6 ⁇ 10 6 CAR-T cells.
  • the first dose and the first subsequent and additional subsequent doses are separated in time from each other by at least four weeks.
  • the dosing regimen can vary over time.
  • an initial candidate dosage can be about 1.1 to about 2.3 ⁇ 10 5 cells/kg CAR-T cells.
  • a typical flat dosage of CAR-T cells might range from about any of about 1 ⁇ 10 4 to 1 ⁇ 10 5 to 1 ⁇ 10 6 to 1 ⁇ 10 7 , to 1 ⁇ 10 7 , to 1 ⁇ 10 8 cells or more, depending on the factors mentioned above.
  • An exemplary dosing regimen comprises administering a first dose of about 0.3 ⁇ 10 4 cells, about 0.5 ⁇ 10 4 cells, about 1 ⁇ 10 4 cells, about 1.5 ⁇ 10 4 cells, about 2 ⁇ 10 4 cells, about 2.5 ⁇ 10 4 cells, about 3 ⁇ 10 4 cells, about 3.5 ⁇ 10 4 cells, about 4 ⁇ 10 4 cells, about 4.5 ⁇ 10 4 cells, about 5 ⁇ 10 4 cells, about 5.5 ⁇ 10 4 cells, about 6 ⁇ 10 4 cells, about 6.5 ⁇ 10 4 cells, about 7 ⁇ 10 4 cells, about 7.5 ⁇ 10 4 cells, about 8 ⁇ 10 4 cells, about 8.5 ⁇ 10 4 cells, about 9 ⁇ 10 4 cells, about 9.5 ⁇ 10 4 cells, about 1 ⁇ 10 5 cells, about 1.5 ⁇ 10 5 cells, 2 ⁇ 10 5 cells, about 2.5 ⁇ 10 5 cells, about 3 ⁇ 10 5 cells, about 3.5 ⁇ 10 5 cells, about 4 ⁇ 10 5 cells, about 4.5 ⁇ 10 5 cells, about 5 ⁇ 10 5 cells, about 5.5 ⁇ 10 5 cells, about 6 ⁇ 10 5 cells, about 6.5 ⁇ 10 5 cells, about 7 ⁇
  • a subsequent dose is administered monthly. In some embodiments, a subsequent dose is administered every other month. In some embodiments, a subsequent dose is administered about every three months. In some embodiments, a subsequent dose is administered about every four months. In some embodiments, a subsequent dose is administered about every five months. In some embodiments, a subsequent dose is administered about every six months. In some embodiments, a subsequent dose is administered about every seven months. In some embodiments, a subsequent dose is administered about 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, a subsequent dose is administered every other month.
  • a dosing regimen comprises administering to a subject a first dose of about 6 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110. In some embodiments, a dosing regimen comprises administering to a subject a first dose of about 1 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110. In some embodiments, a dosing regimen comprises administering to a subject a first dose of about 2 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110.
  • a dosing regimen comprises administering to a subject a first dose of about 3 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110. In some embodiments, a dosing regimen comprises administering to a subject a first dose of about 4 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110. In some embodiments, a dosing regimen comprises administering to a subject a first dose of about 5 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110.
  • a dosing regimen comprises administering to a subject a first dose of about 7 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110. In some embodiments, a dosing regimen comprises administering to a subject a first dose of about 8 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110. In some embodiments, a dosing regimen comprises administering to a subject a first dose of about 1 ⁇ 10 6 CAR-T cells at D0, and a subsequent dose of about 9 ⁇ 10 6 CAR-T cells at between about D30 to about D110. In some embodiments, a dosing regimen comprises administering to a subject a first dose of about 1 ⁇ 10 7 CAR-T cells at D0, and a subsequent dose of about 6 ⁇ 10 6 CAR-T cells at between about D30 to about D110.
  • a dosing regimen comprises administering to a subject a lymphodepletion regimen at D-7 to D-2, followed by a first dose of about 6 ⁇ 10 6 CAR-T cells at D0, followed by an optional lymphodepletion regimen at D60 to D100, followed by a subsequent dose of 6 ⁇ 10 6 CAR-T cells at D63 to D104.
  • a dosing regimen comprises administering to a subject a lymphodepletion regimen at D-7 to D-2, followed by a first dose of about 6 ⁇ 10 6 CAR-T cells at D0, followed by an optional lymphodepletion regimen at D60 to D90, followed by a subsequent dose of 6 ⁇ 10 6 CAR-T cells at D63 to D104.
  • a dosing regimen comprises administering to a subject a lymphodepletion regimen (LD) at D-7, followed by a first dose of about 6 ⁇ 10 6 CAR-T cells at D0, followed by reduced intensity (as compared to the previous LD) LD at D70, followed by a subsequent dose of 6 ⁇ 10 6 CAR-T cells at D99.
  • a dosing regimen comprises administering to a subject a lymphodepletion regimen at D-15, followed by a first dose of about 6 ⁇ 10 6 CAR-T cells at D0, followed by a lymphodepletion regimen at D60, followed by a subsequent dose of 6 ⁇ 10 6 CAR-T cells at D75.
  • a dosing regimen comprises administering to a subject a lymphodepletion regimen at D-7 to D-2, followed by a first dose of about 6 ⁇ 10 6 CAR-T cells at D0, followed by an optional lymphodepletion regimen at D60 to D90, followed by a subsequent dose of 6 ⁇ 10 6 CAR-T cells at D63 to D104.
  • a dosing regimen comprises administering to a subject a lymphodepletion regimen at D-7 to D-2, followed by a first dose of about 6 ⁇ 10 6 CAR-T cells at D0, followed by an optional lymphodepletion regimen at D60 to D90, followed by a subsequent dose of 6 ⁇ 10 6 CAR-T cells at D63 to D104.
  • a dosing regimen comprises administering to a subject a lymphodepletion regimen at D-5 to D-3, followed by a first dose of about 20 ⁇ 10 6 cells, about 40 ⁇ 10 6 cells, about 80 ⁇ 10 6 cells, about 120 ⁇ 10 6 cells, about 240 ⁇ 10 6 cells, or about 360 ⁇ 10 6 cells, followed by an optional subsequent dose of CAR-T cells.
  • the lymphodepletion regimen comprises fludarabine administered at a dosage of about 30 mg/m 2 ; cyclophosphamide administered at about 300 mg/m 2 ; and optionally a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10) administered at a dosage of about 10 to about 13 mg.
  • a CD52 antibody e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10
  • the first dose and the first subsequent and additional subsequent doses are separated in time from each other by at least four weeks.
  • the dosing regimen can vary over time.
  • the methods involve administering CAR-T cells which specifically bind to antigen associated with a disease and result in a response, such as an immune response against such molecules upon binding to such antigen.
  • Any allogeneic CAR-T cells may be used with the methods described herein, unless specifically mentioned to be a method associated with a specific type of allogeneic CAR-T cells (e.g. UCART19 cells).
  • the methods and compositions of the instant disclosure are practiced using an allogeneic CAR-T cell specific for a tumor-specific antigen.
  • the methods and compositions of the instant disclosure are practiced using an allogeneic CAR-T cell specific for CD19.
  • the CAR-T cell expresses a CAR comprising a CD19 binding domain.
  • the CD19 binding domain comprises a single chain variable fragment (scFv).
  • the scFv is derived from and anti-CD19 antibody (e.g., the 4G7 antibody).
  • the scFv comprises a VH and a VL isolated or derived from an anti-CD19 antibody (e.g., the 4G7 antibody).
  • the scFv comprises an HCDR1, HCDR2, HCDR3, LCDR1, LDCR2, and LCDR3 isolated or derived from an anti-CD19 antibody (e.g., the 4G7 antibody).
  • the CAR-T cell expresses a CAR comprising a CD19 binding domain, a 4-1BB domain, and/or a CD3zeta domain.
  • the CAR-T cell further expresses a “safety switch.”
  • Safety switches may comprise an epitope which enables selection of transduced cells and an epitope which enables cells expressing the polypeptide to be detected and/or deleted.
  • a safety switch comprises a mimitope. Exemplary safety switches are disclosed in, for example, WO 2013/153391, which is incorporated by reference herein in its entirety.
  • the safety switch binds to rituximab.
  • the safety switch is expressed in trans with the CAR (e.g. as a separate polypeptide on the same cell).
  • the safety switch is RQR8.
  • the RQR8 containing safety switch is expressed in trans. Full length and component sequences for RQR8 are shown in Table 6, below.
  • the safety switch is R2.
  • the CAR-T cells do not comprise a safety switch.
  • a sequence encoding a safety switch is provided on the same vector comprising a sequence encoding a CAR. In some embodiments, a sequence encoding a safety switch is provided on a different vector from the vector comprising a sequence encoding a CAR.
  • a sequence encoding a safety switch is provided on the same polypeptide comprising the CAR (i.e., provided in cis). In some embodiments, the safety switch is provided on a different polypeptide from the CAR (i.e, provided in trans).
  • the safety switch is expressed in trans with the CAR (e.g. as a separate polypeptide on the same cell).
  • the safety switch is RQR8.
  • the RQR8 containing safety switch is expressed in trans.
  • the safety switch is expressed in cis with the CAR, and comprises a mimotope, specific for an antibody, as described in WO 2013/153391.
  • the CAR-T cell further comprises an inactivated CD52 gene.
  • a CAR-T cell is CD52-deficient.
  • CAR-T cells for administration to a patient comprise a mixture of CD52-deficient and CD52-positive cells.
  • a composition of CAR-T cells for administration to a patient comprises a mixture of cells with different genotypes.
  • the composition comprises CAR-T cells wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the cells have an inactivated CD52 gene.
  • the composition comprises CAR-T cells wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the cells have an inactivated TRAC gene.
  • the composition comprises CAR-T cells wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the cells have an inactivated CD52 gene, and further wherein about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the cells have an inactivated TRAC gene.
  • the CAR-T cells are UCART19 CAR-T cells.
  • UCART19 is an allogeneic engineered human T-cell medicinal product being developed for the treatment of CD19-expressing B-cell leukemias, including young adult, adult and pediatric acute lymphoblastic leukemia (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.
  • UCART19 expresses a CAR directed against human CD19 together with the safety switch RQR8 combining epitopes from both CD34 and CD20 antigens.
  • UCART19 comprises an anti-human CD19 4-1BB/CD3zeta CAR, made up of one or more of the peptides shown in Table 7, and/or as described in U.S. Pat. App. Pub. No. 2016/0145337, which is incorporated by reference herein in its entirety.
  • UCART19 has additionally undergone the simultaneous disruption of the TRAC and CD52 genes followed by depletion of remaining TCR ⁇ + cells.
  • UCART19 comprises a single-chain variable fragment (scFv) derived from the murine anti-human CD19 4G7 hybridoma, a CD8 hinge and transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain.
  • scFv single-chain variable fragment
  • the Drug Substance (DS) of UCART19 is defined as allogeneic genetically modified CD19CAR/RQR8+_TCR ⁇ _T-cells: (i) containing an integrated self-inactivating (SIN) recombinant lentiviral delivery vector that expresses an anti-CD19 CAR to redirect T-cells to the CD19+ tumor cells and eliminate them, (ii) expressing an RQR8 safety switch, conferring susceptibility to rituximab, (iii) that are TCR ⁇ -negative via disruption of the TRAC gene using mRNA-based TALEN® gene-editing followed by depletion of the remaining TCR ⁇ + cells during the manufacturing process, and (iv) that are a mix of CD52-knockout and CD52-positive cells via disruption of the CD52 gene using mRNA-based TALEN® gene-editing to allow administration of UCART19 to patients previously treated or being treated with an anti-CD52 antibody (e.g., an antibody comprising the sequence
  • FIG. 4A Exemplary flows for UCART19 manufacturing are shown in FIG. 4A and in FIG. 4B .
  • UCART19 refers to UCART19 cells expressing a CD19 CAR and a RQR8 safety switch.
  • the cells also comprise an inactivated TRAC gene.
  • the cells comprise an inactivated CD52 gene.
  • UCART19 refers to UCART19 cells expressing a CD19 CAR and a R2 safety switch.
  • the cells also comprise an inactivated TRAC gene.
  • the cells comprise an inactivated CD52 gene.
  • UCART19 refers to UCART19 cells expressing a CD19 CAR.
  • the cells also comprise an inactivated TRAC gene.
  • the cells comprise an inactivated CD52 gene.
  • the CAR-T cells are allogeneic CAR-T cell expressing the amino acid sequence shown in SEQ ID NO: 1.
  • compositions comprising one or more additional agents.
  • These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
  • suitable excipients such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
  • the compositions and methods disclosed herein can be used alone or in combination with other conventional methods of treatment.
  • the CAR-T cells can be administered to a subject via any suitable route. It should be apparent to a person skilled in the art that the examples described herein are not intended to be limiting but to be illustrative of the techniques available. Accordingly, in some embodiments, the CAR-T cells are administered to a subject in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, transdermal, subcutaneous, intra-articular, sublingually, intrasynovial, via insufflation, intrathecal, oral, inhalation or topical routes.
  • intravenous administration e.g., as a bolus or by continuous infusion over a period of time
  • intramuscular, intraperitoneal, intracerebrospinal transdermal
  • subcutaneous, intra-articular sublingually
  • intrasynovial via insufflation, intrathecal,
  • Administration can be systemic, e.g., intravenous administration, or localized.
  • Commercially available nebulizers for liquid formulations including jet nebulizers and ultrasonic nebulizers are useful for administration.
  • Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution.
  • CAR-T cells and a pharmaceutically acceptable excipient may be in various formulations.
  • Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance.
  • an excipient can give form or consistency, 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.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may comprise buffers such as 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; catechol; 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,
  • the pharmaceutical kit comprises CAR-T cells and a CD52 antibody.
  • the pharmaceutical kit comprises UCART19 cells and an antibody.
  • the pharmaceutical kit comprises a first container comprising CAR-T cells (e.g., UCART19 cells) and a second container comprising a CD52 antibody (e.g., an antibody comprising the sequence of SEQ ID NO: 8 and/or SEQ ID NO: 10).
  • the CAR-T cells e.g., UCART19 cells
  • the kit comprises multiple containers comprising CAR-T cells, wherein the amount of CAR-T cells in each container is either the same or different.
  • the first and/or second containers are flexible cell infusion bags.
  • the first and/or second containers are vials or tubes (e.g., glass or plastic vials or tubes).
  • the pharmaceutical kit may further comprise a label or package insert comprising instructions for administering the CAR-T cells and the CD52 antibody to the subject.
  • articles of manufacture comprising a plurality of sealable containers, each individually comprising a unit dose of allogeneic chimeric antigen receptor (CAR)-T cells for administration to a subject, said unit dose comprising about 1 ⁇ 10 6 to about 5 ⁇ 10 8 cells; packaging material; and a label or package insert comprising instructions for administering a plurality of said unit doses to the subject by carrying out a first administration and a subsequent administration, said first administration comprising delivering one of said unit doses to the subject and said subsequent administration comprising administering one or a plurality of said unit doses to the subject.
  • CAR chimeric antigen receptor
  • a method of treating a subject who has refractory and/or relapsed Non-Hodgkin's Lymphoma comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR, wherein the at least one dose is about 20 ⁇ 10 6 cells/dose to about 360 ⁇ 10 6 cells/dose.
  • CAR allogeneic chimeric antigen receptor
  • any one of embodiments 1 to 3, wherein the at least one dose is selected from the group consisting of about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 80 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, about 240 ⁇ 10 6 cells/dose, and about 360 ⁇ 10 6 cells/dose.
  • any one of embodiments 1 to 4 wherein the at least one dose is selected from the group consisting of about 20 ⁇ 10 6 cells/dose, about 40 ⁇ 10 6 cells/dose, about 120 ⁇ 10 6 cells/dose, and about 360 ⁇ 10 6 cells/dose when the subject has a weight of greater than 50 kg.
  • the first lymphodepletion regimen comprises administering fludarabine, cyclophosphamide, and an anti-CD52 antibody.
  • a pharmaceutical kit for treating a patient suffering from cancer comprising:
  • kits of any one of embodiments 40 to 43, wherein the kit comprises a first container comprising the CAR-T cells, and a second container comprising the CD52 antibody.
  • kit of any one of embodiments 40 to 45, wherein the kit further comprises a label or package insert comprising instructions for administering the CAR-T cells and the CD52 antibody to the subject.
  • a method of treatment comprising:
  • the method of embodiment 49, wherein the first dose comprises about 6 ⁇ 10 5 total cells, about 6 ⁇ 10 6 total cells, about 6 ⁇ 10 7 total cells, about 8 ⁇ 10 7 total cells, about 1.8 ⁇ 10 8 total cells, about 2.4 ⁇ 10 8 total cells, or about 5 ⁇ 10 8 total cells.
  • step (b) The method of any one of embodiments 47 to 55, wherein the interim lymphodepletion regimen comprises administering fludarabine, cyclophosphamide, and CD52 antibody to the subject between about 0 to 14 days prior to step (b).
  • the method of embodiment 47, wherein the first lymphodepletion regimen comprises administering fludarabine, cyclophosphamide, and CD52 antibody to the subject.
  • a cytokine release syndrome (CRS)-related outcome in the subject at day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration in (b) is not detectable or is reduced by about 20% to about 99% as compared to a method comprising an alternative dosing regimen wherein the subject is administered the cells in (b) without having been administered the first dose.
  • CRS cytokine release syndrome
  • the tumor or cancer is acute lymphoblastic leukemia (ALL), is chronic lymphocytic leukemia (CLL), HNSCC, non-Hodgkin's lymphoma, acute myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung, liver, breast, prostate, ovarian, skin, melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, or mes
  • ALL acute lymphoblastic leukemia
  • a method of treatment comprising administering a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells to a subject previously administered a first dose of allogeneic CAR-T cells, wherein: the subsequent dose of cells is administered at a time point that is at least or more than about 5 weeks after and less than about 24 weeks after initiation of the first dose.
  • CAR allogeneic chimeric antigen receptor
  • a method of treatment comprising administering to a subject a subsequent dose of allogeneic chimeric antigen receptor (CAR)-T cells, wherein: prior to said administration, the subject has received a previous dose of the CAR-T cells in an amount sufficient to demonstrate clinical benefit in the subject; and at the time of administration, the subject does not exhibit a detectable adaptive host immune response specific for the CAR-T cells; and/or the time between said previous and subsequent doses is greater than about 5 weeks and less than about 24 weeks.
  • CAR allogeneic chimeric antigen receptor
  • compositions comprising allogeneic chimeric antigen receptor (CAR)-T cells for manufacture of a medicament for treatment of a disease in a subject previously treated with the CAR-T cells, wherein: the composition is for use between about 5 to about 24 weeks after the previous treatment; and/or the composition is formulated for administration of a subsequent dose in an amount sufficient for amelioration of a disease in the subject having been previously treated with the CAR-T cells.
  • CAR allogeneic chimeric antigen receptor
  • a composition comprising allogeneic chimeric antigen receptor (CAR)-T cells for use in treating a disease in a subject previously treated with the CAR-T cells, wherein: the cells are for use between about 5 and 24 weeks after the previous treatment; and the cells are formulated for administration of a subsequent dose in an amount sufficient for amelioration of a disease in the subject having been previously treated with the CAR-T cells.
  • CAR allogeneic chimeric antigen receptor
  • CAR-T cells Use of allogeneic chimeric antigen receptor (CAR)-T cells in the manufacture of a medicament for use in a method for treating a disease, said method comprising: administering to a subject having the disease a first dose of the CAR-T cells, said first dose comprising between about 1 ⁇ 10 6 to about 5 ⁇ 10 8 total cells and; administering to the subject a subsequent dose of the CAR-T cells at a time point that is at least or more than about 4 weeks after and less than about 24 weeks after initiation of said administration in (a).
  • a allogeneic chimeric antigen receptor
  • a plurality of sealable containers each individually comprising a unit dose of allogeneic chimeric antigen receptor (CAR)-T cells for administration to a subject, said unit dose comprising about 1 ⁇ 10 6 to about 5 ⁇ 10 8 cells; packaging material; and a label or package insert comprising instructions for administering a plurality of said unit doses to the subject by carrying out a first administration and a subsequent administration, said first administration comprising delivering one of said unit doses to the subject and said subsequent administration comprising administering one or a plurality of said unit doses to the subject.
  • CAR chimeric antigen receptor
  • a method of treating an adult subject who has refractory and/or relapsed CD19+ B-cell acute lymphoblastic leukemia comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR, wherein the at least one dose is selected from the group consisting of about 6 ⁇ 10 5 cell/dose, 6 ⁇ 10 6 cells/dose, about 6-8 ⁇ 10 7 cells/dose, and about 1.8-2.4 ⁇ 10 8 cells/dose.
  • CAR allogeneic chimeric antigen receptor
  • a method of treating an pediatric subject who has refractory and/or relapsed CD19+ B-cell acute lymphoblastic leukemia comprising administering to the subject at least one dose of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) comprising an anti-human CD19 4-1BB/CD3zeta CAR, wherein the at least one dose is about 2-8 ⁇ 10 7 cells/dose.
  • CAR allogeneic chimeric antigen receptor
  • the first lymphodepletion regimen comprises administering fludarabine, cyclophosphamide, and an anti-CD52 antibody.
  • a method of producing a population of allogeneic chimeric antigen receptor (CAR)-T cells (CAR-T cells) directed to a target of interest comprising:
  • step (c) is carried out about 3-4 days following step (b).
  • step (d) is carried out about 2 days following step (c).
  • step (f) is carried out about 10-12 days following step (d).
  • This example illustrates multiple dose treatment with allogeneic CAR-T cells.
  • a lymphodepletion regimen Prior to administration of the CAR-T cells, subjects with disease are administered a lymphodepletion regimen.
  • Such regimen may comprise, e.g., treatment with fludarabine (range total dose about 30 to 150 mg/m 2 ) and cyclophosphamide (range total dose about 300 to 4000 mg/m 2 ), with an anti-CD52 drug (e.g., a CD52 antibody, such as an antibody comprising the sequence of SEQ ID NO: 8 and SEQ ID NO: 10) (range total dose about 0.3 to 1 mg/kg).
  • Subjects are administered a first dose of CAR-T cells of between about 1 ⁇ 10 4 to about 5 ⁇ 10 8 cells at D0.
  • CAR-T cells are assessed.
  • the number of CAR-T cells present in peripheral blood of treated subjects is determined by performing flow cytometry of a cell sample for surface expression of CAR-specific marker, CD4, and/or CD8. Positive MRD and/or loss of persistence suggest subsequent dosing with CAR-T cells.
  • a lymphodepletion regimen Prior to subsequent dosing with CAR-T cells, subjects with MRD are administered a lymphodepletion regimen.
  • Such regimen may comprise, e.g., treatment with fludarabine (range total dose about 30 to 150 mg/m 2 ) and cyclophosphamide (range total dose about 300 to 4000 mg/m 2 ), with or without an anti-CD52 (range total dose about 0.3 to 1 mg/kg).
  • a dose of CAR-T cells of between about 1 ⁇ 10 4 to about 1 ⁇ 10 8 cells. Response to the dose of CAR-T cells is assessed, and the number of CAR-T cells present in peripheral blood of treated subjects is determined. Positive MRD and/or loss of persistence suggest subsequent dosing with CAR-T cells. Multiple doses of allogeneic CAR-T may be administered until MRD negativity is achieved.
  • Example 2 Use of an Allogeneic Anti-CD19 CAR-T Cell Product (UCART19) in Adult Patients with CD19+ Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia
  • B-ALL is incurable in ⁇ 60% of adult patients. At relapse, prognosis is very poor ( ⁇ 10% overall survival). Standard therapy involves combination chemotherapy ⁇ allogeneic SCT.
  • Subjects were administered an allogeneic, universal, adoptive T-cell therapy targeting CD19+ malignancies.
  • UCART19 comprises an anti-CD19 scFv and an intracellular domain comprising CD3 ⁇ +4-1BB.
  • the CAR-T cell further comprises a RQR8 (CD20 mimotope) safety switched expressed in trans.
  • the CAR-T cells were prepared by further knockouts, using TALEN-based technologies. Specifically the TRAC was knocked out, to prevent TCR mediated recognition of patient's HLA antigens. The CD52 was knocked out to permit CD52 antibody use in lymphodepletion.
  • An illustrative UCART19 cell as used here (CD19CAR/RQR8+_TCR ⁇ _T-cells) is provided in FIG. 5 .
  • Objective #1 Evaluation of safety and tolerability of UCART19 at different doses; and determination of the maximum tolerated dose (MTD). ( FIG. 3B ).
  • Objective #3 Evaluate expansion and persistence of UCART19.
  • FIG. 3B shows the study plan, having the following features:
  • Data presented here are for 12 patients treated, with six patients at the first dose level of 6 ⁇ 10 6 total cells (approximately 1 ⁇ 10 5 cells per kilogram) and six patients at the second dose level with 6 to 8 ⁇ 10 7 total cells (approximately 1 ⁇ 10 6 cells per kilogram). No patients yet were treated at the third and final dose level of 1.8 to 2.4 ⁇ 10 8 total cells.
  • allo-SCT allogeneic stem-cell transplant
  • CRS CRS UCART19 related adverse event
  • Tocilizumab was administered in 6/11 patients.
  • CRS correlated with serum cytokine increase (IL-6, IL-10 and IFNgamma) and UCART19 expansion in blood in all patients.
  • Viral reactivations occurred in 4 patients (G1 to G3).
  • the first case occurred at the first dose level and was a grade 4 CRS related to UCART19, and associated with grade 5 neutropenic sepsis related to lymphodepletion and UCART19. Death occurred on day 15 after UCART19 infusion.
  • the second case a grade 4 prolonged cytopenia, occurred at the second dose level and was reported as related to both lymphodepletion and UCART19.
  • This patient underwent allo-SCT and had an unrelated grade 5 pulmonary hemorrhage in the setting of infection on day 19 following allo-SCT or day 82 after UCART19 infusion.
  • Grade 5 adverse events have been reported in other autologous anti-CD19 CAR T cell therapy trials in part due to advanced stage of disease and accompanying confounding conditions.
  • FIG. 7A shows the study status.
  • FIG. 7B illustrates response, duration of remission and re-dosing of UCART19 in this trial (anti-leukemic activity).
  • MRD-CR occurs when a patient achieves a CR and there is no evidence of ALL cells in the marrow when using sensitive tests such as polymerase chain reaction or flow cytometry.
  • CR or CRi rates are the typical regulatory standard, but studies in both children and adults with ALL have demonstrated a strong correlation between minimal residual disease (MRD+) and risks for relapse.
  • CR defined as the absence of any evidence or symptoms of cancer, or CR with incomplete blood count recovery (CRi). Seven patients achieved MRD-CR.
  • CAR T cell expansion was detected in blood from day 7 after UCART19 infusion, reaching the peak expansion between day 10 and day 17.
  • One patient at the second dose level showed the highest peak linked to a long persistence up to day 42 still ongoing at the data cutoff.
  • dose level two the longest persistence observed as of the data cutoff occurred on day 56.
  • FIG. 8A and FIG. 8B show the flow cytometry PK profile and UCART19 kinetics in adult patients with CD19+ relapsed/refractory B-cell acute lymphoblastic leukemia.
  • preliminary data on flow cytometry at DL1 and DL2 show that UCART19 was detectable in blood from D3 to D14 with a proliferation peak between D10 and D17.
  • One patient at DL2 showed the highest peak linked to longest persistence.
  • at DL1 1 patient showed UCART19 persistence up to D42; at DL2: 2 patients showed persistence up to D42 and ongoing persistence at D56.
  • Example 3 Re-Dosing of UCART19 in a Patient with Relapsed/Refractory CD19 Positive B-ALL
  • This example illustrates treatment with UCART19, wherein the treatment comprises a subsequent dose of UCART19 administered after a first dose of UCART19, as administered in Example 2.
  • a lymphodepletion regimen (fludarabine 122 mg/m 2 ; cyclophosphamide 1230 mg/m 2 ; and CD52 antibody 1 mg/kg) was administered over a period of six days prior to UCART19 infusion. No major toxicity was observed.
  • the patient then received a reduced intensity lymphodepletion regimen over a period of six days (fludarabine and cyclophosphamide without CD52 antibody).
  • a reduced intensity lymphodepletion regimen over a period of six days (fludarabine and cyclophosphamide without CD52 antibody).
  • the patient was re-dosed with the same dose (6 ⁇ 10 6 total cells) of UCART19.
  • the patient achieved MRD negativity at D28 after infusion of the second dose of UCART19.
  • dosing of UCART19 after relapse after molecular remission after treatment with a first dose of UCART19 was effective to achieve MRD negativity.
  • Patient proceeded to allogeneic stem cell transplantation 6 weeks after the second dose.
  • UCART19 Male patient, 22 years old, B-ALL diagnosis. Patient received the 1st infusion of UCART19 of 1.8 ⁇ 10 8 cells (dose level 3) after a lymphodepletion combining fludarabine (90 mg/m 2 total dose), cyclophosphamide (1500 mg/m 2 total dose) and CD52 antibody (40 mg total dose) (FCA). No UCART19 were detectable and no mechanism of action related toxicity was reported. At Day 28 (28 days post UCART19 infusion), no anti-leukemic activity was observed (medullary biopsy presented 15% blasts).
  • Example 4 Use of an Allogeneic Anti-CD19 CAR-T Cell Product (UCART19) in High-Risk Pediatric Patients with CD19+ Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia
  • B-cell acute lymphoblastic leukemia is the most common malignant disease in children and accounts for 80% of childhood leukemias. The most common between age is 2-10 years (peaks at 3-4 years), survival is ⁇ 90%. Relapse can occur in 10% of children, with about 40%-50% surviving, unless high risk, in which case about 10%-30% survive.
  • FIG. 9 shows the study design.
  • the lymphodepletion regimen started from D-7 (during the week preceding UCART19 infusion) and combined: cyclophosphamide (C) (60 mg/kg/day for 2 days), fludarabine (F) (30 mg/m 2 /day for 5 days), and w/wo CD52 antibody (A) (0.2 mg/kg/day for 5 days).
  • C cyclophosphamide
  • F fludarabine
  • A w/wo CD52 antibody
  • BMA Safety assessment was performed at D28 post UCART19 administration.
  • BMA was performed at baseline, D-1, D14 (optional), D28, D56, D84 or ahead of allo-SCT conditioning regimen initiation or at the withdrawal visit (at the investigator's discretion).
  • a BMA will be performed at M1, M2, M3, M6 and M12 post allograft for the disease assessment.
  • the BMA will be performed optionally according to investigator's judgment.
  • CMV cytomegalovirus
  • BK virus adenovirus
  • metapneumovirus adenovirus
  • mice experienced transient grade 1 skin GvHD.
  • Two patients died from disease recurrence following allo-SCT and one patient died from complications of allo-SCT. There were no treatment-related deaths. This is presented in Table 12.
  • N 6 Time to onset (median and range in days) 7 (5-9) Duration (median and range in days) 7.5 (4-13) Cytokine elevation (IL-6, IFN- ⁇ , IL-10, CRP) 2 Specific treatment 2/6 Tocilizumab 2 Outcome - Number of complete resolution 6
  • FIG. 10 shows the study status.
  • FIG. 11 illustrates response and duration of remission (anti-leukemic activity).
  • UCART19 vector copy number (VCN) was measured in blood and bone marrow by qPCR. Preliminary data showed that for 5 out of 6 patients, UCART19 was detectable in blood by D7, with a proliferation peak observed around D14. No UCART19 was detected for one patient who subsequently relapsed. For 3 out of 5 patients, UCART19 persisted in blood until D28. For 2 out of 5 patients, UCART19 remained detectable in blood on D42. Persistence beyond D42 was not measured since UCART19 was eliminated by conditioning regimen for allo-SCT.
  • Cytokine kinetics were assessed. 2 out of 6 patients had IL-6 and IFN ⁇ elevation. No cytokine elevation was observed in 4 out of 6 patients. All 6 patients experienced CRS. CRS G3 was observed in patient 1 and CRS G2 in patient 8. Time to onset of first CRS symptoms ranged between D5 and D9.
  • T-cell donor chimerism was assessed ( FIG. 13A , FIG. 13B , FIG. 13C ).
  • UCART19 was detectable in blood from D7 to at least D42 in all patients but one by molecular signatures of T-cell donor chimerism.
  • Example 5 Use of an Allogeneic Anti-CD19 CAR-T Cell Product (UCART19) in Patients with Relapsed/Refractory Large B-Cell and/or Follicular Lymphoma
  • Diffuse large B-cell lymphoma which comprises 20% to 30% of B-cell Non-Hodgkin lymphoma (B-NHL), is fatal if not cured.
  • Primary mediastinal large B-cell lymphoma (PMBCL) and transformed follicular lymphoma (FL) are typically treated along a DLBCL paradigm.
  • DLBCL includes a group of molecularly diverse aggressive lymphomas that not only differ in their chromosomal alterations, but also signaling pathway activation and clinical outcome. The disease itself may be de novo or may result from transformation of indolent B-lymphomas such as from transformed follicular lymphoma. In the US, the annual incidence rate of DLBCL is 6.9 per 100,000 and it is the most common form of B-NHL.
  • This study will be a single-arm, open-label, multicenter Phase 1/2 study evaluating 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 a dose-escalation (Phase 1) and a dose-expansion phase (Phase 2).
  • Phase 1 of the study CAR-T cells comprising an RQR8 safety switch will be used (CD19CAR/RQR8+_TCR ⁇ _T-cells).
  • Phase 2 of the study it one or more patients may receive CAR-T cells that do not comprise an RQR8 safety switch (CD19CAR/TCR ⁇ _T-cells or CD19CAR/R2+_TCR ⁇ _T-cells).
  • Criteria for inclusion in the study may include one or more of the following:
  • phase 1 and phase 2 studies study will be divided into different periods including screening, lymphodepletion, treatment, and follow-up.
  • a single cycle is defined as the combination of one lymphodepletion and one treatment period.
  • Screening will start after the informed consent form is signed. Patient's eligibility criteria will be checked. The Screening period will last up to 28 days. Patients who meet all eligibility criteria at the end of the Screening period will then enter the lymphodepletion period.
  • Lymphodepletion will start on Day ⁇ 5 and end on UCART19 infusion at Day 0.
  • UCART19 Prior to treatment with UCART19, all patients will receive intravenous lymphodepletion with fludarabine (30 mg/m 2 /day), cyclophosphamide (300 mg/m 2 /day), and CD52 antibody (13 mg/day) on an outpatient setting on Day ⁇ 5, Day ⁇ 4 and Day ⁇ 3.
  • the CD52 antibody will be administered over approximately 4 hours and patients must be closely monitored up to 2 hours after the completion of infusion. Premedication comprising high-dose corticosteroids is required prior to CD52 antibody administration.
  • CD52 antibody-related dose-limiting toxicities are observed at the starting dose level, a lower dose of 30 mg total (10 mg/day given over 3 days) will be evaluated. At the end of the lymphodepletion period, eligibility criteria allowing UCART19 administration should be assessed to ensure patients' safety.
  • Treatment period starting from Day 0, will end at Day 56 post-UCART19 infusion. Patients will receive UCART19 as an intravenous infusion on Day 0 over approximately 5 minutes. To closely manage toxicities associated with UCART19, patients will either be hospitalized for a minimum of 5 days from Day 0, or until UCART19 related non-hematological toxicities return to Grade or receive UCART19 in an outpatient setting in those investigational sites allowing direct patient admission.
  • one optional retreatment with UCART19 may be administered to patients.
  • Treatment with UCART19 may be administered at the highest dose level deemed safe in Phase 1, or at the RP2D in Phase 2.
  • Treatment may use a batch and/or donor different from the initial dose.
  • Each retreatment with UCART19 must be at least 4 weeks apart from the initial dose after planned tumor assessments on Day 56, Month 4, and Month 6 and must be following a lymphodepletion with Flu/Cy or Flu/Cy and CD52 antibody.
  • Patients with undetectable UCART19 in whole blood at D14 may be retreated with subsequent lymphodepletion starting after the completion of the DLT observation window in Phase 1, or Day 28 in Phase 2.

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