WO2021232864A1 - 免疫效应细胞治疗肿瘤 - Google Patents

免疫效应细胞治疗肿瘤 Download PDF

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WO2021232864A1
WO2021232864A1 PCT/CN2021/076518 CN2021076518W WO2021232864A1 WO 2021232864 A1 WO2021232864 A1 WO 2021232864A1 CN 2021076518 W CN2021076518 W CN 2021076518W WO 2021232864 A1 WO2021232864 A1 WO 2021232864A1
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cells
subject
total
car
seq
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PCT/CN2021/076518
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English (en)
French (fr)
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李宗海
汪薇
肖珺
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佧珐药业有限公司
李宗海
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Priority to KR1020227044178A priority Critical patent/KR20230012571A/ko
Publication of WO2021232864A1 publication Critical patent/WO2021232864A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A61K39/001112—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present invention belongs to the field of immunotherapy; specifically, it relates to immune cell therapy that targets and recognizes tumor antigens, triggers activation of immune effector cells, and exerts anti-tumor effects.
  • the purpose of the present invention is to provide a method and use of engineered cells (for example, T cells) and their compositions for the treatment of subjects suffering from CD19-positive hematological tumors, usually including leukemia or lymphoma, especially acute , Chronic lymphocytic leukemia and/or non-Hodgkin's lymphoma (NHL).
  • the methods and uses provide or achieve an improved or longer-lasting response or efficacy and/or a reduced risk of toxicity or other side effects compared to the prior art.
  • the method treats a specific patient population (such as a patient population with a specific stage and/or previous treatment history) by administering a specified number or relative number of engineered cells, pre-treated with a specific lymphocyte depletion therapy.
  • the first aspect of the present invention provides a method for treating or suspected of having CD19-positive hematological tumors, comprising administering to the subject cells expressing anti-CD19 chimeric antigen receptors (CAR), and the dose of the cells is It is (a) about 4.9 ⁇ 10 6 cells/kg subject weight (cells/kg), (b) about 3 ⁇ 10 8 total cells, (c) about 1 ⁇ 10 7 cells/kg, (d) about 6 ⁇ 10 8 cells Total cells, (a) not more than about 4.9 ⁇ 10 6 cells/kg, (b) not more than about 3 ⁇ 10 8 total cells, (c) not more than about 1 ⁇ 10 7 cells/kg, (d) not more than about 6 ⁇ 10 8 Total cells, (e) about 1.5x10 5 cells/kg to about 4.9x10 6 cells/kg, (f) about 1.5x10 5 cells/kg to about 1x10 7 cells/kg, (g) about 1x10 7 total cells to about 3 ⁇ 10 8 total cells, and/or (h) about 1 ⁇ 10
  • the dose of cells administered is measured according to the number of cells given per kilogram of the subject's body weight. In this article, each cell per kilogram of the subject's body weight is uniformly expressed as "cells/kg”. In this article, the total dose of cells administered is measured according to the number of total cells administered by each subject, and each total cell/subject is uniformly denoted by "a total cell”.
  • the dose of cells administered is: (a) about 5.8 ⁇ 10 5 cells/kg, (b) about 1.1 ⁇ 10 6 cells/kg, (c) about 1.7 ⁇ 10 6 cells/kg, ( d) about 1.8x10 6 cells/kg, (e) about 1.9x10 6 cells/kg, (f) about 2.0x10 6 cells/kg, (g) about 2.1x10 6 cells/kg, (h) to about 2.6x10 6 cells / kg, (h) from about 3x10 7 total cells, (I) from about 6x10 7 total cells, (j) from about 8x10 7 total cells, (k) approximately 9x10 7 total cells, ( l) About 1x10 8 total cells, (m) about 1.5x10 8 total cells, (n) about 5.8x10 5 cells/kg to about 4.9x10 6 cells/kg, (o) about 1.1x10 6 cells /kg ⁇ about 4.9x10 6 cells/kg, (p) about 1.7x10 6 cells/kg ⁇ about 4.9x10 6
  • the subject during or before the administration of the cell :
  • the subject is identified as having a B-cell malignancy
  • the B-cell malignant tumor is selected from: acute lymphocytic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), and/or non-Hodgkin's lymphoma (NHL); and/or
  • the subject has been identified or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk NHL and/or leukemia;
  • the subject has been identified or has been identified as having high-risk NHL; and/or
  • the NHL is selected from the following group: aggressive NHL, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), large B-cell lymphoma rich in T cells/histiocytosis ( TCHRBCL), Burkitt lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL); and/or
  • the subject is an adult and/or is over 20, 30, 40, 50, 60, or 70 years old or over about 20, 30, 40, 50, 60, or 70 years old .
  • the subject has received two or more previous therapies for the hematological tumors other than CD19CAR-expressing cells before administering the cells, optionally 2, 3 or 4 or more
  • previous therapies include kinase inhibitor treatment optionally ibrutinib, or monoclonal antibody treatment optionally rituximab, or CHOP therapy, and/or hematopoietic stem cell transplantation (HSCT) treatment;
  • HSCT hematopoietic stem cell transplantation
  • the lymphocyte depletion therapy includes:
  • the cell infusion and/or lymphocyte depletion therapy administration is delivered out of clinic.
  • the cell dose is administered parenterally, optionally intravenously, in 1, 2, 3, 4 or more doses within 3 or 4 days.
  • At least 30% of subjects treated according to the method achieve complete remission (CR) and/or overall remission (OR).
  • At least 50% of subjects treated according to the method and achieving complete remission (CR) exhibit a progression-free survival (PFS) and/or overall survival (OS) of more than 12 months ;
  • PFS progression-free survival
  • OS overall survival
  • subjects treated according to the method exhibit an average PFS or OS of more than 6, 12, or 18 months, or more than about 6, 12, or 18 months; and/or The subject exhibited post-treatment PFS or OS for at least 6, 12, 18, or more months, or at least about 6, 12, 18, or more months.
  • none of the subjects treated according to the method exhibited grade 3 or higher CRS and neurotoxicity.
  • the CAR includes a scFv that binds to CD19, a transmembrane domain, a costimulatory domain of 4-1BB or CD28, and an intracellular domain of CD3 ⁇ ;
  • the scFv includes the sequence shown in SEQ ID NO: 2.
  • the scFv includes the heavy chain variable region shown in SEQ ID NO: 8 or 11 and the light chain variable region shown in SEQ ID NO: 9 or 12; or
  • the CD19-binding scFv has the sequence shown in SEQ ID NO: 1 or 10; or
  • the CAR includes the amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20; and/or
  • the costimulatory domain comprises SEQ ID NO: 24 or 25, or a variant thereof, and the variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; and/or
  • the intracellular domain of CD3 ⁇ comprises SEQ ID NO: 26, and they have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the cell is an immune effector cell, preferably a T cell, more preferably an autologous or allogeneic T cell, and most preferably a primary T cell obtained from a subject.
  • the second aspect of the present invention provides a composition comprising cells expressing a chimeric antigen receptor (CAR) that specifically binds to CD19-positive hematological tumors, characterized in that the composition is used to treat patients with or suspected For a subject suffering from a CD19-positive hematological tumor, the treatment includes administering to the subject one or more doses of cells expressing the CAR, and the dose (a) is about 4.9 ⁇ 10 6 cells/kg.
  • CAR chimeric antigen receptor
  • the dose of cells administered is: (a) about 5.8 ⁇ 10 5 cells/kg, (b) about 1.1 ⁇ 10 6 cells/kg, (c) about 1.7 ⁇ 10 6 cells/kg, ( d) about 1.8x10 6 cells/kg, (e) about 1.9x10 6 cells/kg, (f) about 2.0x10 6 cells/kg, (g) about 2.1x10 6 cells/kg, (h) to about 2.6x10 6 cells / kg, (h) from about 3x10 7 total cells, (I) from about 6x10 7 total cells ,, (j) from about 8x10 7 total cells, (k) approximately 9x10 7 total cells, (l) about 1 ⁇ 10 8 total cells, (m) about 1.5 ⁇ 10 8 total cells, (n) about 5.8 ⁇ 10 5 cells/kg to about 4.9 ⁇ 10 6 cells/kg, (o) about 1.1 ⁇ 10 6 cells Cells/kg ⁇ about 4.9x10 6 cells/kg, (p) about 1.7x10 6 cells/kg ⁇ about 4.9
  • it further comprises a drug for lymphocyte clearance therapy, and optionally, the drug includes fludarabine and cyclophosphamide.
  • the subject during or before the administration of the cell :
  • the subject is identified as having a B-cell malignancy
  • the B-cell malignant tumor is selected from: acute lymphocytic leukemia (ALL), adult ALL, chronic lymphocytic leukemia (CLL), and/or non-Hodgkin's lymphoma (NHL); and/or
  • the subject has been identified or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk NHL and/or leukemia;
  • the subject has been identified or has been identified as having high-risk NHL; and/or
  • the NHL is selected from the following group: aggressive NHL, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), large B-cell lymphoma rich in T cells/histiocytosis ( TCHRBCL), Burkitt lymphoma, mantle cell lymphoma (MCL) and/or follicular lymphoma (FL); and/or
  • the subject is an adult and/or is over 20, 30, 40, 50, 60, or 70 years old or over about 20, 30, 40, 50, 60, or 70 years old .
  • the subject has received two or more previous therapies for the hematological tumors other than the CD19CAR-expressing cells before the cells are administered, optionally 2, 3 Or 4 or more, optionally, the previous therapies include kinase inhibitor treatment optionally ibrutinib, or monoclonal antibody treatment optionally rituximab, or CHOP therapy, and/or hematopoietic stem cell transplantation (HSCT) treatment; the previous therapies are intolerable, ineffective, or relapse and refractory after remission after treatment.
  • the previous therapies include kinase inhibitor treatment optionally ibrutinib, or monoclonal antibody treatment optionally rituximab, or CHOP therapy, and/or hematopoietic stem cell transplantation (HSCT) treatment; the previous therapies are intolerable, ineffective, or relapse and refractory after remission after treatment.
  • HSCT hematopoietic stem cell transplantation
  • the lymphocyte depletion therapy :
  • Cyclophosphamide is administered at about 21-59 mg/kg, optionally once a day for 1 day or 2 days or 3 days, or cyclophosphamide is administered at about 250 mg/m 2 ⁇ 500 mg/m 2 /day, any Choose 2 days or 3 days or 4 days, and/or give fludarabine at about 17-27 mg/m 2 /d for 3-4 days.
  • the cells in the composition are administered parenterally, optionally intravenously, in 1, 2, 3, 4 or more times within 3 or 4 days.
  • it is administered in an outpatient setting, and/or when the subject does not need to be hospitalized overnight or for multiple consecutive days, and/or the subject does not need to be hospitalized for one or more days.
  • the cells will be administered.
  • the cell is an immune effector cell, preferably a T cell, more preferably an autologous or allogeneic T cell, and most preferably a primary T cell obtained from a subject.
  • the CAR includes a scFv that binds to CD19, a transmembrane domain, a costimulatory domain of 4-1BB or CD28, and an intracellular domain of CD3 ⁇ ;
  • the scFv includes the sequence shown in SEQ ID NO: 2.
  • the scFv includes the heavy chain variable region shown in SEQ ID NO: 8 or 11 and the light chain variable region shown in SEQ ID NO: 9 or 12; or
  • the CD19-binding scFv has the sequence shown in SEQ ID NO: 1 or 10; or
  • the CAR includes the amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20; and/or
  • the costimulatory domain comprises SEQ ID NO: 24 or 25, or a variant thereof, and the variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; and/or
  • the intracellular domain of CD3 ⁇ comprises SEQ ID NO: 26, and they have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the cell is an immune effector cell, preferably a T cell, more preferably an autologous or allogeneic T cell, and most preferably a primary T cell obtained from a subject.
  • the third aspect of the present invention provides a product for treating or suspected of having CD19-positive hematological tumors, characterized in that the product comprises:
  • kit Instructions for using the kit to treat subjects suffering from or suspected of having CD19-positive hematological tumors.
  • each dose contains cells expressing an anti-CD19 chimeric antigen receptor (CAR), and the instructions specify the dose of cells to be administered to subjects with CD19-positive hematological tumors; and the instructions specify Administering a certain number of CD19 CAR-expressing cells, or administering one or more preparations corresponding to the specified number of cells or a certain amount or volume containing the specified number of cells.
  • CAR anti-CD19 chimeric antigen receptor
  • the "dosing interval" as used herein refers to the time elapsed between the administration of multiple courses of immune effector cell therapy and the administration of pretreatment drugs to a subject. Therefore, the dosing interval can be indicated as a range.
  • the "dose” mentioned herein can be expressed as a dose calculated on the basis of weight or a dose calculated on the basis of body surface area (BSA).
  • the dose calculated on the basis of weight is the dose given to the patient calculated on the basis of the weight of the patient, such as mg/kg, the number of immune effector cells/kg, etc.
  • the dose calculated on the basis of BSA is the dose given to the patient calculated based on the surface area of the patient, such as mg/m 2 , and the number of immune effector cells/m 2 .
  • the "number of administrations" as used herein refers to the frequency of administration of immune effector cells or pretreatment drug doses within a given period of time.
  • the number of doses can be indicated as the number of doses per given time.
  • fludarabine can be administered as follows: once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day for administration.
  • Cyclophosphamide can be administered as follows: once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day.
  • This application relates to adoptive cells or immune effector cells to treat tumors, including the administration of cells for one or more courses of treatment, and methods, compositions and products used therefor.
  • Cells generally express chimeric antigen receptors such as chimeric antigen receptors (CAR) or other transgenic receptors such as T cell receptors (TCR).
  • CAR chimeric antigen receptors
  • TCR T cell receptors
  • the present invention provides therapeutic methods and compositions for treating diseases (such as tumors) related to CD19 expression.
  • the present invention provides a method for treating tumors in a subject using adoptive cells or immune effector cells expressing genetically engineered (recombined) chimeric receptors.
  • the method includes a single course of reinfusion of adoptive cells or immune effector cells, or a multiple course of reinfusion.
  • the method includes administering engineered cells that can recognize and/or specifically bind to CD19 molecules on tumor cells and generate an immune response against CD19.
  • Receptors may include chimeric receptors such as chimeric antigen receptor (CAR) and T cell receptor (TCR).
  • the method includes treating patients suffering from acute or chronic lymphocytic leukemia (ALL/CLL) or non-Hodgkin’s lymphoma (NHL) with a dose of antigen receptor expressing cells (such as CAR expressing cells). Examiner.
  • ALL/CLL acute or chronic lymphocytic leukemia
  • NHL non-Hodgkin’s lymphoma
  • Examiner a dose of antigen receptor expressing cells (such as CAR expressing cells).
  • the subject has been pretreated with immune clearance (e.g., lymphocyte clearance) therapy.
  • immune clearance e.g., lymphocyte clearance
  • Pretreatment of the subject with immune depletion (eg, lymphocyte depletion) therapy can improve the effect of the cell therapy of the present invention.
  • Pretreatment with lymphocyte scavengers (including a combination of cyclophosphamide and fludarabine) effectively improves the efficacy of metastatic tumor infiltrating lymphocytes (TIL) in cell therapy, including improving the response and/or persistence of metastatic cells .
  • the method includes administering a chemotherapeutic agent to reduce tumor burden before administering the agent of CD19 CAR-T cells.
  • the method includes administering a pretreatment agent such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof. In some embodiments, the method includes administering fludarabine and optionally another chemotherapeutic agent other than fludarabine.
  • the subject may be given lymphocyte depletion therapy at least 2 days, such as at least 3, 4, 5, 6, or 7 days before administering the agent of cells.
  • the lymphocyte depletion therapy is administered or started at least 48 hours or at least about 48 hours or 48 hours or about 48 hours or at least 144 hours or at least about 144 hours prior to administration of the agent of cells. In some embodiments, the lymphocyte depletion therapy is administered or started between 48 hours or about 48 hours and 144 hours or about 144 hours before administering the agent of cells.
  • the method includes administering to the subject a pretreatment agent such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject before the first or subsequent dose.
  • a pretreatment agent such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof.
  • the subject can be administered a pretreatment agent at least 2 days before the first or subsequent dose, such as at least 3, 4, 5, 6, or 7 days.
  • the pretreatment agent is administered to the subject no more than 7 days before the first or subsequent dose, such as no more than 6, 5, 4, 3, or 2 days.
  • the subject is administered cyclophosphamide at a dose of about 20 mg/kg to 100 mg/kg, or about 40 mg/kg to 80 mg/kg, or about 20 mg/kg to about 60 mg/kg Perform pretreatment.
  • the subject is pretreated with cyclophosphamide at about 60 mg/kg.
  • cyclophosphamide can be administered in a single dose or can be administered in multiple doses, such as daily, every other day, or every third day.
  • cyclophosphamide is administered once a day for one or two days.
  • the subject when the lymphocyte scavenger comprises cyclophosphamide, the subject is administered at a dose of about 100 mg/m 2 to 500 mg/m 2 (inclusive), or about 250 mg/m 2 to 500 mg/m every day. 2 (inclusive), or about 200 mg/m 2 to 400 mg/m 2 , or about 250 mg/m 2 to 350 mg/m 2 (inclusive) cyclophosphamide. In some cases, about 250 mg/m 2 to 500 mg/m 2 (inclusive) of cyclophosphamide is administered to the subject once a day for 2 days or 3 days or 4 days.
  • cyclophosphamide can be administered in a single dose or can be administered in multiple doses, such as daily, every other day, or every third day. In some embodiments, cyclophosphamide is administered daily, such as for 1 to 5 days, for example, for 3 to 5 days. In some cases, about 300 mg/m 2 of cyclophosphamide is administered to the subject every day for 3 days before starting cell therapy.
  • the subject when the lymphocyte scavenger comprises fludarabine, the subject is administered at a dose of about 1 mg/m 2 to 100 mg/m 2 , or about 10 mg/m 2 to 75 mg/m 2 , or About 15mg/m 2 ⁇ 50mg/m 2 , or about 20mg/m 2 ⁇ 40mg/m 2 , or about 20mg/m 2 ⁇ 30mg/m 2 , or about 24mg/m 2 ⁇ 35mg/m 2 , or about 24mg /m 2 ⁇ 26mg/m 2 of fludarabine. In some cases, 25 mg/m 2 of fludarabine is administered to the subject.
  • fludarabine in some cases, about 30 mg/m 2 of fludarabine is administered to the subject.
  • fludarabine can be given in a single dose or can be given in multiple doses, such as daily, every other day, or every third day.
  • fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days.
  • the subject is given about 30 mg/m 2 of fludarabine every day for 3 days before starting cell therapy.
  • the lymphocyte scavenger comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Therefore, the combination of agents may include cyclophosphamide under any dose or administration schedule (such as those above) and any dose or administration schedule (such as those above). Fludarabine.
  • the subject is administered 21 to 59 mg/kg (about 1 to 2 g/m2) of cyclophosphamide and 3 to 4 doses of 17 to 27 mg/m 2 of fludarat prior to the administration of the cells. coast.
  • the lymphocyte depletion chemotherapy can be modified by reducing or omitting the dose of cyclophosphamide or administering a lower total dose of cyclophosphamide at the same time as fludarabine to minimize the impact on the subject.
  • the method includes administering the cell or a composition containing the cell to a subject, tissue, or cell, such as having a CD19-positive hematological tumor, at risk of having a CD19-positive hematological tumor, or suspected of having CD19 Subjects, tissues or cells with positive blood tumors.
  • the subject is an adult subject.
  • the subject is over 20, 30, 40, 50, 60, or 70 years old or over about 20, 30, 40, 50, 60, or 70 years old.
  • the provided methods include adoptive cell therapy methods (e.g., CAR+T cells) for the treatment of acute and chronic lymphocytic leukemia (ALL/CLL).
  • Treatments for high-risk CLL include chemotherapy (Hallek et al. (2010) Lancet 376:1164-1174), but recently the BTK inhibitor ibrutinib was first approved for relapsed and refractory diseases, and subsequently used for first-line treatment (Burger et al. (2015) N. Engl J. Med., 373: 2425-2437; Byrd et al. (2013) N. Engl. J. Med., 369: 32-42).
  • ORR overall response rate
  • CR complete response rate
  • the survival period of patients who progress to ibrutinib may be short.
  • the provided methods and uses provide or achieve such as in a particular treatment group of subjects (such as in patients with leukemia (such as ALL/CLL or NHL) Including those with high-risk disease) improved or longer-lasting response or efficacy.
  • the method is performed by administering T cell therapy (e.g., a composition that includes cells for adoptive cell therapy, such as T cell therapy (e.g., CAR expressing T cells)) and lymphocyte depletion therapy (e.g., cyclophosphamide, Fludarabine or a combination thereof) is advantageous.
  • T cell therapy e.g., a composition that includes cells for adoptive cell therapy, such as T cell therapy (e.g., CAR expressing T cells)
  • lymphocyte depletion therapy e.g., cyclophosphamide, Fludarabine or a combination thereof
  • the diagnosis of ALL should use MICM (morphology, immunology, cytogenetics and molecular) diagnostic mode b1, and the diagnostic classification should use the WHO2016 standard.
  • the minimum standard should be cell morphology and immunophenotyping to ensure the reliability of the diagnosis; the ratio of primitive/naive lymphocytes in the bone marrow> 20% can be used to diagnose ALL; the immunophenotyping should use multi-parameter flow cytometry.
  • genetic abnormalities with good prognosis include superdiploid (51-65 chromosomes), t(12;21) (p13; q22) and (or) ETV6-RUNX1; poor prognosis genetic abnormalities Including hypodiploid ( ⁇ 44 chromosomes), t(v; 1lq23) [t(4; 11) and other MLL rearrangements], t(9; 22) (q34; ql 1.2), complex chromosomal abnormalities.
  • Ann Arbor staging Stage I: Invasion of 1 lymph node area or 1 extranodal organ or site.
  • Stage II One side of the diaphragm invades 2 or more lymph node areas or a localized invasion of 1 extranodal organ or site.
  • Stage III Infringement of the lymph node area on both sides of the diaphragm or a localized invasion of an extranodal organ or part or the spleen or both.
  • Stage IV Diffuse or disseminated invasion of one or more extranodal organs or parts, with or without lymph node invasion.
  • a and B are divided into A and B according to the presence or absence of systemic symptoms.
  • the method includes administering cells to a subject selected or identified as having a high-risk NHL.
  • the subject exhibits one or more cytogenetic abnormalities, such as associated with high-risk NHL.
  • the subject is selected or identified based on having a disease or condition that is characterized or determined to be aggressive NHL, diffuse large B-cell lymphoma (DLBCL), primary mediastinum Large B-cell lymphoma (PMBCL), T-cell/histiocytic-rich large B-cell lymphoma (TCHRBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and/or follicular lymphoma (FL) .
  • DLBCL diffuse large B-cell lymphoma
  • PMBCL primary mediastinum Large B-cell lymphoma
  • TCHRBCL T-cell/histiocytic-rich large B-cell lymphoma
  • MCL mantle cell lymphoma
  • FL follicular lymphoma
  • the subject has been previously treated with a therapy or therapeutic agent targeting ALL/CLL or NHL before administering the cells expressing the recombinant antigen receptor.
  • the therapeutic agent is a kinase inhibitor, such as an inhibitor of Bruton's tyrosine kinase (Btk), for example, ibrutinib.
  • the therapeutic agent is an inhibitor of B-cell lymphoma-2 (Bcl-2), such as venetoclax.
  • the therapeutic agent is an antibody (e.g., monoclonal antibody) that specifically binds to an antigen expressed by cells of ALL/CLL or NHL, such as from CD20, CD19, CD22, ROR1, CD45, CD21, CD5 , CD33, Ig ⁇ , Ig ⁇ , CD79a, CD79b or CD30 any one or more of the antigen.
  • the therapeutic agent is an anti-CD20 antibody, such as rituximab.
  • the therapeutic agent is wasting chemotherapy, which is a combination therapy including rituximab, such as a combination therapy of fludarabine and rituximab or anthracycline and rituximab Anti-combination therapy or CHOP combination therapy (cyclophosphamide, doxorubicin, vincristine, prednisone) or R-CHOP combination therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone).
  • the subject has previously been treated with hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT or autologous HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the subject has been treated or has previously received at least or about at least 1, 2, 3, or 4 other than lymphocyte clearance therapy and/or cells expressing antigen receptors for the agent Therapies to treat NHL or ALL/CLL. In some embodiments, the subject has been previously treated with chemotherapy or radiation therapy.
  • the subject is refractory or unresponsive to other therapies or therapeutic agents.
  • the subject suffers from persistent or recurrent disease, for example, after treatment with another therapy or therapeutic intervention (including chemotherapy or radiation).
  • cell therapy such as adoptive cell therapy, such as adoptive T cell therapy
  • adoptive cell therapy is performed by autologous transfer, in which it is isolated from a subject to be subjected to cell therapy or from a sample derived from such subject and/or otherwise Way to prepare cells. Therefore, in some aspects, the cells are derived from the subject (e.g., patient) in need of treatment, and the cells are administered to the same subject after isolation and processing.
  • cell therapy such as adoptive cell therapy, such as adoptive T cell therapy
  • adoptive cell therapy is carried out by allogeneic transfer, wherein separation from a subject other than the subject to receive or ultimately receive cell therapy, such as the first subject And/or prepare cells in other ways.
  • the cells are then administered to a different subject of the same species, such as a second subject.
  • the first and second subjects are genetically the same.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject.
  • the cells can be administered by any suitable method, such as by bolus infusion, by injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection , Intrachoroidal injection, anterior chamber injection, subconjunctival injection, subconjunctival injection, suboculofascial sac injection, retrobulbar injection, peribulbar injection or posterior scleral delivery. In some embodiments, they are administered parenterally, intrapulmonarily, and intranasally, and (if needed for local treatment) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • a given dose is administered by a single bolus administration of cells. In some embodiments, a given dose is administered by multiple boluses of cells, for example, over a period of no more than 3 days, or by continuous infusion of cells.
  • the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are given for prevention or treatment purposes, previous treatments, and subjects being tested.
  • the patient s clinical history and response to the cells, as well as the decision of the attending physician.
  • the composition and cells are suitable for administration to a subject at one time or over a series of treatments.
  • the biological activity of the engineered cell population is measured by any of a number of known methods.
  • the parameters to be evaluated include the specific binding of engineered or natural T cells or other immune cells to the antigen, which is evaluated in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry.
  • any suitable method known in the art can be used to measure the ability of engineered cells to destroy target cells, as described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) And Herman et al. J. Immunological Methods, 285(1): 25-40 (2004) described in the cytotoxicity assay.
  • the biological activity of cells can also be measured by measuring the expression and/or secretion of certain cytokines such as CD107a, IFN ⁇ , IL-2 and TNF.
  • biological activity is measured by evaluating clinical outcomes such as tumor burden or reduction in burden.
  • the toxicity results, the persistence and/or expansion of the cells, and/or the presence or absence of the host immune response are assessed.
  • the engineered cell is modified in many ways so that its therapeutic or preventive efficacy is increased.
  • the engineered CAR or TCR expressed by the population can be conjugated to the targeting moiety directly or indirectly through a linker.
  • the practice of conjugating a compound such as a CAR or TCR to a targeting moiety is known in the art.
  • the cell is administered as part of a combination therapy, such as concurrently with another therapeutic intervention such as an antibody or engineered cell or receptor or agent (such as a cytotoxic agent or therapeutic agent) or sequentially in any order give.
  • another therapeutic intervention such as an antibody or engineered cell or receptor or agent (such as a cytotoxic agent or therapeutic agent) or sequentially in any order give.
  • the cell is co-administered with one or more additional therapeutic agents or co-administered with another therapeutic intervention (simultaneously or sequentially in any order).
  • the cell is co-administered with another therapy in close enough time so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa.
  • the cell is administered before one or more additional therapeutic agents.
  • the cell is administered after one or more additional therapeutic agents.
  • the one or more additional agents include cytokines such as IL-2, for example to enhance persistence.
  • the administration of a given "dose” includes administration of a certain number of cells in a single composition and/or a single uninterrupted administration (for example, in a single injection or continuous infusion), and is also included in no more than 4
  • a certain number of cells are administered in divided doses provided in multiple separate compositions or infusions over a specified period of day. Therefore, in some cases, dose refers to a single or continuous administration of a certain number of cells, given or started at a single point in time. However, in some cases, the dose is given by multiple injections or infusions within a period of no more than 4 days, such as once a day for 4 days or 3 days or 2 days or by multiple infusions within a day .
  • the dose of cells is administered as a single pharmaceutical composition. In some embodiments, the dose of cells is administered in multiple compositions that collectively contain the dose of cells.
  • a dose of cells is administered to the subject according to the provided methods.
  • the size or timing of the dose is determined according to the particular disease or condition of the subject.
  • administering to the subject from about 105 cells to about 107 kg of subject body weight range cells /, for example, from about 105 to about 106 cells (e.g., about 1x10 5, about 2x10 5 , about 3x10 5 , about 4x10 5 , about 5x10 5 , about 6x10 5 , about 7x10 5 , about 8x10 5 , about 9x10 5 , about 1x10 6 or the range defined by any two of the foregoing), about 10 6 to about 107 cells (e.g., about 1x10 6, about 2x10 6, about 3x10 6, about 4x10 6, about 5x10 6, about 6x10 6, about 7x10 6, about 8x10 6, about 9x10 6, about 1x10 7, or by the aforementioned Any two values defined in the range), or any value between these ranges and/or per kilogram of the subject's body weight.
  • 105 to about 106 cells e.g., about 1x10 5, about 2x10 5 , about 3x10 5 , about
  • the dosage may vary depending on the attributes specific to the disease or condition and/or patient and/or other treatments. In some embodiments, these values refer to the number of recombinant receptor CD19-CAR expressing cells; in other embodiments, they refer to the number of T cells or PBMCs or total cells administered.
  • the cell therapy involves the administration of a dose comprising a plurality of cells, the cells of at least or at least about 0.1x10 6 cells / kg body weight of the subject, 0.2x10 6 cells /kg,0.3x10 6 cells / kg , 0.4x10 6 cells/kg, 0.5x10 6 cells/kg, 1x10 6 cells/kg, 2x10 6 cells/kg, 3x10 6 cells/kg, 4x10 6 cells/kg, 5x10 6 cells/ kg, 6x10 6 cells / kg, 7x10 6 cells / kg, 8x10 6 cells / kg, 9x10 6 cells / kg or 1x10 7 cells / kg, or of 0.1x10 6 cells /kg,0.2x10 6 /kg,0.3x10 6 cells cells cells cells cells /kg,0.4x10 6 /kg,0.5x10 6 cells / kg, 1x10 6 cells / kg, 2x10 6 cells / kg, 3x10 6 cells / kg, 4 ⁇ 10 6 cells/kg,
  • cell therapy includes administering a dose comprising a certain number of cells, the dose comprising (a) about 4.9 ⁇ 10 6 cells/kg of subject’s body weight (cells/kg), (b) about 3 ⁇ 10 8 total cells , (C) about 1 ⁇ 10 7 cells/kg, (d) about 6 ⁇ 10 8 total cells, (a) not more than about 4.9 ⁇ 10 6 cells/kg, (b) or not more than about 3 ⁇ 10 8 total cells, ( c) no more than about 1x10 7 cells/kg, (d) no more than about 6x10 8 total cells, (e) about 1.5x10 5 cells/kg to about 4.9x10 6 cells/kg, (f) about 1.5 x10 5 cells/kg to about 1x10 7 cells/kg, (g) about 1x10 7 total cells to about 3x10 8 total cells, and/or (h) about 1x10 7 total cells to about 6x10 8 total cells Cell (including each endpoint).
  • the agent contains (a) about 5.8 ⁇ 10 5 cells/kg, (b) about 1.1 ⁇ 10 6 cells/kg, (c) about 1.7 ⁇ 10 6 cells/kg, (d) about 1.8x10 6 cells/kg, (e) about 1.9x10 6 cells/kg, (f) about 2.0x10 6 cells/kg, (g) about 2.1x10 6 cells/kg, (h) about 2.6x10 106 cells / kg, (h) from about 3x10 7 total cells, (i) from about 6x10 7 total cells ,, (j) from about 8x10 7 total cells, (k) approximately 9x10 7 total cells, (l) About 1x10 8 total cells, (m) about 1.5x10 8 total cells, (n) about 5.8x10 5 to about 4.9x10 6 cells/kg, (o) about 1.1x10 6 to about 4.9x10 6 cells/ kg, (p) about 1.7x10 6 /kg to about 4.9x10 6 cells/kg, (q) about 1.8x10
  • the agent of cells comprises administering (a) about 4.9 ⁇ 10 6 cells/kg of subject’s body weight (cells/kg), (b) about 3 ⁇ 10 8 total cells, and (c) about 1 ⁇ 10 7 cells /kg, (d) about 6 ⁇ 10 8 total cells, (a) not more than about 4.9 ⁇ 10 6 cells/kg, (b) or not more than about 3 ⁇ 10 8 total cells, (c) or not more than about 1 ⁇ 10 7 Cells/kg, (d) or no more than about 6x10 8 total cells, (e) or about 1.5x10 5 to about 4.9x10 6 cells/kg, (f) or about 1.5x10 5 to about 1x10 7 cells/ kg, (g) or about 1 ⁇ 10 7 to about 3 ⁇ 10 8 total cells, and/or (h) or about 1 ⁇ 10 7 to about 6 ⁇ 10 8 total cells (including each endpoint).
  • Standard dose refers to the case where the dose of the entire course of treatment is divided into multiple doses to the subject within a course of treatment, and the dose of a single administration is also considered to be a single dose.
  • the agent can be administered in divided doses.
  • the dosage may be administered to the subject within 2 days or 3 days or 4 days.
  • An exemplary method for divided administration includes 10% of the total dose on the first day, 30% of the total dose on the second day, and 60% of the total dose on the third day.
  • 12.5% of the total dose can be given on the first day
  • 37.5% of the total dose can be given on the second day
  • 50% of the total dose can be given on the fourth day.
  • 30% of the total dose is given on the first day and 70% of the total dose is given on the second day.
  • the divided dose does not exceed 4 days or 3 days or 2 days.
  • the agent cells are generally large enough to effectively reduce disease burden.
  • the size of the dose is determined based on one or more criteria, such as the subject’s response to existing treatments such as chemotherapy, the subject’s disease burden such as tumor load, volume, size or degree, degree of metastasis, or Type, stage, and/or likelihood or incidence of toxic results in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or against the cells and/or recombinant receptors administered The host immune response.
  • the subject s response to existing treatments such as chemotherapy
  • the subject’s disease burden such as tumor load, volume, size or degree, degree of metastasis, or Type, stage, and/or likelihood or incidence of toxic results in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or against the cells and/or recombinant receptors administered The host immune response.
  • the method further includes administering one or more additional doses of chimeric antigen receptor (CAR)-expressing cells and/or lymphocyte depletion therapy, and/or repeating one or more steps of the method.
  • the one or more additional doses are the same as the initial dose.
  • the one or more additional doses are different from the initial dose, for example higher, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or higher than the initial dose. 10 times or more, or lower such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times or more times lower than the initial dose.
  • the administration of one or more additional doses is determined based on the subject’s response to the initial treatment or any existing treatment, the subject’s disease burden such as tumor load, volume, size, or Degree, degree or type of metastasis, stage, and/or likelihood or incidence of toxic results in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or targeting the cells administered And/or the host immune response of the recombinant recipient.
  • the subject s disease burden such as tumor load, volume, size, or Degree, degree or type of metastasis, stage, and/or likelihood or incidence of toxic results in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or targeting the cells administered And/or the host immune response of the recombinant recipient.
  • At least or about at least 30%, 40%, 50%, 60%, 70%, 80%, 90% of subjects treated according to the method achieve complete remission (CR) and/or partial remission ( PR) and/or achieve objective response (OR).
  • administration according to the provided methods generally reduces or prevents the spread or burden of the disease or condition in the subject.
  • the method generally reduces tumor size, volume, metastasis, percentage of blasts in bone marrow or molecularly detectable cancer and/or improves prognosis or survival or other related tumor burden symptom.
  • progression-free survival is described as the length of time during and after treatment of a disease (such as cancer) that a subject survives with the disease without the disease getting worse.
  • an objective response is described as a measurable response.
  • the objective response rate is described as the proportion of patients who achieve CR or PR.
  • overall survival is described as the length of time from the date of diagnosis or start of treatment of a disease (such as cancer) to when a subject diagnosed with the disease is still alive.
  • event-free survival is described as the length of time after the end of cancer treatment that a subject remains free of certain complications or events that the treatment is intended to prevent or delay. These events may include the recurrence of cancer or the onset of certain symptoms, such as bone pain caused by cancer that has spread to the bone, or death.
  • alternative administration regimens are used, such as alternative administration methods in which the subject receives one or more alternative therapeutic agents and/or the subject does not receive a dose of cells and/or according to the provided methods
  • This method reduces the burden of the disease or condition, for example, tumors, to a greater extent and/or over a longer period of time than the reduction observed in comparable methods of alternative administration methods of lymphocyte scavengers.
  • the burden of a disease or condition in a subject is detected, evaluated, or measured.
  • the disease burden can be detected in some aspects by detecting the total number of disease or disease-related cells, such as tumor cells, in the subject or in the subject's organs, tissues, or body fluids (such as blood or serum).
  • the subject's survival, survival within a specific period of time, degree of survival, existence or duration of event-free or asymptomatic survival, or recurrence-free survival is assessed.
  • any symptoms of the disease or condition are assessed.
  • the event-free survival rate or probability of subjects treated by the method at 6 months after the dose is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70% , Greater than about 80%, greater than about 90%, or greater than about 95%.
  • the overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%.
  • the subject treated with the method exhibits event-free survival, recurrence-free survival, or survival for at least 6 months or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. year.
  • the time to progression is improved, such as the time to progression is greater than 6 months or greater than about 6 months or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the scavenger method reduces the probability of recurrence after treatment by this method.
  • the probability of recurrence at 6 months after the first dose is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, Less than about 20% or less than about 10%.
  • Disease burden/disease burden may include the total number of diseased cells in the subject or in the subject's organ, tissue, or body fluid (such as the organ or tissue of a tumor or another location, such as a location that will indicate metastasis).
  • tumor cells can be detected and/or quantified in blood or bone marrow in certain hematological malignancies.
  • the disease burden may include the quality of the tumor, the number or extent of metastases, and/or the percentage of primitive cells present in the bone marrow.
  • response assessment utilizes any of clinical, hematological, and/or molecular methods.
  • CR Complete remission
  • PR partial remission
  • PD Progressive disease
  • the subject Show CR or PR.
  • the subject has leukemia.
  • the degree of disease burden can be determined by evaluating residual leukemia in the blood or bone marrow.
  • the subject if greater than or equal to 5% of blasts are present in the bone marrow, for example by light microscopy, the subject exhibits a morphological disease, such as greater than or equal to 10% of blasts in the bone marrow, or greater than or equal to 10% in the bone marrow. 20% blast cells, 30% blast cells in the bone marrow, 40% blast cells in the bone marrow, or 50% blast cells in the bone marrow. In some embodiments, if less than 5% blasts are present in the bone marrow, the subject exhibits complete or clinical remission.
  • the subject may exhibit complete remission, but there is a small portion of residual leukemia cells that are morphologically (by light microscopy techniques) undetectable. If the subject exhibits less than 5% of the original cells in the bone marrow and exhibits a molecularly detectable cancer, the subject is said to exhibit minimal residual disease (MRD).
  • MRD minimal residual disease
  • any of a variety of molecular techniques that allow sensitive detection of a small number of cells can be used to assess molecularly detectable cancer. In some aspects, such techniques include PCR assays, which can determine unique Ig/T cell receptor gene rearrangements or fusion transcripts resulting from chromosomal translocations.
  • flow cytometry can be used to identify cancer cells based on the leukemia-specific immunophenotype.
  • molecular detection of cancer can detect only 1 leukemia cell in 10,000 normal cells or only 1 leukemia cell in 100,000 normal cells.
  • the subject if at least or more than 1 leukemia cell in 10,000 cells or 1 leukemia cell in 100,000 cells is detected, such as by PCR or flow cytometry, the subject exhibits Molecularly detectable MRD.
  • the subject's disease burden is molecularly undetectable or MRD-, so that in some cases leukemia cells in the subject cannot be detected using PCR or flow cytometry techniques.
  • the bone marrow of the subject or in the bone marrow of greater than 50%, 60%, 70%, 80%, 90%, or more subjects treated according to the method
  • Index cloning to leukemia such as ALL/CLL.
  • Lugano criteria are used to assess responses (Cheson et al., J CO Se ptem be r 20, 2014vol.32no.273059-3067; Johnson et al., (2015) Imaging for staging and response assessment in lymphoma.Radiology 2:323 -338). Lugano criteria include assessment by imaging, tumor volume measurement, and assessment of spleen, liver, and bone marrow invasion.
  • the response assessed using Lugano criteria includes the use of positron emission tomography (PET) computer tomography (CT) and/or CT (as the case may be) for imaging evaluation.
  • PET-CT assessment may further include the use of fluorodeoxyglucose (FDG) to assess FDG absorption in FDG lymphoma.
  • FDG fluorodeoxyglucose
  • FDG lymphomas include Hodgkin’s lymphoma (HL) and certain non-Hodgkin’s lymphomas (NHL), which include diffuse large B-cell lymphoma (DLBCL), marginal zone NHL with aggressive transformation, and FDG Lymph node lymphoma (except basically all histological types: chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, lymphoplasmacytic lymphoma/macroglobulinemia, and mycosis fungoides).
  • CLL chronic lymphocytic leukemia
  • small lymphocytic lymphoma small lymphocytic lymphoma
  • lymphoplasmacytic lymphoma/macroglobulinemia and mycosis fungoides.
  • CT is the preferred imaging method.
  • post-treatment scans are performed for as long as possible after the treatment is given.
  • a post-treatment scan is performed after treatment for a minimum of 3 weeks, such as 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, and 12 weeks after treatment. Scanning after a week or more of treatment.
  • a 5-point scale such as the Deauville 5-point scale (Deauville 5ps) can be used for assessment or staging.
  • the Dauville score is based on the visual interpretation of the fluorodeoxyglucose (FDG) absorption visible on PET/CT scans for each lesion compared to the two reference organs, the mediastinum (ie blood pool) and the liver.
  • FDG fluorodeoxyglucose
  • An assessment is performed before treatment (initial staging), and a second round of FDG PET/CT scans are used during and/or after treatment to assess residual quality (compared to FDG updates in the reference organ).
  • the scale ranges from 1 to 5, with 1 being the best and 5 being the worst.
  • the 5-point scale includes the following criteria: 1. No absorption above background; 2. Absorption ⁇ mediastinum; 3. Absorption> mediastinum but ⁇ liver; 4. Moderate absorption> liver; 5. Absorption is significantly high In the liver (for example, the maximum standard absorption value (SUVMAX>2 livers; 5a) and/or new lesions that may be associated with lymphoma (based on response assessment) (5b); X, new lesions that are unlikely to be associated with lymphoma Absorption area.
  • a Deauville score of 1 or 2 is considered to indicate a complete metabolic response (CMR) in the middle and end of treatment.
  • a Deauville score of 3 also indicates CMR, but the interpretation of a score of 3 depends on the time of evaluation, clinical background, and treatment.
  • a mid-term Deauville score of 4 or 5 is considered to indicate a partial metabolic response. However, if absorption decreases from baseline, a Deauville score of 4 or 5 at the end of treatment indicates residual metabolic disease; if there is no change in absorption from baseline, it indicates no metabolic response (NMR); and if absorption from baseline increases and/or New lesions indicate progressive metabolic disease (PMD). In the middle and end of treatment, NMR or PMD indicates treatment failure.
  • the complete response at the end of the treatment includes complete metabolic response and complete radiation response at various measurable sites.
  • these sites include lymph nodes and extralymphatic sites, where when PET-CT is used, CR is described as a score of 1, 2 or 3 on a 5-point scale, with or without residual mass.
  • absorption may be greater than normal mediastinum and/or liver.
  • CT is used to assess the response in the lymph nodes, where CR is described as an extralymphatic site without disease, and the longest transverse diameter (LDi) of the lesion of the target lymph node/lymph node mass must be restored to ⁇ 1.5 cm.
  • Other assessment sites include bone marrow, where PET-CT-based assessments should show the lack of evidence of FDG disease in the bone marrow, and CT-based assessments should show normal morphology, or IHC-negative if uncertain.
  • Other sites may include an assessment of organ enlargement, which should return to normal.
  • unmeasured lesions and new lesions are evaluated, which should not be present in the case of CR.
  • the method does not cause or reduce toxicity or toxic consequences (such as cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, at least 38 degrees Celsius, or at least About 38 degrees Celsius fever for three or more days and a plasma CRP level of at least 20 mg/dL or at least about 20 mg/dL), neurotoxicity and/or neurotoxicity potential.
  • CRS cytokine release syndrome
  • sCRS severe CRS
  • macrophage activation syndrome tumor lysis syndrome
  • tumor lysis syndrome at least 38 degrees Celsius, or at least About 38 degrees Celsius fever for three or more days and a plasma CRP level of at least 20 mg/dL or at least about 20 mg/dL
  • at least 50% of the subjects treated according to the method e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects treated
  • have no Exhibiting toxic results e.g. CRS or neurotoxicity
  • severe toxic results e.g. severe CRS or severe neuro
  • adoptive T cell therapy such as treatment with T cells expressing chimeric antigen receptors can induce toxic effects or results, such as cytokine release syndrome and neurotoxicity. In some instances, this effect or result is parallel to high levels of circulating cytokines, which may be the basis for the observed toxicity.
  • the toxic result is cytokine release syndrome (CRS) or severe CRS (sCRS) or is associated with cytokine release syndrome (CRS) or severe CRS (sCRS) or is indicative of cytokine release syndrome (CRS) or severe CRS (sCRS).
  • CRS is caused by an excessive systemic immune response mediated, for example, by T cells, B cells, NK cells, monocytes, and/or macrophages. Such cells can release a large number of inflammatory mediators such as cytokines and chemokines. Cytokines can trigger an acute inflammatory response and/or induce endothelial organ damage, which may lead to microvascular leakage, heart failure or death. Severe life-threatening CRS can cause lung infiltration and lung damage, renal failure, or diffuse intravascular coagulation. Other severely life-threatening toxicities can include cardiotoxicity, respiratory distress, neurotoxicity, and/or liver failure.
  • Anti-inflammatory therapies such as anti-IL-6 therapy (e.g., anti-IL-6 antibodies, such as tocilizumab or antibiotics) can be used to treat CRS.
  • anti-IL-6 therapy e.g., anti-IL-6 antibodies, such as tocilizumab or antibiotics
  • the results, signs, and symptoms of CRS are known and include those described herein.
  • a specific dosage regimen or administration affects or does not affect a given CRS-related result, sign, or symptom
  • a specific result, sign, and symptom and/or amount or degree can be specified.
  • CRS usually occurs 6-20 days after the infusion of the CAR expressing cells. In some cases, CRS occurs less than 6 days or more than 20 days after CAR T cell infusion.
  • the incidence and time of CRS may be related to the baseline cytokine level or tumor burden at the time of infusion.
  • CRS includes elevated serum levels of interferon (IFN)- ⁇ , tumor necrosis factor (TNF)- ⁇ , and/or interleukin (IL)-2.
  • IFN interferon
  • TNF tumor necrosis factor
  • IL interleukin
  • Other cytokines that can be rapidly induced in CRS are IL-1 ⁇ , IL-6, IL-8 and IL-10.
  • Exemplary results related to CRS include fever, chills, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, elevated ALT/AST, renal failure, heart disease, hypoxia, neurological disorders, and die.
  • Nervous system complications include delirium, epileptiform activity, confusion, difficulty finding words, aphasia, and/or becoming dull.
  • Other results associated with CRS include fatigue, nausea, headache, epilepsy, tachycardia, myalgia, skin rash, acute vascular leakage syndrome, liver damage, and kidney failure.
  • CRS is associated with an increase in one or more factors (such as serum ferritin, d-dimer, aminotransferase, lactate dehydrogenase, and triglycerides), or with low fibrinogen blood or liver Splenomegaly is related.
  • factors such as serum ferritin, d-dimer, aminotransferase, lactate dehydrogenase, and triglycerides
  • the results associated with CRS include one or more of the following: sustained fever, such as fever at a specified temperature (eg, greater than 38 degrees Celsius or greater than about 38 degrees Celsius) for two or more days, such as three or more Days, such as four or more days or at least three consecutive days; fever greater than 38 degrees Celsius or greater than about 38 degrees Celsius; and at least two cytokines (e.g., at least two of the following group, the group consisting of : Interferon gamma (IFN gamma), GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine and IL-5 and/or tumor necrosis factor alpha (TNF alpha) levels before treatment are increased compared to cytokines , Such as the greatest fold change such as at least 75-fold or at least about 75-fold, or the greatest fold change of at least one such cytokine, such as at least 250-fold or at least about 250-fold; and/or at least one clinical sign of toxicity such as hypotension (
  • Exemplary CRS-related results include increased or high serum levels of one or more factors including cytokines and chemokines and other factors associated with CRS. Exemplary results further include an increase in the synthesis or secretion of one or more such factors. This synthesis or secretion can be through T cells or cells that interact with T cells (such as innate immune cells or B cells).
  • CRS-related serum factors or CRS-related results include inflammatory cytokines and/or chemokines
  • the inflammatory cytokines and/or chemokines include interferon gamma (IFN- gamma), TNF -a, IL-1 ⁇ , IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Ra, granulocyte macrophage colony stimulating factor (GM-CSF), macro Phage Inflammatory Protein (MIP)-1, Tumor Necrosis Factor ⁇ (TNF ⁇ ), IL-6 and IL-10, IL-1 ⁇ , IL-8, IL-2, MIP-1, Flt-3L, fracktalkine and/ Or IL-5.
  • IFN- gamma interferon gamma
  • TNF -a interferon gamma
  • IL-1 ⁇ IL-2
  • IL-6, IL-7 IL-8
  • IL-10 IL-12
  • sIL-2Ra
  • the factor or result includes C-reactive protein (CRP).
  • CRP C-reactive protein
  • CRP is also a marker of cell expansion.
  • it is measured that subjects with high levels of CRP (eg, ⁇ 15 mg/dL) have CRS.
  • CRS it is measured that subjects who have high levels of CRP do not have CRS.
  • the measurement of CRS includes a measurement of CRP and another factor indicative of CRS.
  • one or more inflammatory cytokines or chemokines are monitored before, during, or after CAR treatment.
  • the one or more cytokines or chemokines include IFN- ⁇ , TNF- ⁇ , IL-2, IL-1 ⁇ , IL-6, IL-7, IL-8, IL-10, IL- 12.
  • IFN- ⁇ , TNF- ⁇ , and IL-6 are monitored.
  • a subject is considered to have developed "severe CRS" ("sCRS") in response to or secondary to cell therapy or the administration of a dose of cells, provided that the subject shows: (1) Fever of at least 38 degrees Celsius for at least three days; (2) Increase in cytokines, including (a) The maximum fold change of at least two of the following seven cytokines is at least 75 compared with the level immediately after administration Times: Interferon gamma (IFN gamma), GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine and IL-5 and/or (b) compared with the level immediately after administration, the following seven The maximum fold change of at least one of the cytokines is at least 250-fold: interferon gamma (IFN gamma), GM-CSF, IL-6, IL-10, Flt
  • CRS includes a combination of: (1) persistent fever (fever at least 38 degrees Celsius for at least three days) and (2) the serum level of CRP is at least 20 mg/dL or at least about 20 mg/dL.
  • CRS includes hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation.
  • the toxic result is neurotoxicity or is related to neurotoxicity.
  • symptoms associated with the clinical risk of neurotoxicity include confusion, delirium, expressive aphasia, retardation, myoclonus, drowsiness, altered mental status, convulsions, epileptiform activities, epilepsy (optionally via brain Electrogram [EEG] confirmed), elevated beta amyloid (A beta) levels, elevated glutamate levels, and elevated oxygen free radical levels.
  • neurotoxicity is graded based on severity (e.g., using a 1-5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010)); National Cancer Institute-Common Toxicity Standard Section Version 4.03 (NCI-CTCAE v4.03)).
  • this method reduces symptoms associated with neurotoxicity compared to other methods.
  • subjects treated according to this method may have reduced neurotoxic symptoms such as weakness or numbness of limbs, loss of memory, vision and/or intelligence, uncontrollable obsessive-compulsiveness, and / Or compulsory behavior, delusions, headaches, cognitive and behavioral problems (including loss of motor control, cognitive deterioration and autonomic nervous system dysfunction, and sexual dysfunction).
  • subjects treated in accordance with the methods of the present invention may have reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, hypoesthesia, neuralgia, or paresthesia.
  • the method reduces the consequences associated with neurotoxicity, including damage to the nervous system and/or brain, such as the death of neurons. In some aspects, the method reduces the levels of factors associated with neurotoxicity, such as amyloid beta (A ⁇ ), glutamate, and oxygen free radicals.
  • a ⁇ amyloid beta
  • glutamate glutamate
  • oxygen free radicals oxygen free radicals
  • the cells used in or administered with the provided methods contain or are engineered to contain engineered receptors such as engineered antigen receptors such as chimeric antigen receptors (CAR) or T Cell Receptor (TCR).
  • engineered receptors such as engineered antigen receptors such as chimeric antigen receptors (CAR) or T Cell Receptor (TCR).
  • CAR chimeric antigen receptors
  • TCR T Cell Receptor
  • compositions containing such cells and/or enriched in such cells such as where a certain type of cells such as T cells or CD8+ or CD4+ cells are enriched or selected.
  • pharmaceutical compositions and formulations for administration such as adoptive cell therapy.
  • the cell includes one or more nucleic acids introduced via genetic engineering to express recombinant or genetically engineered products of such nucleic acids.
  • gene transfer is accomplished by first stimulating the cell, such as by combining the cell with a stimulus that induces a response (such as proliferation, survival, and/or activation) measured, for example, by the expression of a cytokine or activation marker, followed by The activated cells are transduced and expanded in culture to sufficient numbers for clinical applications.
  • Cells usually express recombinant receptors such as antigen receptors (including functional non-TCR antigen receptors such as chimeric antigen receptors (CAR)) and other antigen binding receptors such as transgenic T cell receptors (TCR). There are other chimeric receptors in the receptors.
  • antigen receptors including functional non-TCR antigen receptors such as chimeric antigen receptors (CAR)
  • CAR chimeric antigen receptors
  • TCR transgenic T cell receptors
  • the immune effector cells express a chimeric antigen receptor that recognizes CD19 or its variants.
  • the chimeric antigen receptor (CAR) usually includes an extracellular antigen binding domain, such as a part of an antibody molecule, which is usually a variable of an antibody.
  • the heavy (VH) chain region and/or the variable light (VL) chain region for example, scFv antibody fragments.
  • CD19 includes, but is not limited to, variants, isotypes, and species homologs of human CD19.
  • the CD19-CAR of the present invention can recognize CD19 of species other than humans.
  • the complete amino acid sequence of an exemplary human CD19 has SwissPort accession number P15391 (SEQ ID NO: 31).
  • CD19 is also known as B cell surface antigen B4, B cell antigen CD19, CD19 antigen or Leu-12.
  • Human CD19 is called Gene ID: 930 by Entrez Gene, and HGNC: 1633 by HGNC.
  • CD19 can be coded by a gene called CD19.
  • the use of "human CD19” herein covers all known or undiscovered alleles and polymorphic forms of human CD19.
  • the heavy chain variable region and the light chain variable region of the antibody have: HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6 and HCDR2 shown in SEQ ID NO: 7 HCDR3 shown in SEQ ID NO: 2, LCDR1 shown in SEQ ID NO: 2, LCDR2 shown in SEQ ID NO: 3, LCDR3 shown in SEQ ID NO: 4, or at least 85%, 86%, 87%, 88 with the above sequence %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences;
  • the antibody’s The heavy chain variable region and the light chain variable region have the amino acid sequence shown in SEQ ID NO: 8 and SEQ ID NO: 9, or have at least 85%, 86%, 87%, 88%, 89%, and the above sequence.
  • the heavy chain of the antibody is variable
  • the region and the light chain variable region have the amino acid sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the antibody has the sequence of the scFv shown in SEQ ID NO: 1 or 10, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the above sequence. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the chimeric antigen receptor has an amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20, or at least 85%, 86% of the above sequence. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the antibody portion of the chimeric antigen receptor also includes a linking sequence, which may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region, such as , IgG4 hinge region and/or CH1/CL and/or Fc region and/or CD8 hinge region.
  • a hinge region such as , IgG4 hinge region and/or CH1/CL and/or Fc region and/or CD8 hinge region.
  • the constant region or portion is of human IgG, such as IgG4 or IgG1.
  • the CD8 hinge region has the amino acid sequence shown in SEQ ID NO: 21, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the above sequence. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the antigen recognition domain is generally connected to one or more intracellular signal transduction moieties, for example, in the case of CAR, through an antigen receptor complex (such as a TCR complex) mimicking an activated signal transduction moiety, and/or through Signal from another cell surface receptor. Therefore, in some embodiments, the antigen binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signal transduction domains. In some embodiments, the transmembrane domain is fused to the extracellular domain.
  • an antigen receptor complex such as a TCR complex
  • the transmembrane domain is derived from natural or synthetic sources.
  • the domain is derived from any membrane-bound or transmembrane protein.
  • the transmembrane region includes ⁇ , ⁇ or ⁇ chains derived from T-cell receptors, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 , CD137, CD 154, and/or transmembrane regions include those functional variants (for example, those that substantially retain their structural parts (for example, transmembrane structural parts) and properties) (that is, include at least their transmembrane regions).
  • the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, for example, CD8 ⁇ or a functional variant thereof.
  • the transmembrane domain is synthetic.
  • the synthetic transmembrane domain mainly contains hydrophobic residues such as leucine and valine.
  • the CD8 transmembrane domain has the amino acid sequence shown in SEQ ID NO: 22, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the above sequence. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the CD28 transmembrane domain has the amino acid sequence shown in SEQ ID NO: 23, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the above sequence. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the receptor for example, CAR, generally includes at least one of one or more intracellular signal transduction moieties.
  • the receptor includes the intracellular component of the TCR complex, such as the TCRCD3+ chain that mediates T-cell activation and cytotoxicity, for example, the CD3 ⁇ chain. Therefore, in some aspects, the antigen binding moiety is connected to one or more cell signal transduction modules.
  • the cell signal transduction module includes a CD3 transmembrane domain, a CD3 intracellular signal transduction domain, and/or other CD transmembrane domains.
  • the receptor for example, CAR, also includes portions of one or more other molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16.
  • CAR or other chimeric antigen receptors include chimeric molecules between CD3 ⁇ (CD3- ⁇ ) or Fc receptor ⁇ and CD8, CD4, CD25, or CD16.
  • the cytoplasmic domain or intracellular signal transduction domain of the receptor activates at least one of the normal effector function or response of the immune cell, for example, it is engineered T cells modified to express CAR.
  • CAR induces T cell functions, such as cytolytic activity or helper T cell activity, such as secretion of cytokines or other factors.
  • an antigen receptor portion or a truncated portion of the intracellular signal transduction domain of a costimulatory molecule is used to replace the complete immunostimulatory chain, for example, if it transduces effector function signals.
  • the intracellular signal transduction domain includes the cytoplasmic sequence of the T cell receptor (TCR), and in some aspects, it also includes those co-receptors that exist in nature in concert with these receptors. Signal transduction is initiated after antigen receptor engagement.
  • TCR T cell receptor
  • the CAR does not include components used to generate costimulatory signals.
  • other CARs are expressed in the same cell and provide components for generating a second or costimulatory signal.
  • T cell activation is described as being mediated by two types of cytoplasmic signal transduction sequences: those that initiate antigen-dependent first activation by TCR (the first cytoplasmic signal transduction sequence), and those that are antigen-independent. Those that act to provide a second or costimulatory signal (second cytoplasmic signal transduction sequence).
  • the CAR includes one or both of such signal transduction components.
  • the antibody portion of the chimeric antigen receptor (such as CAR) further includes a signal peptide, such as a signal peptide including CD8 or a variant thereof, such as shown in SEQ ID NO: 27 at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity.
  • a signal peptide such as a signal peptide including CD8 or a variant thereof, such as shown in SEQ ID NO: 27 at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity.
  • the CAR includes a major cytoplasmic signal transduction sequence that regulates the initial activation of the TCR complex.
  • the first cytoplasmic signal transduction sequence that acts in a stimulating manner may include a signal transduction motif, which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM include cytoplasmic signal transduction sequences including those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b, and CD66d.
  • the cytoplasmic signal transduction molecule in the CAR comprises a cytoplasmic signal transduction domain, part of which or a sequence derived from CD3 ⁇ .
  • the CAR includes the transmembrane portion and/or signal transduction domain of a costimulatory receptor, such as CD28, CD137, OX40, DAP10, and ICOS.
  • a costimulatory receptor such as CD28, CD137, OX40, DAP10, and ICOS.
  • the same CAR includes both activation and costimulatory components.
  • the activation domain is included in one CAR, and the costimulatory component is provided by another CAR that recognizes another antigen.
  • the CAR includes activating or stimulating CAR and co-stimulating CAR, all of which are expressed on the same cell (see WO2014/055668).
  • the cell includes one or more stimulating or activating CARs and/or co-stimulating C A R.
  • the cell also includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl.
  • the activation signal delivered by the disease-targeted CAR is reduced or inhibited by the binding of the inhibitory CAR to its ligand, for example, to reduce off-target effects.
  • the intracellular signal transduction portion of the chimeric antigen receptor comprises a CD3 ⁇ intracellular domain and a costimulatory signal transduction region.
  • the intracellular signal transduction domain comprises a CD28 transmembrane and signal transduction domain, which is connected to the CD3 (eg, CD3- ⁇ ) intracellular domain.
  • the intracellular signal transduction domain comprises a chimeric CD28 and/or CD137 (4-1BB, TNFRSF9) costimulatory domain, which is linked to the CD3 ⁇ intracellular domain.
  • the CD28 costimulatory domain has the amino acid sequence shown in SEQ ID NO: 24, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the above sequence. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the CD137 costimulatory domain has the amino acid sequence shown in SEQ ID NO: 25, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the above sequence. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the CD3 ⁇ intracellular domain has the amino acid sequence shown in SEQ ID NO: 26, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the above sequence. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the CAR encompasses one or more, for example, two or more, a costimulatory domain and an activation domain, for example, the initial activation domain in the cytoplasmic portion.
  • exemplary CARs include anti-CD19 scfv, CD28 transmembrane region, CD137 and CD3 ⁇ intracellular domains.
  • CARs are referred to as first, second and/or third generation CARs.
  • the first-generation CAR is a CAR that only provides CD3 chain induction signals when antigen is bound; in some aspects, the second-generation CAR is a CAR that provides such signals and co-stimulatory signals, for example, including those derived from co-stimulatory receptors ( For example, the intracellular signal transduction domain of CD28 or CD137); in some aspects, the third-generation CAR is a CAR that includes multiple costimulatory domains of different costimulatory receptors.
  • the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signal transduction domain. In some embodiments, the antibody or fragment includes scFv, and the intracellular domain includes ITAM. In some aspects, the intracellular signal transduction domain includes the signal transduction domain of the zeta chain of the CD3- ⁇ chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain that connects the extracellular domain and the intracellular signal transduction domain. In some aspects, the transmembrane domain comprises the transmembrane portion of CD28.
  • the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule.
  • the extracellular domain and the transmembrane domain can be directly or indirectly connected.
  • the extracellular domain and transmembrane pass through a linking sequence.
  • the receptor comprises the extracellular portion of the molecule from which the transmembrane domain is derived, such as the CD28 extracellular portion.
  • the chimeric antigen receptor comprises an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, for example, between a transmembrane domain and an intracellular signal transduction domain.
  • the T cell costimulatory molecule is CD28 or 41BB.
  • the CAR contains an antibody, such as an antibody fragment, a transmembrane domain containing a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signal containing a signal transduction portion or functional variant of CD28.
  • the CAR contains an antibody, such as an antibody fragment, a transmembrane domain containing a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signal transduction structure containing a signal transduction portion of CD137 or a functional variant thereof Domain, and the signal transduction portion of CD3 ⁇ or its functional variants.
  • the receptor further includes a linking sequence that includes a portion of an Ig molecule (e.g., a human Ig molecule), such as an Ig hinge, such as an IgG4 hinge, such as a hinge-only linking sequence.
  • an Ig molecule e.g., a human Ig molecule
  • an Ig hinge such as an IgG4 hinge, such as a hinge-only linking sequence.
  • the chimeric antigen receptor has: (i) antibodies that specifically recognize tumor antigens, CD28 or CD8 transmembrane regions, CD28 costimulatory signal domains, and CD3 ⁇ ; or (ii) specific Antibodies that specifically recognize tumor antigens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD137, and CD3 ⁇ ; or (iii) antibodies that specifically recognize tumor antigens, the transmembrane region of CD28 or CD8, and the costimulatory signal of CD28 Domain, the costimulatory signal domain of CD137 and CD3 ⁇ .
  • the CAR includes antibodies, such as antibody fragments, including scFv, linking sequences, such as linking sequences containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more heavy chain constant regions,
  • linking sequences such as linking sequences containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more heavy chain constant regions,
  • the CAR includes antibodies or fragments, such as scFv, linking sequences, such as any linking sequence containing Ig-hinge, CD28-derived transmembrane domain, CD137-derived intracellular signal transduction domain and CD3 ⁇ -derived signal Transduction domain.
  • linking sequences such as any linking sequence containing Ig-hinge, CD28-derived transmembrane domain, CD137-derived intracellular signal transduction domain and CD3 ⁇ -derived signal Transduction domain.
  • the methods described herein include administering an adoptive cell or immune effector cell to the subject. In some embodiments, the methods described herein include administering two or more types of adoptive cells or immune effector cells to the same tumor antigen to the subject in different courses of treatment. In some embodiments, the methods described herein include administering two or more types of adoptive cells or immune effector cells to different tumor antigens to the subject in different courses of treatment. In some embodiments, the methods described herein include administering two or more types of adoptive cells or immune effector cells to the same epitope of the same tumor antigen to the subject in different courses of treatment.
  • the methods described herein include administering two or more adoptive cells or immune effector cells to different epitopes of the same tumor antigen to the subject in different courses of treatment. In some embodiments, the methods described herein include administering two or more types of adoptive cells or immune effector cells to the subject in different courses of treatment to treat tumors at the same site. In some embodiments, the methods described herein include administering two or more kinds of adoptive cells or immune effector cells to the subject in different courses of treatment to treat tumors at different sites. In some embodiments, at least one of the adoptive cells or immune effector cells used in the methods described herein targets CD19-targeted CAR-T cells.
  • At least one of the adoptive cells or immune effector cells used in the methods described herein is the CD19-targeting CAR-T cells described herein.
  • a person skilled in the art such as a clinician, can determine the number of administrations and doses of the CD19-CAR-T cells of the present invention according to the previous treatment.
  • the genetically engineered antigen receptor includes a recombinant T cell receptor (TCR) and/or TCR cloned from a naturally occurring T cell.
  • TCR recombinant T cell receptor
  • high-affinity T cell clones directed against the target antigen eg, cancer antigen
  • TCR clones for the target antigen have been generated in transgenic mice engineered with human immune system genes (eg, human leukocyte antigen system or HLA).
  • the cells provided by the methods described herein are immune effector cells expressing chimeric antigen receptors.
  • the cells are generally mammalian cells, and usually human cells.
  • the cells are derived from blood, bone marrow, lymph, or lymphoid organs, and are cells of the immune system, such as cells of innate or adaptive immunity, for example, bone marrow or lymphoid cells, including lymphocytes, generally T cells and / Or NK cells.
  • Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSC).
  • the cells are generally primary cells, such as those cells isolated directly from the subject and/or isolated and frozen from the subject.
  • the cells include one or more subgroups of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells and subgroups thereof, such as by function, activation state, maturation, differentiation potential, Those defined by expansion, recycling, localization, and/or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • the cells may be allogeneic and/or autologous. Methods include ready-made methods.
  • the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSC).
  • the method includes isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same patient before or after cryopreservation.
  • T cells and/or CD4+ and/or CD8+ T cells include: naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM) , Central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxicity T cells, mucosal-associated non-variant T (MAIT) cells, naturally occurring and adoptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 Cells, follicular helper T cells, ⁇ / ⁇ T cells, and ⁇ / ⁇ T cells.
  • TN naive T
  • TEFF effector T cells
  • TCM stem cell memory T
  • TCM Central memory T
  • TEM effector memory T
  • TIL
  • the cell is a natural killer (NK) cell.
  • the cells are monocytes or granulocytes, for example, bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils Granulocytes.
  • the cell includes one or more nucleic acids introduced by genetic engineering, and thereby expresses a recombinant or genetically engineered product of the nucleic acid.
  • the nucleic acid is heterologous, that is, it is not normally present in a cell or a sample obtained from the cell, such as a sample obtained from another organism or cell, such as a cell that is not normally under engineering And/or found in organisms from which such cells are derived.
  • the nucleic acid is not naturally occurring, for example, the nucleic acid is not found in nature, including chimeric combinations comprising nucleic acids encoding various domains from multiple different cell types.
  • the present invention also provides methods, compositions and kits for producing genetically engineered cells expressing chimeric antigen receptors.
  • Genetic engineering generally involves introducing the nucleic acid encoding the recombinant or engineered part into a cell, such as introducing into the cell through viral transduction, electrotransduction, or the like.
  • gene transfer is performed by first, stimulating the cell, for example, by combining it with a stimulus that induces a response, such as proliferation, survival, and/or activation, for example, by cytokine or The expression of the activation marker is detected, and then the activated cells are transduced, and expanded in culture to a sufficient number for clinical application.
  • a stimulus that induces a response such as proliferation, survival, and/or activation, for example, by cytokine or
  • the expression of the activation marker is detected, and then the activated cells are transduced, and expanded in culture to a sufficient number for clinical application.
  • the cells are also engineered to promote the expression of cytokines or other factors.
  • cytokines e.g., IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, etc.
  • CARs antigen receptors
  • Exemplary methods include those used to transfer nucleic acid encoding a receptor, including via virus, for example, retrovirus or lentivirus, transduction, transposon, and electroporation.
  • recombinant infectious virus particles are used to transfer recombinant nucleic acid into cells, for example, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV).
  • SV40 simian virus 40
  • AAV adeno-associated virus
  • recombinant lentiviral vectors or retroviral vectors such as gamma-retroviral vectors, are used to transfer recombinant nucleic acids into T cells.
  • the preparation of engineered cells includes one or more culturing and/or one or more preparation steps.
  • Cells used to introduce nucleic acid encoding a transgenic receptor can be isolated from a sample (e.g., a biological sample, such as a sample obtained or derived from a subject).
  • a sample e.g., a biological sample, such as a sample obtained or derived from a subject.
  • the subject from which the cells are isolated is a subject suffering from a certain disease or condition or in need of cell therapy or who will be given cell therapy.
  • the subject is a person in need of specific therapeutic intervention, for example, a person in need of adoptive cell therapy or effector cell therapy, and the cells used for the therapy are isolated, processed, and/or engineered.
  • the cell is a primary cell, for example, a primary human cell.
  • the sample includes tissues, body fluids and other samples taken directly from the subject, as well as obtained from one or more processing steps, such as separation, centrifugation, genetic engineering (such as transduction with viral vectors), washing and/or Incubate the sample.
  • the biological sample may be a sample obtained directly from a biological source or a processed sample.
  • Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived from them.
  • Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosal-associated lymphoid tissue, spleen , Other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs, and/or cells derived therefrom.
  • samples include samples from autologous and allogeneic sources.
  • the separation of cells includes one or more preparation and/or non-affinity-based cell separation steps.
  • the cells are washed, centrifuged, and/or incubated in the presence of one or more substances, for example, to remove unwanted components, enrich required components, lyse or remove specific substances.
  • Sensitive cells cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and/or resistance to specific components.
  • cells from the circulating blood of the subject are obtained, for example, by apheresis or leukopenia.
  • the sample includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated blood leukocytes, red blood cells, and/or platelets.
  • the blood cells collected from the subject are washed, for example, to remove the plasma fraction, and the cells are placed in a suitable buffer or medium for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the cleaning solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • the cleaning step is performed by a semi-automated "flow-through" centrifugation method (for example, COBE 2991 cell processor, BaXter) according to the manufacturer's instructions.
  • the cleaning step is accomplished by intangential flow filtration (TFF) in accordance with the manufacturer's instructions.
  • the cells after washing, the cells are resuspended in a variety of biocompatible buffers, for example, Ca++/Mg++-free PBS.
  • biocompatible buffers for example, Ca++/Mg++-free PBS.
  • blood cell sample components are removed, and the cells are directly resuspended in the culture medium.
  • the method includes a density-based cell separation method, for example, by lysing red blood cells or not lysing red blood cells and centrifuging the peripheral blood by Percoll or Ficoll gradient or single sampling or leukocyte ablation sample preparation to obtain a single peripheral blood Nuclear cells (PBMC).
  • PBMC peripheral blood Nuclear cells
  • the separation method includes separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, for example, surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method of separation based on such markers can be used. In some embodiments, the separation is based on affinity or based on immunoaffinity. For example, in some aspects, the separation includes separating cells and cell populations based on the expression or expression levels of one or more markers (usually cell surface markers) of the cells, for example, by specifically binding to them The antibody or binding partner of the class marker is incubated, usually followed by a washing step, and cells that have bound the antibody or binding partner are separated from those cells that have not yet bound to the antibody or binding partner.
  • surface markers for example, surface proteins, intracellular markers, or nucleic acids.
  • any known method of separation based on such markers can be used.
  • the separation is based on affinity or based on immunoaffinity.
  • the separation includes separating cells and cell populations based on the expression or expression levels
  • Such separation steps may be based on positive selection, where cells that have bound the agent are retained for further use, and/or, negative selection, where cells that have not yet bound to the antibody or binding partner are retained.
  • a specific subset of T cells such as one or more surface marker-positive cells or cells expressing high levels of one or more surface markers, for example, CD3+, CD28+, CD62L+, CCR7+ , CD27+, CD127+, CD4+, CD8+, CD45RA+ and/or CD45RO+ T cells, separated by positive or negative selection techniques.
  • CD3+ and CD28+ T cells can be positively selected using CD3/CD28-connected magnetic beads (for example, DYNA bead M-450CD3/CD28 T cell expander).
  • T cells are isolated from a PBMC sample by negative selection of markers (eg, CD14) expressed on non-T cells (eg, B cells, monocytes, or other blood leukocytes).
  • markers eg, CD14
  • non-T cells eg, B cells, monocytes, or other blood leukocytes.
  • a CD4+ or CD8+ selection step is used to isolate helper CD4+ and CD8+ cytotoxic T cells.
  • the monoclonal antibody mixture in order to enrich CD4+ cells by negative selection, usually includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • the antibody or binding partner is bound to a solid support or matrix, such as magnetic beads or paramagnetic beads, to allow separation of cells for positive and/or negative selection.
  • the preparation method includes a freezing step, for example, freezing the cells before or after isolation, incubation, and/or engineering.
  • the freezing and subsequent thawing steps remove granulocytes and, to some extent, remove monocytes in the cell population.
  • the cells are suspended in a freezing solution.
  • any of a variety of known freezing solutions and parameters can be used.
  • PBS or other suitable cell freezing medium containing 20% DMSO and 8% human serum albumin (HAS). Then, it was diluted 1:1 with medium, so that the final concentrations of DMSO and HSA were 10% and 4%, respectively.
  • the cells are generally frozen to -80°C or -90°C according to a predetermined procedure or principle, such as a rate of 1°/min, with a programmable cooling device, and stored in the vapor phase of a liquid nitrogen storage tank.
  • a predetermined procedure or principle such as a rate of 1°/min
  • compositions comprising cells for administration, including pharmaceutical compositions and preparations, such as a composition from a unit dosage form containing the number of cells for administration in a given dose or part thereof.
  • the pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients.
  • the composition includes at least one other therapeutic agent.
  • pharmaceutical preparation refers to a form of preparation that allows the biological activity of the active ingredients contained therein to be effective and does not contain additional ingredients that have unacceptable toxicity to the subject to which the preparation is to be administered.
  • “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical preparation that is not an active ingredient and is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the choice of vehicle is determined in part by the particular cell and/or method of administration. Therefore, there are a variety of suitable formulations.
  • the pharmaceutical composition may include a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is usually present in an amount of about 0.0001% to about 2% (based on the total weight of the composition).
  • the carrier is described in, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).
  • pharmaceutically acceptable carriers are generally non-toxic to the recipient, including but not limited to: buffers such as phosphate, citrate and other organic acid buffers; antioxidants, including ascorbic acid and Methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexaalkyl quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; p-hydroxy Alkyl benzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) Polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid, such as glycine,
  • the composition includes a buffer.
  • Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts.
  • a mixture of two or more buffering agents is used.
  • the buffering agent or mixture thereof is usually present in an amount of about 0.001% to about 4% (based on the total weight of the composition). Methods for preparing administrable pharmaceutical compositions are known.
  • the formulation may comprise an aqueous solution.
  • the preparation or composition may also contain more than one active ingredient, which can be used for the specific indication, disease or condition to be treated with the cell, preferably those with complementary activity to the cell, wherein the corresponding activity
  • the agents do not negatively affect each other.
  • Such active ingredients are suitably present in combination in an amount effective for the desired purpose.
  • the pharmaceutical composition further comprises other pharmaceutically active substances or drugs, such as chemotherapeutics, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin Bicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • chemotherapeutics for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin Bicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • the pharmaceutical composition contains an amount effective to treat or prevent a disease or condition, such as a therapeutically effective or preventively effective amount of cells.
  • a disease or condition such as a therapeutically effective or preventively effective amount of cells.
  • the efficacy of treatment or prophylaxis is monitored by regularly evaluating the treated subjects.
  • the required dose can be delivered by administering the cells by a single pill, by administering the cells by multiple pills, or by administering the cells by continuous infusion.
  • the composition comprises an amount effective to reduce the burden of the disease or condition, and/or an amount that does not cause CRS or severe CRS in the subject and/or an amount that achieves any other results of the methods described herein Cell.
  • the cells and compositions can be administered using standard administration techniques, formulations and/or devices.
  • the administration of the cells can be autologous or heterologous.
  • immunosuppressive cells or progenitor cells can be obtained from one subject and administered to the same subject or different compatible subjects.
  • Peripheral blood-derived immunosuppressive cells or their progeny can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration.
  • a therapeutic composition e.g., a pharmaceutical composition containing genetically modified immunosuppressive cells
  • it is usually formulated in a unit-dose injectable form (solution, suspension, emulsion).
  • Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, mucosal, sublingual or suppository administration.
  • the cell population is administered parenterally.
  • parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration.
  • the cells are administered to the subject by intravenous, intraperitoneal or subcutaneous injection using peripheral systemic delivery.
  • the composition is provided in the form of a sterile liquid formulation, for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects can be buffered to a selected pH.
  • a sterile liquid formulation for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects can be buffered to a selected pH.
  • Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions.
  • liquid compositions are somewhat easier to administer, especially by injection.
  • the viscous composition can be formulated within a suitable viscosity range to provide a longer contact time with specific tissues.
  • the liquid or viscous composition may include a carrier, which may be a solvent or dispersion medium, which contains, for example, water, saline, phosphate buffered saline, polyhydroxy compounds (for example, glycerol, propylene glycol, liquid polyethylene glycol), and Suitable mixture.
  • a carrier which may be a solvent or dispersion medium, which contains, for example, water, saline, phosphate buffered saline, polyhydroxy compounds (for example, glycerol, propylene glycol, liquid polyethylene glycol), and Suitable mixture.
  • Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, and the like.
  • the composition may contain auxiliary substances such as wetting, dispersing, or emulsifying agents (for example, methyl cellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or pigments, depending on The desired route of administration and preparation.
  • auxiliary substances such as wetting, dispersing, or emulsifying agents (for example, methyl cellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or pigments, depending on The desired route of administration and preparation.
  • the present invention also provides products, such as kits and devices, for administering cells to subjects according to the provided methods for adoptive cell therapy or immune effector cell therapy, and for storing and administering the cells and compositions.
  • the article of manufacture includes one or more containers, generally multiple containers, packaging materials, and a label or package insert combined with or on one or more containers and/or packaging, and generally includes instructions for administering cells to a subject.
  • the container generally contains the cells to be administered, for example, one or more unit doses thereof.
  • Articles of manufacture generally include multiple containers, each containing a single unit dose of cells.
  • the unit dose may be the amount or number of cells to be administered to the subject of the immune effector cells of the previous course of treatment or twice (or more) the number of cells to be administered with the immune effector cells of the first or subsequent course of treatment. It may be the lowest dose or lowest possible dose given to the subject's cells in relation to the method of administration.
  • the unit dose is the number of cells or the minimum number of cells to be administered to any subject or any subject with a specific disease or condition in a unit dose according to the method of the present invention.
  • the unit dose may include the minimum amount of cells that will be administered to a subject with a lower body weight and/or a lower disease burden, such as administering one or more immune effector cells to a given subject with a previous course of treatment. In some cases more than one unit dose and one or more unit doses are administered to a given subject in one or more subsequent course of immune effector cells, for example, in accordance with the methods provided.
  • the number of cells in a unit dose is the number of immune effector cells that need to be administered to a specific subject with a previous course of treatment, such as the number of chimeric antigen receptor-expression or CAR-expression in a cell-derived subject. Number of cells.
  • the cells are derived from the subject to be treated by the methods provided herein.
  • each container individually contains a unit dose of cells, for example, including the same or substantially the same number of cells. Therefore, in some embodiments, each container contains the same or approximately or substantially the same number of cells or chimeric antigen receptor-expressing cells.
  • the unit dose includes less than about 1 ⁇ 10 10 , less than about 1 ⁇ 10 9 , less than about 1 ⁇ 10 8, or less than about 1 ⁇ 10 7 engineered cells, total cells, T cells, or PBMC/kg to be treated and/or cell-derived Of subjects.
  • Suitable containers include, for example, bottles, vials, syringes, and flexible bags such as freezer bags.
  • the container is a bag, for example, a flexible bag, such as those suitable for infusion of cells into a subject, for example, a flexible plastic or PVC bag or EVA or ULPDE, and/or an IV solution bag.
  • the bag is sealable and/or sterilizable to provide sterile solutions and delivery of cells and compositions.
  • the container for example, the volume of the bag is equal to or about or at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 ml Volume, such as equal to or about 10 to equal to or about 100 mL or equal to or about 10 to equal to or about 500 mL volume.
  • the container for example, the bag is and/or made of a material that is stable at one or more different temperatures and/or provides stable storage and/or maintenance of cells, such as at low temperatures, for example, low temperature At or about or equal to or about -20°C, -80°C, -120°C, 135°C, -196°C and/or a temperature suitable for cryopreservation, and/or other temperatures, such as a temperature suitable for freezing and thawing cells and Body temperature, such as equal to or about 37°C or -38°C, or -39°C, or -40°C, to allow freezing and thawing prior to treatment, for example, at the subject's site or treatment site.
  • low temperatures for example, low temperature At or about or equal to or about -20°C, -80°C, -120°C, 135°C, -196°C and/or a temperature suitable for cryopreservation, and/or other temperatures, such as a temperature suitable for freezing and thawing cells
  • the article may also include a package insert or label, one or more pieces of which display usage information and/or instructions.
  • the information or instructions show what can or should be used to treat a particular disease or condition, and/or provide instructions for it.
  • the label or package insert may show the contents of the article to be used to treat the disease or condition.
  • the label or package insert provides instructions for treating a subject, for example, a subject for which the cells have been derived, by including cells that administer the first and one or more immune effector cells for subsequent courses of treatment, for example, The method according to any of the provided method embodiments.
  • the instructions specify that a unit dose is given to the immune effector cells of the previous course of treatment, for example, the contents of a single container of the product, and thereafter it is detected at a specified time point or within a specified time window and/or The presence or absence or amount or degree of one or more factors or results in the subject is then administered to one or more immune effector cells for a subsequent course of treatment.
  • the article of manufacture includes one or more, usually multiple containers containing a cell-containing composition, such as a separate unit dosage form thereof, and also includes one or more other containers containing the composition, which The composition includes other agents, such as cytotoxic or other therapeutic agents, which, for example, will be combined with the cells, for example, administered simultaneously or at once in any order.
  • the article of manufacture may also include another or the same container containing a pharmaceutically acceptable buffer. It may also include other materials, such as other buffers, diluents, filters, tubes, needles, and/or syringes.
  • package insert refers to the instructions often included in the commercial packaging of therapeutic products, the instructions containing information about the instructions, usage, dosage, administration, combination therapy, contraindications, and/or warnings about the use of such therapeutic products.
  • PBMC Peripheral blood mononuclear cells
  • T cells from human subjects suffering from tumors are cultured and transduced with viral vectors encoding chimeric antigen receptors (CAR) based on "Demonocytic Isolation" Cells, the chimeric antigen receptor (CAR) specifically binds to an antigen expressed by a tumor in a subject, which is a tumor-associated or tumor-specific antigen, such as CD19.
  • the cells are cryopreserved in an infusion medium in a separate flexible freezing bag, each containing a single unit dose of cells, which is about 1 ⁇ 10 5 cells to 1 ⁇ 10 9 cells. Prior to infusion, the cells are maintained at a temperature of about below -130°C or about below -175°C.
  • tumor necrosis factor alpha TNF ⁇
  • IFN ⁇ interferon gamma
  • IL-6 IL-6
  • tumor burden can optionally be assessed by measuring the size or quality of the solid tumor, for example by PET or CT scan.
  • the resuscitation is carried out by raising the temperature to about 38°C, and the subject is given the cells of the immune effector cells of the present invention by multiple infusions.
  • the infusion is a continuous intravenous (IV) infusion within about 1-20 ml/min.
  • the subject After administering a certain dose of immune effector cells, the subject undergoes a physical examination and monitors any symptoms of toxicity or toxicity, such as fever, hypotension, hypoxia, neurological disorders, or inflammatory cytokines or C-reactive protein (CRP) Elevated serum levels.
  • toxicity or toxicity such as fever, hypotension, hypoxia, neurological disorders, or inflammatory cytokines or C-reactive protein (CRP) Elevated serum levels.
  • CRP C-reactive protein
  • the presence or absence of an anti-CAR immune response in the subject is optionally detected, for example, by qPCR, ELISA, ELISPOT, cell-based antibody assay and/or mixed lymphocyte reaction.
  • the percentage of tumor burden reduction achieved by the infusion of immune effector cells of the present invention can optionally be measured one or more times after the previous course of immune effector cells are administered in solid tumor patients by scanning (e.g., PET and CT scan), And/or by quantifying disease-positive cells in the blood or tumor site.
  • scanning e.g., PET and CT scan
  • the subjects are monitored regularly.
  • the tumor burden is measured, and/or CAR-expressing cells are detected by flow cytometry and quantitative polymerase chain reaction (qPCR) to measure the in vivo proliferation and persistence of the administered cells, and/or evaluate anti- The development of the CAR immune response.
  • qPCR quantitative polymerase chain reaction
  • the purpose of the present invention is to provide a method and use of engineered cells (for example, T cells) and compositions thereof for the treatment of subjects suffering from or suspected of having CD19-positive hematological tumors, usually including leukemia or lymphoma , Especially acute and chronic lymphocytic leukemia and/or non-Hodgkin’s lymphoma (NHL).
  • the methods and uses provide or achieve an improved or longer-lasting response or efficacy and/or a reduced risk of toxicity or other side effects compared to the prior art.
  • the present invention adopts anti-CD19 CAR-T cells in different doses (the dose range is 1.5 ⁇ 10 5 to 4.9 ⁇ 10 6 CAR-T cells/kg, or the total dose is 1 ⁇ 10 7 to 3 ⁇ 10 8 CAR-T cells)
  • Treatment of CD19-positive hematological tumors the ORR of the subjects reached 67% (8/12 subjects), and the CR reached 50% (6/12 subjects); PFS of 6 subjects out of 12 subjects Over 6 months, the PFS of 3 subjects with CRS curative effect was more than 1 year up to nearly 3 years and the follow-up was up to now, and the PFS of 1 subject with PR curative effect was more than 500 days and the follow-up was so far; and it is safe
  • the 12 subjects did not have grade 3 or higher CRS and neurotoxicity.
  • the most common adverse events related to the anti-CD19CAR-T cells of the present invention were fever, followed by a decrease in platelets and a decrease in white blood cells.
  • Supportive treatments are all in remission or in remission.
  • subjects with the highest infusion dose of 4.9 ⁇ 10 6 CAR-T cells/kg or the total infusion dose of 3 ⁇ 10 8 CAR-T cells achieved CR after cell infusion, and have been followed up to about 19
  • the curative effect evaluations in several months are all CR, and the follow-up is still underway; the subjects only experienced adverse reactions such as decreased lymphocyte count, decreased white blood cell count, and increased lactate dehydrogenase after CAR-T cell infusion, and alleviated spontaneously.
  • the anti-CD19 CAR of the present invention can be well implanted in CD19-positive blood tumor subjects and has strong amplification ability in the body and lasts for several months.
  • the infusion dose is as high as 4.9 ⁇ 10 6 CAR-T cells/kg or the total infusion dose is as high as 3 ⁇ 10 8 CAR-T cells. It is also safe and has significant curative effect.
  • the present invention adopts anti-CD19 CAR-T cells in different doses (the dose range is 1.5 ⁇ 10 5 to 4.9 ⁇ 10 6 CAR-T cells/kg, or the total dose is 1 ⁇ 10 7 to 3 ⁇ 10 8 CAR-T cells)
  • the ORR of the subjects reached 56% (5/9 subjects), and the CR reached 33% (3/9 subjects); PFS of 5 subjects out of 9 subjects exceeded At 6 months, the PFS of 3 subjects with CRS curative effect was more than 1 year up to nearly 3 years and the follow-up was up to now, and the PFS of 1 subject with PR curative effect was more than 500 days and the follow-up was so far; and safety High, none of the 9 NHL subjects developed grade 3 or higher CRS and neurotoxicity.
  • the most common adverse event related to the anti-CD19 CAR-T cells of the present invention was fever, followed by a decline in platelets and a decline in white blood cells.
  • Symptomatic and supportive treatment have been alleviated or in remission.
  • subjects with the highest infusion dose of 4.9 ⁇ 10 6 CAR-T cells/kg or the total infusion dose of 3 ⁇ 10 8 CAR-T cells achieved CR after cell infusion, and have been followed up to about 19
  • the curative effect evaluations in several months are all CR, and the follow-up is still underway; the subject only experienced adverse reactions such as decreased lymphocyte count, decreased white blood cell count, and increased lactate dehydrogenase after CAR-T cell infusion, and alleviated spontaneously.
  • the anti-CD19 CAR of the present invention can be well implanted in CD19-positive NHL subjects and has strong expansion ability in vivo and can last for several months.
  • the infusion dose is as high as 4.9 ⁇ 10 6 CAR-T cells/kg or the total infusion dose is as high as 3 ⁇ 10 8 CAR-T, which is also safe and has significant curative effect.
  • the present invention adopts anti-CD19 CAR-T cells in different doses (the dose range is 1.8 ⁇ 10 6 to 2.6 ⁇ 10 6 CAR-T cells/kg, or the total dose is 1 ⁇ 10 8 to 1.5 ⁇ 10 8 CAR-T cells)
  • the best curative effect evaluation of the 3 ALL subjects was CR, and the PFS was 120-432 days; none of them had Grade 3 or higher CRS and neurotoxicity, and the most common was the anti-CD19 CAR of the present invention.
  • -T cell-related adverse events are fever, followed by decreased platelets and decreased white blood cells. After symptomatic and supportive treatment, they have alleviated or are in remission.
  • reagents can be purchased from commercial channels.
  • Exemplary antigen receptors including CARs, and methods for engineering such receptors and introducing such receptors into cells, for example, Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, CN 108884459 A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468803279A, CN109503275A, CN110, 082,105, CN110837275A, CN110837275A, CN110 Patent Application Publication Numbers WO2017186121A1, WO2018006882A1, WO2015172339A
  • the amino acid sequence of the scFv portion of the chimeric antigen receptor is shown in SEQ ID NO: 10, and the scFv has the heavy chain variable region shown in SEQ ID NO: 11 and SEQ ID NO: 11.
  • the light chain variable region shown in ID NO: 12 and the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 19.
  • the CAR containing the aforementioned scFv may also have other intracellular domains. Therefore, the sequence of the CAR may also be the sequence shown in SEQ ID NO: 17, 18 or 20.
  • the scFv shown in SEQ ID NO: 10 has HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 7, and LCDR1 shown in SEQ ID NO: 2. , LCDR2 shown in SEQ ID NO: 3, LCDR3 shown in SEQ ID NO: 4.
  • Example 1 Treatment of subjects with relapsed or refractory CD19-positive hematological tumors with CD19 CAR
  • CD19-positive hematological tumors Twelve subjects who were given relapsed or refractory CD19-positive hematological tumors were infused with autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR).
  • the age of the subjects ranged from 22 to 62 years, with an average age of 43 years.
  • the subjects exhibited multiple disease types, including 6 cases of diffuse large B-cell lymphoma (DLBCL), 1 case of mantle cell lymphoma (MCL), 1 case of follicular lymphoma, and 1 case of B lymphoblastic lymphoma /Acute lymphocytic leukemia (ALL) and 3 cases of acute lymphocytic leukemia (ALL).
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • ALL B lymphoblastic lymphoma /Acute lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • anti-CD19 CAR-T cells prepared by PBMC from the donor (prepared according to the method of Chinese patent application CN201711330443.7).
  • the subject receives lymphocytic depletion therapy, infusion of about 17-27 mg/m 2 /d fludarabine 3 or 4 times, and 21-59mg/kg cyclophosphamide (administered in 1 or 2 or 3 doses).
  • CAR-T cell infusion 1.5 ⁇ 10 5 , 5.8 ⁇ 10 5 , 1.1 ⁇ 10 6 , 1.7 ⁇ 10 6 , 1.8 ⁇ 10 6 , 1.9 ⁇ 10 6 , 2.0 ⁇ 10 6 , 2.1 ⁇ 10 6 , 2.6 ⁇ 10 6 or 4.9 ⁇ 10 6 CAR-T cells/kg subject’s body weight were infused into the subject, and the infusion dose of CAR-T cells was 1.8 ⁇ 10 6 , 1.9 ⁇ 10 6 CAR-T cells/kg There are 2 subjects in each kg, and the remaining dose is 1 subject; or the total dose is 1 ⁇ 10 7 , or 3 ⁇ 10 7 , or 6 ⁇ 10 7 , or 8 ⁇ 10 7 , or 9 ⁇ 10 7 , or 1 ⁇ 10 8 , or 1.5 ⁇ 10 8 or 3 ⁇ 10 8 CAR-T cells are infused into the subject, and the total dose of CAR-T cell infusion is 1 ⁇ 10 8 CAR-T cells is 2 There were 4 subjects, 1.5 ⁇ 10 8 CAR-T cells were 4 subjects, and the remaining doses were
  • the patient Before and 4 weeks after the administration of anti-CD19 CAR-T cells, the patient underwent PET-CT whole body imaging and diagnostic quality CT scan or bone marrow cell morphology and biopsy histopathology to assess the response to treatment.
  • Morphological analysis of bone marrow subjects with at least 5% of primitive and naive cells in the bone marrow are considered to have morphological diseases (MD).
  • the lymph node tumor volume was evaluated as the sum of the cross-sectional areas of the 6 largest index lymph nodes identified in the diagnostic quality CT scan. Lymphoma burden was determined quantitatively based on the SPD value of target lesions according to Lugano criteria (Cheson et al., JCO September 20, 2014 vol. 32 no. 273059-3067). According to the results of imaging examination and bone marrow aspiration/biopsy, the efficacy was evaluated according to the Lugano2014 standard. According to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.03, the severity of adverse events was graded. Cytokine release syndrome (CRS) is graded as described in 2014 Lee DW and 2019 ASTCT.
  • ORR overall response rate
  • the ORR was 67% (8/12 subjects) and the CR was 50% (6/12 subjects).
  • the PFS of the CR group tended to be higher than that of the non-CR group.
  • the continuous survival period of anti-CD19 CAR-T cells in the body is detected, that is, the period during which CAR-T cells are "implanted” in the body. From the end of the initial infusion (day 0), Q-PCR is used at each visit point.
  • the probe used is Probe (see SEQ ID NO: 28CGTGGAGGCCAACGACACCG for the nucleotide sequence); the upstream primer sequence is: Primer (nuclear) The nucleotide sequence is shown in SEQ ID NO: 29TTCACCCTGACCATCAACCC; the downstream primer sequence is: Primer (nucleotide sequence is shown in SEQ ID NO: 30CTGCTGGCAGTAGTAGTTGG), and the number of copies of anti-CD19 CAR DNA in peripheral blood is detected until any 2 consecutive detections If it is negative, it is recorded as the continuous survival period of anti-CD19 CAR-T cells.
  • the test results showed that anti-CD19 CAR-T cells proliferated in all subjects, and the CAR copy number was detected about 1-7 days after cell infusion, and reached a peak about 9-22 days, about 5 to 5 days. Undetectable for 6 months.
  • neurotoxicity neurotoxicity complications, including confusion symptoms, aphasia, seizures, convulsions, lethargy and/or altered mental state
  • graded according to severity use 1-5
  • Guido Cavaletti and Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010), of which grade 3 (severe symptoms), 4 (life-threatening symptoms) or 5 (death) are considered serious Neurotoxicity.
  • Grade 1 (mild)-not life-threatening, only systemic treatments such as antipyretics and antiemetics are required (for example, fever, nausea, fatigue, headache, myalgia, malaise);
  • Oxygen demand ⁇ 40%, or high-dose single vasopressor drugs for example, norepinephrine ⁇ 20ug/kg/min, dopamine ⁇ 10ug/kg/min, phenylephrine ⁇ 200ug/kg/min, or adrenal gland Low blood pressure ⁇ 10ug/kg/min), or a variety of vasopressor drugs (for example, antidiuretic + one of the above reagents, or vasopressor equal to norepinephrine ⁇ 20ug/kg/min) (Drug combination) hypotension, or grade 3 organ toxicity, or grade 4 transaminase elevation (through CTCAE v4.0);
  • Grade 4 life-threatening-requires ventilator support, or Grade 4 organ toxicity (excludes elevated transaminase);
  • Level 2 (moderate)-there are symptoms that restrict active daily activities (ADL), such as cooking, shopping for vegetables or clothes, using the phone, and managing money;
  • ADL active daily activities
  • Level 3 (Severe)-There is restrictive self-management ADL, such as symptoms of bathing, dressing or undressing, eating, using the toilet, and taking medication;
  • the present invention adopts anti-CD19 CAR-T cells in different doses (the dose range is 1.5 ⁇ 10 5 to 4.9 ⁇ 10 6 CAR-T cells/kg, or the total dose is 1 ⁇ 10 7 to 3 ⁇ 10 8 CAR-T cells)
  • Treatment of CD19-positive hematological tumors the ORR of the subjects reached 67% (8/12 subjects), and the CR reached 50% (6/12 subjects); PFS of 6 subjects out of 12 subjects Over 6 months, the PFS of 3 subjects with CRS curative effect was more than 1 year up to nearly 3 years and the follow-up was up to now, and the PFS of 1 subject with PR curative effect was more than 500 days and the follow-up was so far; and it is safe
  • the 12 subjects did not have grade 3 or higher CRS and neurotoxicity.
  • the most common adverse events related to the anti-CD19CAR-T cells of the present invention were fever, followed by a decrease in platelets and a decrease in white blood cells. , Supportive treatments are all in remission or in remission.
  • the subjects with the highest infusion dose of 4.9 ⁇ 10 6 CAR-T cells/kg or the total infusion dose of 3 ⁇ 10 8 CAR-T cells after cell infusion The curative effect evaluation reached CR, and several curative effect evaluations for about 19 months have been CR, and the follow-up is still ongoing; the subject only experienced a decrease in lymphocyte count, a decrease in white blood cell count, and lactate loss after CAR-T cell infusion.
  • Adverse reactions such as elevated hydrogenase alleviate spontaneously.
  • the anti-CD19 CAR of the present invention can be well implanted in CD19-positive hematological tumor subjects and has a strong amplification ability in the body and lasts for several months.
  • the infusion dose is as high as 4.9 ⁇ 10 6 CAR-T cells/kg or the total infusion dose is as high as 3 ⁇ 10 8 CAR-T cells. It is also safe and has significant curative effect.
  • the subject receives lymphocyte depletion chemotherapy, infusion of about 17-24 mg/m 2 /d fludarabine 3 or 4 times, and 21-59mg/kg cyclophosphamide (administered in 1 or 2 or 3 doses).
  • the anti-CD19 CAR-T cells described in Example 1 were used for infusion: respectively at doses of 1.5 ⁇ 10 5 , 5.8 ⁇ 10 5 , 1.1 ⁇ 10 6 , 1.7 ⁇ 10 6 , 1.8 ⁇ 10 6 , 1.9 ⁇ 10 6 , 2.0 ⁇ 10 6 , 2.1 ⁇ 10 6 or 4.9 ⁇ 10 6 CAR-T cells/kg subject weight, infusion into the subject; or in a total dose of 1 ⁇ 10 7 , or 3 ⁇ 10 7 , or 6 ⁇ 10 7 , or 8 ⁇ 10 7 , or 1 ⁇ 10 8 , or 1.5 ⁇ 10 8 or 3 ⁇ 10 8 CAR-T cells are infused into the subject, and the total dose of CAR-T cell infusion is 1.5 ⁇ 10 8 CAR-T cells were 3 subjects, and the remaining doses were 1 subject.
  • CAR-T cell infusion can be a single infusion, or two or three infusions.
  • CRS Cytokine release syndrome
  • the therapeutic efficacy of the subject is monitored by measuring ORR.
  • the ORR was 56% (5/9 subjects) and the CR was 33% (3/9 subjects).
  • the PFS of the CR group tended to be higher than that of the non-CR group.
  • the present invention adopts anti-CD19 CAR-T cells in different doses (the dose range is 1.5 ⁇ 10 5 to 4.9 ⁇ 10 6 CAR-T cells/kg, or the total dose is 1 ⁇ 10 7 to 3 ⁇ 10 8 CAR-T cells)
  • the ORR of the subjects reached 56% (5/9 subjects), and the CR reached 33% (3/9 subjects); PFS of 5 subjects out of 9 subjects exceeded At 6 months, the PFS of 3 subjects with CRS curative effect was more than 1 year up to nearly 3 years and the follow-up was up to now, and the PFS of 1 subject with PR curative effect was more than 500 days and the follow-up was so far; and safety High, none of the 9 NHL subjects developed grade 3 or higher CRS and neurotoxicity.
  • the most common adverse event related to the anti-CD19 CAR-T cells of the present invention was fever, followed by a decline in platelets and a decline in white blood cells. Symptomatic and supportive treatment have been alleviated or in remission.
  • the anti-CD19 CAR of the present invention can be well implanted into CD19-positive NHL subjects and has a strong ability to expand in vivo and last for several months.
  • the infusion dose is as high as 4.9 ⁇ 10 6 CAR-T cells/kg or the total infusion dose is as high as 3 ⁇ 10 8 CAR-T is also safe and has a significant effect.
  • CD19-positive diffuse large B-cell lymphoma Four subjects in the high-dose group of relapsed or refractory CD19-positive diffuse large B-cell lymphoma were infused with autologous T cells expressing anti-CD19 chimeric antigen receptor (CAR).
  • CAR anti-CD19 chimeric antigen receptor
  • the subject received lymphocyte depletion chemotherapy, and infused about 21-27 mg/m2/d fludarabine 3 or 4 times, and 28-31 mg/kg Cyclophosphamide (administered in 2 doses).
  • the anti-CD19 CAR-T cells described in Example 1 were used for infusion: a total dose of 3 ⁇ 10 8 CAR-T cells was infused into the subject; CAR-T cell infusion was performed once or divided into two Infusions.
  • CRS Cytokine release syndrome
  • the most common adverse event related to the anti-CD19 CAR-T cells of the present invention was fever, followed by decreased platelets and decreased white blood cells. After symptomatic and support The treatments are all in remission or in remission.
  • ALL relapsed or refractory CD19-positive acute lymphoblastic leukemia
  • CAR anti-CD19 chimeric antigen receptor
  • the subject receives lymphocyte depletion chemotherapy, infusion of about 21-27 mg/m 2 /d fludarabine 3 or 4 times, and 28-31mg/kg cyclophosphamide (administered in 2 doses).
  • the anti-CD19 CAR-T cells described in Example 1 were used for infusion: the subjects were infused at doses of 1.8 ⁇ 10 6 , 1.9 ⁇ 10 6 , and 2.6 ⁇ 10 6 CAR-T cells per kg of the subject’s body weight. Note; or the total dose of 9 ⁇ 10 7 , or 1 ⁇ 10 8 or 1.5 ⁇ 10 8 CAR-T cell infusion to the subject; CAR-T cell infusion is a single infusion.
  • the patient Before and 4 weeks after the administration of anti-CD19 CAR-T cells, the patient underwent bone marrow cell morphology and biopsy histopathology to assess the response to treatment. According to the results of bone marrow aspiration/biopsy, the curative effect was evaluated according to NCCN clinical practice guidelines. According to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.03, the severity of adverse events was graded.
  • NCI National Cancer Institute
  • CCAE Common Terminology Criteria for Adverse Events
  • the present invention adopts anti-CD19 CAR-T cells in different doses (the dose range is 1.8 ⁇ 10 6 to 2.6 ⁇ 10 6 CAR-T cells/kg, or the total dose is 1 ⁇ 10 8 to 1.5 ⁇ 10 8 CAR-T cells)
  • the best curative effect evaluation of the 3 ALL subjects was CR, and the PFS was 120-432 days; none of them had Grade 3 or higher CRS and neurotoxicity, and the most common was the anti-CD19 CAR of the present invention.
  • -T cell-related adverse events are fever, followed by decreased platelets and decreased white blood cells. After symptomatic and supportive treatment, they have alleviated or are in remission.
  • the CAR shown in SEQ ID NO: 19 is selected as an example.
  • other CARs that target CD19 can also be applied to the technical solutions described in this application.
  • the CAR scFv shown in SEQ ID NO: 13, 14, 15 and 16 has the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region shown in SEQ ID NO: 9
  • the amino acid sequence of the region, the CDR regions are: HCDR1 shown in SEQ ID NO: 5, HCDR1 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 7, and LCDR1 shown in SEQ ID NO: 2.
  • LCDR2 shown in SEQ ID NO: 3 LCDR3 shown in SEQ ID NO: 4.
  • the CAR scFv shown in SEQ ID NO: 17, 18, and 20 has the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12.
  • the amino acid sequence and the CDR regions are: HCDR1, SEQ ID NO: 5, HCDR2, SEQ ID NO: 6, HCDR3, SEQ ID NO: 7, and LCDR1, SEQ ID NO: 2 ID NO: LCDR2 shown in 3, LCDR3 shown in SEQ ID NO: 4.

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Abstract

提供一种免疫效应细胞的治疗方法，包括给予个体特异性识别CD19的免疫效应细胞。先进行预处理后再给予本发明的免疫效应细胞时，能够显著提高免疫效应细胞的肿瘤治疗疗效。

Description

免疫效应细胞治疗肿瘤
相关申请引用
本申请要求申请号CN202010419953.7，发明名称为免疫效应细胞治疗肿瘤案件的优先权。
技术领域
本发明属于免疫治疗领域；具体地，涉及靶向识别肿瘤抗原、引发免疫效应细胞活化且发挥抗肿瘤效应的免疫细胞治疗。
背景技术
肿瘤免疫疗法近年来得到广泛的关注和应用，尤其是CAR-T技术的出现，使人类对肿瘤的控制得到了里程碑式的发展。临床应用中需要改进给药方案来增强疗效。
发明内容
本发明的目的在于提供一种工程化细胞(例如，T细胞)及其组合物用于治疗患有CD19阳性的血液肿瘤的受试者的方法和用途，通常包括白血病或淋巴瘤，尤其是急性、慢性性淋巴细胞白血病和/或非霍奇金淋巴瘤(NHL)。在一些方面，与现有技术相比，该方法和用途提供或实现改善的或更持久的应答或功效和/或降低的毒性风险或其他副作用。在一些实施例中，所述方法通过给予指定数量或相对数量的工程化细胞，用特定淋巴细胞清除疗法预处理，治疗特定患者群体(如具有特定分期和/或先前治疗历史患者群体)。
本发明第一方面，提供了一种治疗患有或怀疑患CD19阳性血液肿瘤的方法，包括向所述受试者给予表达抗CD19嵌合抗原受体(CAR)的细胞，所给予细胞的剂量为(a)约4.9x10 6个细胞/公斤受试者体重(细胞/kg)，(b)约3x10 8个总细胞，(c)约1x10 7个细胞/kg，(d)约6x10 8个总细胞，(a)不超过约4.9x10 6个细胞/kg，(b)不超过约3x10 8个总细胞，(c)不超过约1x10 7个细胞/kg，(d)不超过约6x10 8个总细胞，(e)约1.5x10 5个细胞/kg～约4.9x10 6个细胞/kg，(f)约1.5x10 5个细胞/kg～约1x10 7个细胞/kg，(g)约1x10 7个总细胞～约3x10 8个总细胞，和/或(h)约1x10 7个总细胞～约6x10 8个总细胞；所述细胞能结合肿瘤细胞上表达的CD19。进一步优选CD19是如SEQ ID NO：31所示的人来源的CD19。
在本文中，所给予细胞的剂量按照公斤受试者体重给与的细胞个数来计量，在本文中，个细胞/公斤受试者体重统一表示为“细胞/kg”。在本文中，所给予细胞的总剂量按照每个受试者给予的总细胞的个数来计量，个总细胞/个受试者统一用“个总细胞”来表示。
在具体实施方式中，所给予细胞的剂量为：(a)约5.8x10 5个细胞/kg，(b)约1.1x10 6 个细胞/kg，(c)约1.7x10 6个细胞/kg，(d)约1.8x10 6个细胞/kg，(e)约1.9x10 6个细胞/kg，(f)约2.0x10 6个细胞/kg，(g)约2.1x10 6个细胞/kg，(h)约2.6x10 6个细胞/kg，(h)约3x10 7个总细胞，(i)约6x10 7个总细胞，(j)约8x10 7个总细胞，(k)约9x10 7个总细胞，(l)约1x10 8个总细胞，(m)约1.5x10 8个总细胞，(n)约5.8x10 5个细胞/kg～约4.9x10 6个细胞/kg，(o)约1.1x10 6个细胞/kg～约4.9x10 6个细胞/kg，(p)约1.7x10 6个细胞/kg～约4.9x10 6个细胞/kg，(q)约1.8x10 6个细胞/kg～约4.9x10 6个细胞/kg，(r)约1.9x10 6个细胞/kg～约4.9x10 6个细胞/kg，(s)约2x10 6个细胞/kg～约4.9x10 6个细胞/kg，(t)约2.6x10 6个细胞/kg～约4.9x10 6个细胞/kg，(u)约3x10 7个总细胞～约3x10 8个总细胞，(v)约6x10 7个总细胞～约3x10 8个总细胞，(w)约8x10 7个总细胞～约3x10 8个总细胞，(x)约1x10 8个总细胞～约3x10 8个总细胞，和/或(y)约1.5x10 8个总细胞～约3x10 8个总细胞；其中在细胞给予之前所述受试者接受淋巴细胞清除疗法。
在具体实施方式中，所述受试者在给予所述细胞时或给予之前：
所述受试者被鉴定出患有B细胞恶性肿瘤；和/或
所述B细胞恶性肿瘤选自：急性淋巴细胞白血病(ALL)、成人ALL、慢性淋巴细胞白血病(CLL)、和/或非霍奇金淋巴瘤(NHL)；和/或
所述受试者被鉴定出或已经被鉴定出具有一种或多种细胞遗传学异常，任选地与高风险NHL和/或白血病相关；
所述受试者被鉴定出或已经被鉴定出患有高风险NHL；和/或
所述NHL选自下组：侵袭性NHL、弥漫性大B细胞淋巴瘤(DLBCL)、原发性纵隔大B细胞淋巴瘤(PMBCL)、富含T细胞/组织细胞的大B细胞淋巴瘤(TCHRBCL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)和/或滤泡性淋巴瘤(FL)；和/或
所述受试者是成年人和/或年龄超过20岁、30岁、40岁、50岁、60岁或70岁或者超过约20岁、30岁、40岁、50岁、60岁或70岁。
在具体实施方式中，在给予所述细胞之前所述受试者已经接受表达CD19CAR细胞以外的其他两种或更多种针对所述血液肿瘤的先前疗法，任选2、3或4种或更多种，任选地，先前疗法包括激酶抑制剂治疗任选依鲁替尼，或单克隆抗体治疗任选利妥昔单抗，或CHOP疗法，和/或造血干细胞移植(HSCT)治疗；所述先前疗法不耐受、无效、或治疗后出现缓解后复发难治。
在具体实施方式中，所述淋巴细胞清除疗法包括：
(i)给予氟达拉滨，任选地为还包括给予环磷酰胺；
(ii)在给予细胞前至少48小时或至少约48小时或在48小时、或约48小时和144小时或
约144小时之间的时间开始；并且(iii)以约21-59mg/kg给予环磷酰胺，任选每天1次持续1天或2天或3天，或以约250mg/m 2～500mg/m 2/日给予环磷酰胺，任选2天或3天或4天，和/或以约17-27mg/m 2/d给予氟达拉滨，持续3-4天。
在具体实施方式中，所述细胞输注和/或淋巴细胞清除疗法施与由门诊递送。
在具体实施方式中，所述细胞剂量分1、2、3、4或以上次在3或4天内经由胃肠外给予，任选静脉内给予。
在具体实施方式中，根据所述方法治疗的受试者中的至少30％实现完全缓解(CR)和/或总体缓解(OR)。
在具体实施方式中，根据所述方法治疗并实现完全缓解(CR)的受试者中的至少50％展现出超过12个月的无进展生存期(PFS)和/或总生存期(OS)；平均而言，根据该方法治疗的受试者展现出超过6个月、12个月或18个月或者超过约6个月、12个月或18个月的平均PFS或OS；和/或该受试者展现出至少6、12、18或更多个月或者至少约6、12、18或更多个月的治疗后PFS或OS。
在具体实施方式中，根据所述方法治疗的受试者均没有出现3级或更高级的CRS及神经毒性。
在具体实施方式中，所述CAR包含结合CD19的scFv、跨膜结构域、4-1BB或CD28的共刺激结构域、以及CD3ζ胞内结构域；所述scFv包含SEQ ID NO：2所示的LCDR1、SEQ ID NO：3所示的LCDR2、SEQ ID NO：4所示的LCDR3、及SEQ ID NO：5所示的HCDR1、SEQ ID NO：6所示的HCDR2、SEQ ID NO：7所示的HCDR3；或所述scFv包含SEQ ID NO：8或11所示的重链可变区以及SEQ ID NO：9或12所示的轻链可变区；或
所述结合CD19的scFv具有SEQ ID NO：1或10所示序列；或
所述CAR包含SEQ ID NO：13、14、15、16、17、18、19或20所示的氨基酸序列；和/或
所述共刺激结构域包含SEQ ID NO:24或25、或其变体，该变体与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性；和/或
所述CD3ζ胞内结构域包含SEQ ID NO:26，它们具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性。
在具体实施方式中，所述细胞是免疫效应细胞，优选地为T细胞，更优选地为自体同源或同种异体T细胞，最优选地为从受试者获得的原代T细胞。
本发明第二方面提供了一种包含表达特异性地结合至CD19阳性血液肿瘤的嵌合抗原受体(CAR)的细胞的组合物，其特征在于，所述组合物用于治疗患有或怀疑患有CD19阳性血液肿瘤的受试者，所述治疗包括向所述受试者给予一剂或多个剂量表达所述CAR的细胞，所述剂量(a)约4.9x10 6个细胞/公斤受试者体重(细胞/kg)，(b)约3x10 8个总细胞，(c)约1x10 7个细胞/kg，(d)约6x10 8个总细胞，(a)不超过约4.9x10 6个细胞/kg，(b)不超过约3x10 8个总细胞，(c)不超过约1x10 7个细胞/kg，(d)不超过约6x10 8个总细胞，(e)约1.5x10 5 个细胞/kg～约4.9x10 6个细胞/kg，(f)约1.5x10 5个细胞/kg～约1x10 7个细胞/kg，(g)约1x10 7个总细胞～约3x10 8个总细胞，和/或(h)约1x10 7个总细胞～约6x10 8个总细胞；所述细胞能结合肿瘤细胞上表达的CD19。
在具体实施方式中，所给予细胞的剂量为：(a)约5.8x10 5个细胞/kg，(b)约1.1x10 6个细胞/kg，(c)约1.7x10 6个细胞/kg，(d)约1.8x10 6个细胞/kg，(e)约1.9x10 6个细胞/kg，(f)约2.0x10 6个细胞/kg，(g)约2.1x10 6个细胞/kg，(h)约2.6x10 6个细胞/kg，(h)约3x10 7个总细胞，(i)约6x10 7个总细胞，，(j)约8x10 7个总细胞，(k)约9x10 7个总细胞，(l)约1x10 8个总细胞，(m)约1.5x10 8个总细胞，(n)约5.8x10 5个细胞/kg～约4.9x10 6个细胞/kg，(o)约1.1x10 6个细胞/kg～约4.9x10 6个细胞/kg，(p)约1.7x10 6个细胞/kg～约4.9x10 6个细胞/kg，(q)约1.8x10 6个细胞/kg～约4.9x10 6个细胞/kg，(r)约1.9x10 6个细胞/kg～约4.9x10 6个细胞/kg，(s)约2x10 6个细胞/kg～约4.9x10 6个细胞/kg，(t)约2.6x10 6个细胞/kg～约4.9x10 6个细胞/kg，(u)约3x10 7个总细胞～约3x10 8个总细胞，(v)约6x10 7个总细胞～约3x10 8个总细胞，(w)约8x10 7个总细胞～约3x10 8个总细胞，(x)约1x10 8个总细胞～约3x10 8个总细胞，和/或(y)约1.5x10 8个总细胞～约3x10 8个总细胞；其中在细胞给予之前所述受试者接受淋巴细胞清除疗法。
在具体实施方式中，还包含用于淋巴细胞清除疗法的药物，任选地，所述药物包括氟达拉滨和环磷酰胺。
在具体实施方式中，所述受试者在给予所述细胞时或给予之前：
所述受试者被鉴定出患有B细胞恶性肿瘤；和/或
所述B细胞恶性肿瘤选自：急性淋巴细胞白血病(ALL)、成人ALL、慢性淋巴细胞白血病(CLL)、和/或非霍奇金淋巴瘤(NHL)；和/或
所述受试者被鉴定出或已经被鉴定出具有一种或多种细胞遗传学异常，任选地与高风险NHL和/或白血病相关；
所述受试者被鉴定出或已经被鉴定出患有高风险NHL；和/或
所述NHL选自下组：侵袭性NHL、弥漫性大B细胞淋巴瘤(DLBCL)、原发性纵隔大B细胞淋巴瘤(PMBCL)、富含T细胞/组织细胞的大B细胞淋巴瘤(TCHRBCL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)和/或滤泡性淋巴瘤(FL)；和/或
所述受试者是成年人和/或年龄超过20岁、30岁、40岁、50岁、60岁或70岁或者超过约20岁、30岁、40岁、50岁、60岁或70岁。
在具体实施方式中，所述受试者在给予所述细胞之前所述受试者已经接受表达CD19CAR细胞以外的其他两种或更多种针对所述血液肿瘤的先前疗法，任选2、3或4种或更多种，任选地，先前疗法包括激酶抑制剂治疗任选依鲁替尼，或单克隆抗体治疗任选利妥昔单抗，或CHOP疗法，和/或造血干细胞移植(HSCT)治疗；所述先前疗法不耐受、无效、或治疗后出现缓解后复发难治。
在具体实施方式中，所述淋巴细胞清除疗法：
(i)在给予细胞前至少48小时或至少约48小时或在48小时、或约48小时和144小时或约144小时之间的时间开始；和/或
(ii)以约21-59mg/kg给予环磷酰胺，任选每天1次持续1天或2天或3天，或以约250mg/m 2～500mg/m 2/日给予环磷酰胺，任选2天或3天或4天，和/或以约17-27mg/m 2/d给予氟达拉滨，持续3-4天。
在具体实施方式中，所述组合物中的细胞分1、2、3、4或以上次在3或4天内通过肠胃外给药，任选静脉内给药。
在具体实施方式中，在门诊环境中、和/或在所述受试者无需住院过夜或连续多天的情况下、和/或所述受试者无需住院一天或多天的情况下给予或将给予所述细胞。
在具体实施方式中，所述细胞是免疫效应细胞，优选地为T细胞，更优选地为自体同源或同种异体T细胞，最优选地为从受试者获得的原代T细胞。
在具体实施方式中，所述CAR包含结合CD19的scFv、跨膜结构域、4-1BB或CD28的共刺激结构域、以及CD3ζ胞内结构域；所述scFv包含SEQ ID NO：2所示的LCDR1、SEQ ID NO：3所示的LCDR2、SEQ ID NO：4所示的LCDR3、及SEQ ID NO：5所示的HCDR1、SEQ ID NO：6所示的HCDR2、SEQ ID NO：7所示的HCDR3；或所述scFv包含SEQ ID NO：8或11所示的重链可变区以及SEQ ID NO：9或12所示的轻链可变区；或
所述结合CD19的scFv具有SEQ ID NO：1或10所示序列；或
所述CAR包含SEQ ID NO：13、14、15、16、17、18、19或20所示的氨基酸序列；和/或
所述共刺激结构域包含SEQ ID NO:24或25、或其变体，该变体与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性；和/或
所述CD3ζ胞内结构域包含SEQ ID NO:26，它们具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性。
在具体实施方式中，所述细胞是免疫效应细胞，优选地为T细胞，更优选地为自体同源或同种异体T细胞，最优选地为从受试者获得的原代T细胞。
本发明第三方面提供了一种用于治疗患有或怀疑患有CD19阳性血液肿瘤的制品，其特征在于，所述制品包含：
1)如本发明所述的组合物；
2)利用所述试剂盒治疗患有或怀疑患有CD19阳性血液肿瘤受试者的给药说明书。
在具体实施方式中，每个剂量包含表达抗CD19嵌合抗原受体(CAR)的细胞，所述说明书规定了向患有CD19阳性血液肿瘤的受试者给予的细胞的剂量；并且该说明书规定了给予一定数量的CD19 CAR表达细胞，或规定了给予对应于所述指定数量的细胞或含 有所述指定数量的细胞的一定量或体积的一种或多种制剂。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现表达。在此基础上完成了本发明。
如果本文所示的定义与引用的专利、公开申请和其它出版物中所示的定义不同或其它情况下不一致，则以本文所示的定义为主。
本发明中，范围形式的描述不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，对具体数值的范围，应当理解为在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围，除非明确地排除所述范围的上下限。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两个的范围。无论范围宽窄，该原则均适用。
本文使用的术语“约”是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中“约”值或参数，包括指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”可意指至多10％(即±10％)的范围。例如，约5mg可包括在4.5mg与5.5mg之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，除非另外指出，否则“约”应为在该特定值或组成的可接受误差范围内。
本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值，除非另外指出。
本文中所述的“给药间隔”是指对受试者给予多个疗程的免疫效应细胞治疗之间以及与给予预处理药物之间所经过的时间。所以，给药间隔可被指示为范围。
本文中所述的“剂量”，可以是以重量为基础计算的剂量或以体表面积(BSA)为基础计算的剂量表示。以重量为基础计算的剂量是以患者体重为基础所计算出的对于患者给予的剂量，例如mg/kg，免疫效应细胞个数/kg等。以BSA为基础计算的剂量是以患者的表面积为基础所计算出的对患者给予的剂量，例如mg/m 2，以及免疫效应细胞个数/m 2等。
本文中所述的“给药次数”是指给定时间内给予免疫效应细胞或预处理药物剂量的频率。给药次数可被指示为每给定时间内剂量数。例如，可以按照下列来给予氟达 拉滨：连续4日每日给予一次剂量、连续3日每日给予一次剂量、连续2日每日给予一次剂量、或1日给予一次剂量。可以按照下列来给予环磷酰胺：连续4日每日给予一次剂量、连续3日每日给予一次剂量、连续2日每日给予一次剂量、或1日给予一次剂量。
本申请涉及过继细胞或免疫效应细胞治疗肿瘤，包括给予一次或者多次疗程的细胞，及其使用的方法、组合物和制品。细胞一般表达嵌合抗原受体例如，嵌合抗原受体(CAR)或其他转基因受体如T细胞受体(TCR)。
本发明的提供用于治疗与CD19表达相关的疾病(例如肿瘤)的治疗方法及组合物。
本发明提供了采用表达遗传工程改造(重组)的嵌合受体的过继细胞或免疫效应细胞治疗受试者的肿瘤的方法。该方法包括过继细胞或免疫效应细胞的单疗程回输、或多疗程回输。
I.用基因工程化细胞进行的细胞疗法的方法和用途
提供了用于治疗CD19阳性肿瘤的方法及用在细胞疗法中的组合物。该方法包括给予能识别和/或特异性地结合至与肿瘤细胞上的CD19分子并产生针对CD19的免疫应答的工程化细胞。受体可包括嵌合受体例如嵌合抗原受体(CAR)和T细胞受体(TCR)。
在一些实施例中，该方法包括用一定剂量的抗原受体表达细胞(例如CAR表达细胞)治疗患有急性或慢性淋巴细胞白血病(ALL/CLL)或非霍奇金淋巴瘤(NHL)的受试者。
在一些实施例中，该受试者已经用免疫清除(例如淋巴细胞清除)疗法预处理。用免疫清除(例如，淋巴细胞清除)疗法预处理受试者可以改善本发明细胞疗法的效果。用淋巴细胞清除剂(包括环磷酰胺和氟达拉滨的组合)预处理有效地改善转移的肿瘤浸润淋巴细胞(TIL)在细胞疗法中的功效，包括改善转移细胞的应答和/或持久性。在一些实施例中，该方法包括在给予该剂CD19 CAR-T细胞之前给予化学治疗剂用以减轻肿瘤负荷。在一些实施例中，该方法包括给予预处理剂如淋巴细胞清除剂或化学治疗剂，如环磷酰胺、氟达拉滨或其组合。在一些实施例中，该方法包括给予氟达拉滨和任选除氟达拉滨以外的另一种化学治疗剂。在一些实施例中，可以在给予该剂细胞之前至少2天如至少3、4、5、6或7天向该受试者给予淋巴细胞清除疗法。在一些实施例中，在给予该剂细胞之前至少48小时或至少约48小时或者48小时或约48小时或者至少144小时或至少约144小时给予或开始淋巴细胞清除疗法。在一些实施例中，在给予该剂细胞之前在48小时或约48小时和144小时或约144小时之间给予或开始淋巴细胞清除疗法。
在一些实施例中，该方法包括在第一或后续剂量之前向受试者给予预处理剂如淋巴细胞清除剂或化学治疗剂，如环磷酰胺、氟达拉滨或其组合。例如，可以在第一或后续剂量之前至少2天如至少3、4、5、6或7天向该受试者给予预处理剂。在一些实施例中，在第一或后续剂量之前不超过7天如不超过6、5、4、3或2天向受试给予预处理剂。
在一些实施例中，将该受试者用在约20mg/kg～100mg/kg、或在约40mg/kg～80mg/kg、 或在约20mg/kg～约60mg/kg的剂量的环磷酰胺进行预处理。在一些方面，将该受试者用约60mg/kg的环磷酰胺进行预处理。在一些实施方案中，环磷酰胺可以以单剂量给予或者可以以多个剂量给予，如每天给药、每隔一天给药或每三天给药。在一些实施方案中，环磷酰胺每天给予一次，持续一天或两天。在一些实施方案中，当淋巴细胞清除剂包含环磷酰胺时，每天向该受试者给予剂量在约100mg/m 2～500mg/m 2(含)、或约250mg/m 2～500mg/m 2(含)、或约200mg/m 2～400mg/m 2、或约250mg/m 2～350mg/m 2(含)环磷酰胺。在一些情况下，向该受试者每天给予约250mg/m 2～500mg/m 2(含)的环磷酰胺，每天给予一次，持续2天或3天或4天。在一些实施方案中，环磷酰胺可以以单剂量给予或者可以以多个剂量给予，如每天给药、每隔一天给药或每三天给药。在一些实施方案中，每天给予环磷酰胺，如持续1～5天，例如持续3至5天。在一些情况下，在开始细胞疗法之前每天向该受试者给予约300mg/m 2的环磷酰胺，持续3天。
在一些实施方案中，当淋巴细胞清除剂包含氟达拉滨时，向该受试者给予剂量在约1mg/m 2～100mg/m 2、或约10mg/m 2～75mg/m 2、或约15mg/m 2～50mg/m 2、或约20mg/m 2～40mg/m 2、或约20mg/m 2～30mg/m 2、或约24mg/m 2～35mg/m 2、或约24mg/m 2～26mg/m 2的氟达拉滨。在一些情况下，向该受试者给予25mg/m 2的氟达拉滨。在一些情况下，向该受试者给予约30mg/m 2的氟达拉滨。在一些实施方案中，氟达拉滨可以以单剂量给予或者可以以多个剂量给予，如每天给药、每隔一天给药或每三天给药。在一些实施方案中，每天给予氟达拉滨，如持续1-5天，例如持续3至5天。在一些情况下，在开始细胞疗法之前每天向该受试者给予约30mg/m 2的氟达拉滨，持续3天。
在一些实施方案中，淋巴细胞清除剂包含药剂的组合，如环磷酰胺和氟达拉滨的组合。因此，药剂的组合可包括任何剂量或给药时间表(如上述那些剂量或给药时间表)下的环磷酰胺以及任何剂量或给药时间表(如上述那些剂量或给药时间表)下的氟达拉滨。例如，在一些方面，在给予该剂细胞之前向该受试者给予21～59mg/kg(约1～2g/m2)的环磷酰胺和3至4剂量的17～27mg/m 2氟达拉滨。在一些实施方案中，可以通过减少或省略环磷酰胺的剂量或给予与氟达拉滨同时给予的较低总剂量的环磷酰胺的方案来修改淋巴细胞清除化学疗法，以最小化对受试者的毒性，如已经接受多个先前化疗周期、先前已经历同种异体移植、骨髓储备不良和/或有其他严重的合并症的受试者。
在一些实施方案中，该方法包括向受试者、组织或细胞给予该细胞或含有该细胞的组合物，如患有CD19阳性血液肿瘤、处于患有CD19阳性血液肿瘤的风险或怀疑患有CD19阳性血液肿瘤的受试者、组织或细胞。在一些实施方案中，受试者是成年受试者。在一些实施方案中，受试者年龄超过20岁、30岁、40岁、50岁、60岁或70岁或超过约20岁、30岁、40岁、50岁、60岁或70岁。
在一些实施方案中，所提供的方法包括过继细胞治疗方法(例如CAR+T细胞)，用于治疗急、慢性淋巴细胞白血病(ALL/CLL)。高风险CLL的治疗包括化学疗法(Hallek等人(2010)Lancet 376:1164-1174)，但最近BTK抑制剂依鲁替尼首先被批准用于复发和难治性 疾病，并随后用于一线治疗(Burger等人(2015)N.Engl J.Med.,373:2425-2437；Byrd等人(2013)N.Engl.J.Med.,369:32-42)。虽然对依鲁替尼的总体应答率(ORR)高，但在一些情况下可能完全应答率(CR；或完全缓解)低，并且针对依鲁替尼进展的患者的生存期可能是短的。
在一些实施方案中，与某些替代方法相比，所提供的方法和用途提供或实现如在特定的治疗的受试者组中(如在患有白血病(如ALL/CLL或NHL)的患者中，包括那些患有高危疾病的患者)改善的或更持久的应答或功效。在一些实施方案中，该方法通过给予T细胞疗法(如包括用于过继性细胞疗法例如T细胞疗法(例如CAR表达T细胞)的细胞的组合物)和淋巴细胞清除疗法(例如环磷酰胺、氟达拉滨或其组合)是有利的。
ALL诊断应采用MICM(形态学、免疫学、细胞遗传学和分子学)诊断模式b1，诊断分型采用WHO2016标准。最低标准应进行细胞形态学、免疫表型检查，以保证诊断的可靠性；骨髓中原始/幼稚淋巴细胞比例>20％才可以诊断ALL；免疫分型应采用多参数流式细胞术。细胞遗传学分组参考NCCN2016建议：预后良好遗传学异常包括超二倍体(51～65条染色体)、t(12；21)(p13；q22)和(或)ETV6一RUNXl；预后不良遗传学异常包括亚二倍体(<44条染色体)、t(v；1lq23)[t(4；11)和其他MLL重排]、t(9；22)(q34；ql 1.2)、复杂染色体异常。
在一些情况下，一种对受试者进行分类的方法是Ann Arbor系统。在一些方面，Ann Arbor分期：I期：侵及1个淋巴结区或1个结外器官或部位。
Ⅱ期：横隔一侧，侵及2个或更多个的淋巴结区或外加局限侵犯1个结外器官或部位。
Ⅲ期：侵犯横隔两侧淋巴结区或外加局限侵犯1个结外器官或部位或脾或二者。
Ⅳ期：弥漫性或播散性侵犯1个或多个的结外器官或部位，同时伴有或不伴有淋巴结侵犯。
另外，按照有无全身症状统一分为A、B两类。
A：无症状。
B：出现6个月内不明原因的体重下降>10％或原因不明的发热(38℃以上，连续3天)或盗汗。
在一些实施方案中，该方法包括向被选择或鉴定为具有高风险NHL的受试者给予细胞。在一些实施方案中，受试者展现出一种或多种细胞遗传学异常，如与高风险NHL相关。在一些实施方案中，基于具有如下疾病或病况来选择或鉴定受试者，该疾病或病况的特征为或被确定为侵袭性NHL、弥漫性大B细胞淋巴瘤(DLBCL)、原发性纵隔大B细胞淋巴瘤(PMBCL)、富含T细胞/组织细胞的大B细胞淋巴瘤(TCHRBCL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)和/或滤泡性淋巴瘤(FL)。
在一些实施方案中，在给予表达重组抗原受体的细胞之前该受试者先前已经用靶向ALL/CLL或NHL的疗法或治疗剂治疗。在一些实施方案中，该治疗剂是激酶抑制剂如布 鲁顿酪氨酸激酶(Btk)的抑制剂，例如依鲁替尼。在一些实施方案中，该治疗剂是B细胞淋巴瘤-2(Bcl-2)的抑制剂，例如venetoclax。在一些实施方案中，该治疗剂是特异性地结合至由ALL/CLL或NHL的细胞表达的抗原的抗体(例如单克隆抗体)，例如来自CD20、CD19、CD22、ROR1、CD45、CD21、CD5、CD33、Igκ、Igλ、CD79a、CD79b或CD30中的任何一种或多种的抗原。在一些实施方案中，该治疗剂是抗CD20抗体，例如利妥昔单抗。在一些实施方案中，该治疗剂是消耗化学疗法，其是包括利妥昔单抗的组合疗法，例如氟达拉滨和利妥昔单抗的组合疗法或蒽环霉素和利妥昔单抗的组合疗法或CHOP组合疗法(环磷酰胺、阿霉素、长春新碱、强的松)或R-CHOP组合疗法(利妥昔单抗、环磷酰胺、阿霉素、长春新碱、强的松)。在一些实施方案中，该受试者先前已经用造血干细胞移植(HSCT)治疗，例如同种异体HSCT或自体HSCT。在一些实施方案中，该受试者已经进行治疗或先前已接受除淋巴细胞清除疗法和/或该剂表达抗原受体的细胞以外的至少或约至少1、2、3或4种其他用于治疗NHL或ALL/CLL的疗法。在一些实施方案中，该受试者先前已经用化学疗法或放射疗法治疗。
在一些方面，该受试者对于其他疗法或治疗剂难以治疗的或无反应的。在一些实施方案中，该受试者例如在用另一种疗法或治疗性干预(包括化学疗法或放射)治疗后患有持续性或复发性疾病。
用于过继细胞疗法的细胞的给予方法是已知的，并且可以与所提供的方法和组合物一起使用。例如，过继T细胞治疗方法描述于例如Gruenberg等人的美国专利申请公开号2003/0170238；Rosenberg的美国专利号4,690,915；Rosenberg(2011)Nat Rev Clin Oncol.8(10):577-85。
在一些实施方案中，细胞疗法例如过继性细胞疗法例如过继T细胞疗法通过自体转移进行，其中从要接受细胞疗法的受试者或从来源于这种受试者的样品分离和/或以其他方式制备细胞。因此，在一些方面，细胞来源于需要治疗的受试者(例如患者)，并且该细胞在分离和加工后被给予同一受试者。
在一些实施方案中，细胞疗法例如过继细胞疗法例如过继T细胞疗法通过同种异体转移进行，其中从除要接受或最终接受细胞疗法的受试者例如第一受试者以外的受试者分离和/或以其他方式制备细胞。在此类实施方案中，细胞随后被给予相同物种的不同受试者例如第二受试者。在一些实施方案中，第一和第二受试者在遗传上是相同的。在一些实施方案中，第一和第二受试者在遗传上是相似的。在一些实施方案中，第二受试者表达与第一受试者相同的HLA类或超型。
该细胞可以通过任何合适的方式给予，例如通过推注输注，通过注射例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房注射、结膜下注射、结膜下注射、眼球筋膜囊下注射、球后注射、球周注射或后巩膜递送。在一些实施方案中，它们通过肠胃外、肺内和鼻内给予以及(如果需要用于局部治疗的话)病灶内给药。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内 或皮下给药。在一些实施方案中，给定剂量通过细胞的单次推注给药来给予。在一些实施方案中，给定剂量通过细胞的多次推注给药例如在不超过3天的时间内来给予，或通过细胞的连续输注给药。
为了预防或治疗疾病，适当的剂量可取决于待治疗的疾病类型、细胞或重组受体的类型、疾病的严重程度和病程、细胞是否针对预防或治疗目的而被给予、先前的治疗、受试者的临床病史和对细胞的应答以及主治医师的决断。在一些实施方案中，该组合物和细胞适合一次或在一系列治疗中给予受试者。
在将该细胞给予受试者(例如人)后，在一些方面中工程化细胞群的生物活性通过许多已知方法中的任何一种来测量。待评估的参数包括工程化或天然T细胞或其他免疫细胞与抗原的特异性结合，其在体内例如通过成像进行评估，或离体例如通过ELISA或流式细胞术进行评估。在某些实施方案中，可以使用本领域已知的任何合适的方法测量工程化细胞破坏靶细胞的能力，如在例如Kochenderfer等人,J.Immunotherapy,32(7):689-702(2009)和Herman等人J.Immunological Methods,285(1):25-40(2004)中描述的细胞毒性测定。在某些实施方案中，还可以通过测定某些细胞因子如CD107a、IFNγ、IL-2和TNF的表达和/或分泌来测量细胞的生物活性。在一些方面，通过评估临床结果如肿瘤负荷或负载的减少来测量生物活性。在一些方面，评估毒性结果、细胞的持久性和/或扩增和/或宿主免疫应答的存在或不存在。
在某些实施方案中，以许多方式修饰工程化细胞，使得其治疗或预防功效增加。例如，由群体表达的工程化CAR或TCR可以直接或通过接头间接缀合至靶向部分。将化合物例如CAR或TCR与靶向部分缀合的实践是本领域已知的。
在一些实施方案中，该细胞作为组合治疗的一部分给予，如与另一种治疗性干预如抗体或工程化细胞或受体或药剂(如细胞毒性剂或治疗剂)同时给予或以任何顺序依次给予。在一些实施方案中，该细胞与一种或多种附加治疗剂共同给予或与另一种治疗性干预联合给予(同时或以任何顺序依次给予)。在一些情况下，该细胞与另一种疗法以足够接近的时间共同给予，使得细胞群增强一种或多种附加治疗剂的作用，或者相反。在一些实施方案中，该细胞在一种或多种附加治疗剂之前给予。在一些实施方案中，该细胞在一种或多种附加治疗剂之后给予。在一些实施方案中，该一种或多种附加药剂包括细胞因子如IL-2，例如用于增强持久性。
A.给药
给定“剂量”的给予包括以单一组合物和/或单次不间断给药的方式(例如以单次注射或连续输注的方式)给予给一定数量的细胞，并且还包括在不超过4天的指定时间段内以在多个单独组合物或输注中提供的分割剂量的方式给予给一定量数量的细胞。因此，在一些情况下，剂量是指一定数量的细胞的单次或连续给药，在单个时间点给予或开始。然而，在一些情况下，剂量在不超过4天的时间内以多次注射或输注的方式给予，如每天一次持续4天或3天或2天或者通过在一天的时间内多次输注。
在一些方面，该剂量的细胞以单一药物组合物施用。在一些实施方案中，该剂量的细胞以共同含有该剂量的细胞的多种组合物施用。
在一些实施方案中，根据所提供的方法将一定剂量细胞给予受试者。在一些实施方案中，剂量的大小或时间安排根据受试者的特定疾病或病况确定。
在某些实施方案中，向该受试者给予约10 5～约10 7细胞/公斤受试者体重的范围的细胞，例如约10 5至约10 6个细胞(例如，约1x10 5、约2x10 5、约3x10 5、约4x10 5、约5x10 5、约6x10 5、约7x10 5、约8x10 5、约9x10 5、约1x10 6或由前述任何两个值限定的范围)、约10 6至约10 7个细胞(例如，约1x10 6、约2x10 6、约3x10 6、约4x10 6、约5x10 6、约6x10 6、约7x10 6、约8x10 6、约9x10 6、约1x10 7或由前述任何两个值限定的范围)、或这些范围之间的任何值和/或每公斤受试者体重。剂量可以根据疾病或病症和/或患者和/或其他治疗特有的属性而变化。在一些实施方案中，这些值是指重组受体CD19-CAR表达细胞的数量；在其他实施方案中，它们是指给予的T细胞或PBMC或总细胞的数量。
在一些实施方案中，细胞疗法包括给予包含许多细胞的剂量，该细胞为至少或至少约0.1x10 6个细胞/kg受试者体重、0.2x10 6个细胞/kg、0.3x10 6个细胞/kg、0.4x10 6个细胞/kg、0.5x10 6个细胞/kg、1x10 6个细胞/kg、2x10 6个细胞/kg、3x10 6个细胞/kg、4x10 6个细胞/kg、5x10 6个细胞/kg、6x10 6个细胞/kg、7x10 6个细胞/kg、8x10 6个细胞/kg、9x10 6个细胞/kg或1x10 7个细胞/kg，或者为0.1x10 6个细胞/kg、0.2x10 6个细胞/kg、0.3x10 6个细胞/kg、0.4x10 6个细胞/kg、0.5x10 6个细胞/kg、1x10 6个细胞/kg、2x10 6个细胞/kg、3x10 6个细胞/kg、4x10 6个细胞/kg、5x10 6个细胞/kg、6x10 6个细胞/kg、7x10 6个细胞/kg、8x10 6个细胞/kg、9x10 6个细胞/kg或1x10 7个细胞/kg。
在一些实施方案中，细胞疗法包括给予包含一定细胞数量的剂量，该剂量包含(a)约4.9x10 6个细胞/公斤受试者体重(细胞/kg)，(b)约3x10 8个总细胞，(c)约1x10 7个细胞/kg，(d)约6x10 8个总细胞，(a)不超过约4.9x10 6个细胞/kg，(b)或不超过约3x10 8个总细胞，(c)不超过约1x10 7个细胞/kg，(d)不超过约6x10 8个总细胞，(e)约1.5x10 5个细胞/kg～约4.9x10 6个细胞/kg，(f)约1.5x10 5个细胞/kg～约1x10 7个细胞/kg，(g)约1x10 7个总细胞～约3x10 8个总细胞，和/或(h)约1x10 7个总细胞～约6x10 8个总细胞(包含每个端点)。
在一些实施方案中，该剂细胞包含(a)约5.8x10 5个细胞/kg，(b)约1.1x10 6个细胞/kg，(c)约1.7x10 6个细胞/kg，(d)约1.8x10 6个细胞/kg，(e)约1.9x10 6个细胞/kg，(f)约2.0x10 6个细胞/kg，(g)约2.1x10 6个细胞/kg，(h)约2.6x10 6个细胞/kg，(h)约3x10 7个总细胞，(i)约6x10 7个总细胞，，(j)约8x10 7个总细胞，(k)约9x10 7个总细胞，(l)约1x10 8个总细胞，(m)约1.5x10 8个总细胞，(n)约5.8x10 5～约4.9x10 6个细胞/kg，(o)约1.1x10 6～约4.9x10 6个细胞/kg，(p)约1.7x10 6/kg～约4.9x10 6个细胞/kg，(q)约1.8x10 6～约4.9x10 6个细胞/kg，(r)约1.9x10 6～或约4.9x10 6个细胞/kg，(s)约2x10 6～约4.9x10 6个细胞/kg，(t)约2.6x10 6～约4.9x10 6个细胞/kg之间，(u)约3x10 7～或约3x10 8个总细胞之间，(v)约6x10 7～约3x10 8个总细胞，(w)约8x10 7～约3x10 8个总细胞，(x)约1x10 8～约3x10 8个总细胞，和/或(y)约 1.5x10 8～约3x10 8个总细胞(包含每个端点)。
在一些实施方案中，给予患者多个剂量，并且每个剂量或总剂量可以在任何前述值内。在一些实施方案中，该剂细胞包括给予(a)约4.9x10 6个细胞/公斤受试者体重(细胞/kg)，(b)约3x10 8个总细胞，(c)约1x10 7个细胞/kg，(d)约6x10 8个总细胞，(a)不超过约4.9x10 6个细胞/kg，(b)或不超过约3x10 8个总细胞，(c)或不超过约1x10 7个细胞/kg，(d)或不超过约6x10 8个总细胞，(e)或约1.5x10 5～约4.9x10 6个细胞/kg，(f)或约1.5x10 5～约1x10 7个细胞/kg，(g)或约1x10 7～约3x10 8个总细胞，和/或(h)或约1x10 7～约6x10 8个总细胞(包含每个端点)。
“分剂量”是指在一个疗程内，在将整个疗程的剂量分为多次给予受试者的情况下，单次给药的剂量，也被认为是单剂量。
因此，该剂细胞可以以分割剂量的形式给予。例如，在一些实施方案中，剂量可以在2天或3天或4天内给予受试者。用于分割给药的示例性方法包括在第一天给予10％的总剂量、第二天给予30％的总剂量、第三天给予60％的总剂量。在其他实施方案中，可以在第一天给予12.5％的总剂量、第二天给予37.5％的总剂量、第四天给予50％的总剂量。在一些方面，在第一天给予30％的总剂量，在第二天给予70％的总剂量。在一些实施方案中，分割剂量不超过4天或3天或2天。
在一些实施方案中，该剂细胞通常足够大以有效减轻疾病负荷。
在一些方面，剂量的大小基于一个或多个标准来确定，如受试者对现有治疗例如化学疗法的反应、受试者的疾病负荷如肿瘤负载、体积、尺寸或程度、转移的程度或类型、分期和/或受试者发生毒性结果的可能性或发生率，例如CRS、巨噬细胞激活综合征、肿瘤溶解综合征、神经毒性和/或针对所给予的细胞和/或重组受体的宿主免疫应答。
在一些实施方案中，该方法还包括给予一个或多个附加剂量的表达嵌合抗原受体(CAR)的细胞和/或淋巴细胞清除疗法，和/或重复该方法的一个或多个步骤。在一些实施方案中，该一个或多个附加剂量与初始剂量相同。在一些实施方案中，该一个或多个附加剂量不同于初始剂量，例如更高如比初始剂量高2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍或10倍或更多倍，或者更低如比初始剂量低2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍或10倍或更多倍。在一些实施方案中，一个或多个附加剂量的给予基于以下各项来确定，如受试者对初始治疗或任何现有治疗的反应、受试者的疾病负荷如肿瘤负载、体积、尺寸或程度、转移的程度或类型、分期和/或受试者发生毒性结果的可能性或发生率，例如CRS、巨噬细胞激活综合征、肿瘤溶解综合征、神经毒性和/或针对所给予的细胞和/或重组受体的宿主免疫应答。
B.应答、功效和存活
在一些实施方案中，至少或约至少30％、40％、50％、60％、70％、80％、90％根据该方法治疗的受试者实现完全缓解(CR)和/或部分缓解(PR)和/或实现客观应答(OR)。
在一些方面，根据所提供的方法的给药通常减少或防止受试者的疾病或病况的扩展或 负荷。例如，在疾病或病况是肿瘤的情况下，该方法通常减少肿瘤尺寸、体积、转移、骨髓中的原始细胞百分比或分子上可检测的癌症和/或改善预后或存活或与肿瘤负荷相关的其他症状。
在一些方面，无进展生存期(PFS)被描述为治疗疾病(如癌症)期间和之后受试者伴随疾病生存但疾病不恶化的时间长度。在一些方面，客观应答(OR)被描述为可测量的应答。在一些方面，客观应答率(ORR)被描述为实现CR或PR的患者的比例。在一些方面，总生存期(OS)被描述为从诊断或开始治疗疾病(如癌症)的日期到被诊断患有该疾病的受试者仍然存活时的时间长度。在一些方面，无事件生存期(EFS)被描述为癌症治疗结束后受试者保持没有该治疗旨在预防或延迟的某些并发症或事件的时间长度。这些事件可包括癌症的复发或某些症状的发作，如已经扩散到骨骼的癌症引起的骨痛，或死亡。
在一些实施方案中，与使用替代给药方案如其中受试者接受一种或多种替代治疗剂的替代给药方法和/或受试者未接受根据所提供的方法的一剂细胞和/或淋巴细胞清除剂的替代给药方法的可比较方法所观察到的减轻相比，该方法在更大的程度上和/或在更长的时间段内减轻疾病或病况的负荷，例如，肿瘤细胞的数量、肿瘤的尺寸、患者存活或无事件存活的持续时间。在一些实施方案中，检测、评估或测量受试者中疾病或病况的负荷。可以在一些方面通过检测受试者中或受试者的器官、组织或体液(如血液或血清)中的疾病或疾病相关细胞例如肿瘤细胞的总数来检测疾病负荷。在一些方面，评估受试者的存活、特定时间段内的存活、存活程度、无事件或无症状存活的存在或持续时间或无复发存活。在一些实施方案中，评估疾病或病况的任何症状。
在一些实施方案中，与其他方法相比，例如其中受试者接受一种或多种替代治疗剂的方法和/或其中受试者未接受根据所提供方法的一剂细胞和/或淋巴细胞清除剂的方法，通过该方法改善受试者的无事件存活率或总存活率。例如，在一些实施方案中，在该剂量后6个月时通过该方法治疗的受试者的无事件存活率或概率大于约40％、大于约50％、大于约60％、大于约70％、大于约80％、大于约90％或大于约95％。在一些方面，总存活率大于约40％、大于约50％、大于约60％、大于约70％、大于约80％、大于约90％或大于约95％。在一些实施方案中，用该方法治疗的受试者展现出无事件存活、无复发存活或存活至少6个月或至少1、2、3、4、5、6、7、8、9或10年。在一些实施方案中，进展的时间得到改善，如进展的时间大于6个月或大于约6个月或至少1、2、3、4、5、6、7、8、9或10年。
在一些实施方案中，与其他方法相比，例如其中受试者接受一种或多种替代治疗剂的方法和/或其中受试者未接受根据所提供方法的一剂细胞和/或淋巴细胞清除剂的方法，在通过该方法治疗后复发的概率降低。例如，在一些实施方案中，在第一剂量后6个月时复发的概率小于约80％、小于约70％、小于约60％、小于约50％、小于约40％、小于约30％、小于约20％或小于约10％。
疾病负荷/疾病负载可以包括受试者中或受试者的器官、组织或体液(如肿瘤的器官或 组织或另一位置，例如将指示转移的位置)中的疾病细胞的总数。例如，可以在某些血液恶性肿瘤环境中在血液或骨髓中检测和/或定量肿瘤细胞。在一些实施方案中，疾病负载可包括肿瘤的质量、转移的数量或程度和/或骨髓中存在的原始细胞的百分比。
在一些方面，应答评估利用临床、血液学和/或分子方法中的任何一种。
1.NCCN临床实践指南标准评估疗效
在一些方面，这些标准描述如下：完全缓解(CR)，其在一些方面要求不存在通过免疫表型分析的外周血克隆淋巴细胞、不存在淋巴结病、不存在肝肿大或脾肿大、不存在全身症状和令人满意的血细胞计数；完全缓解伴随不完全骨髓恢复(CRi)，其在一些方面被描述为上述CR但没有正常的血细胞计数；部分缓解(PR)，其在一些方面被描述为淋巴细胞计数下降≥50％、淋巴结病减少≥50％或肝或脾减少≥50％以及外周血细胞计数改善；进展性疾病(PD)，其在一些方面被描述为淋巴细胞计数增加≥50％至≥5x109/L、淋巴结病增加≥50％、肝或脾尺寸增加≥50％、Richter转化或由于CLL导致新的血细胞减少；和稳定的疾病，其在一些方面被描述为不符合CR、CRi、PR或PD的标准。
在一些实施方案中，如果在给予该剂细胞的1个月内受试者中的淋巴结的尺寸小于20mm或小于约20mm、小于10mm或小于约10mm或小于10mm或小于约10mm，则受试者展现出CR或PR。
在一些实施方案中，受试者患有白血病。疾病负荷的程度可以通过评估血液或骨髓中的残留白血病来确定。
在一些实施方案中，如果骨髓中存在例如通过光学显微镜检测的大于或等于5％原始细胞，则受试者展现出形态学疾病，如骨髓中大于或等于10％原始细胞、骨髓中大于或等于20％原始细胞、骨髓中大于或等于30％原始细胞、骨髓中大于或等于40％原始细胞或骨髓中大于或等于50％原始细胞。在一些实施方案中，如果骨髓中存在少于5％原始细胞，则受试者展现出完全或临床缓解。
在一些实施方案中，受试者可展现出完全缓解，但存在一小部分形态学上(通过光学显微镜技术)不可检测的残留白血病细胞。如果受试者展现在骨髓中小于5％原始细胞并且展现分子可检测的癌症，则称受试者展现最小残留病(MRD)。在一些实施方案中，可以使用允许灵敏检测少量细胞的各种分子技术中的任何一种来评估分子可检测的癌症。在一些方面，此类技术包括PCR测定，其可确定由染色体易位产生的独特Ig/T细胞受体基因重排或融合转录物。在一些实施方案中，流式细胞术可用于基于白血病特异性免疫表型鉴定癌细胞。在一些实施方案中，癌症的分子检测可检测10,000个正常细胞中的仅1个白血病细胞或100,000个正常细胞中的仅1个白血病细胞。在一些实施方案中，如果检测到如通过PCR或流式细胞术检测到10,000个细胞中的至少或大于1个白血病细胞或者检测到100,000个细胞中的1个白血病细胞，则受试者展现出分子可检测的MRD。在一些实施方案中，受试者的疾病负荷是分子不可检测的或MRD-，使得在一些情况下使用PCR或流式细胞术技术不能检测到受试者中的白血病细胞。
在一些实施方案中，在该受试者的骨髓中(或在根据该方法治疗的大于50％、60％、70％、80％、90％或更多的受试者的骨髓中)未检测到白血病例如ALL/CLL的指数克隆。
2.Lugano标准
在一些方面，使用Lugano标准评估应答(Cheson等人,J CO Se ptem be r 20,2014vol.32no.273059-3067；Johnson等人,(2015)Imaging for staging and response assessment in lymphoma.Radiology 2:323–338)。Lugano标准包括通过成像进行评估、肿瘤体积测量以及脾、肝和骨髓侵犯的评估。
在一些方面，使用Lugano标准评估的应答包括使用正电子发射断层扫描(PET)计算机断层扫描(CT)和/或CT(视情况而定)以用于成像评估。PET-CT评估可进一步包括使用氟脱氧葡萄糖(FDG)来评估在嗜FDG淋巴瘤中的FDG吸收。嗜FDG淋巴瘤包括霍奇金淋巴瘤(HL)和某些非霍奇金淋巴瘤(NHL)，其包括弥漫性大B细胞淋巴瘤(DLBCL)、具有侵袭性转化的边缘区NHL和嗜FDG淋巴结淋巴瘤(基本上所有组织学类型除外：慢性淋巴细胞白血病(CLL)、小淋巴细胞淋巴瘤、淋巴浆细胞淋巴瘤/巨球蛋白血症和蕈样真菌病)。在一些情况下，对于非嗜FDG组织学，CT是优选的成像方法。在一些方面，在给予治疗后尽可能长时间地进行治疗后扫描。在一些方面，在治疗后进行最少3周的治疗后扫描，如在给予治疗后进行3周、4周、5周、6周、7周、8周、9周、10周、11周、12周或更长时间的治疗后扫描。
在一些方面，在PET-CT将用于评估嗜FDG组织学中的应答的情况下，5分量表如Deauville五分量表(Deauville 5ps)可用于评估或分期。Dauville得分基于与两个参考器官即纵膈膜(即血池)和肝相比，对每个病变通过PET/CT扫描可见的氟脱氧葡萄糖(FDG)吸收的目视判读。在治疗之前进行一次评估(初始分期)，并且在治疗期间和/或之后使用第二轮FDG PET/CT扫描来评估残余质量(与参考器官中的FDG更新相比)。量表范围为1到5，其中1为最佳且5为最差。每个嗜FDG(或先前嗜FDG)的病变都是独立评定的。在一些方面，5分量表包括以下标准：1，无高于背景的吸收；2，吸收≤纵隔膜；3，吸收>纵膈膜但≤肝；4，中等吸收>肝；5，吸收明显高于肝(例如，最大标准吸收值(SUVMAX>2个肝；5a)和/或可能与淋巴瘤有关的新病变(基于应答评估)(5b)；X，不大可能与淋巴瘤有关的新的吸收区域。
Deauville得分为1或2被认为表示治疗中期和结束时的完全代谢应答(CMR)。
Deauville得分为3也表示CMR，但得分3的解释取决于评估的时间、临床背景和治疗。中期的Deauville得分4或5被认为表示部分代谢应答。然而，如果吸收从基线降低，则在治疗结束时Deauville得分4或5表示残留代谢疾病；如果从基线的吸收没有变化，则表示无代谢应答(NMR)；并且如果从基线的吸收增加和/或有新的病变，则表示进行性代谢疾病(PMD)。在治疗的中期和结束时，NMR或PMD表示治疗失败。
在一些方面，如使用Lugano标准所描述的，在治疗结束时的完全应答包括在各种可测量部位处的完全代谢应答和完全放射应答。在一些方面，这些部位包括淋巴结和淋巴外 部位，其中当使用PET-CT时，在5分量表上CR被描述为得分为1、2或3，其具有或不具有残余质量。在一些方面，在具有高生理吸收或在脾或骨髓内激活(例如，用化学疗法或骨髓集落刺激因子)的Waldeyer环或结外部位中，吸收可能大于正常纵膈膜和/或肝。在这种情况下，如果初始侵犯部位的吸收不大于周围正常组织，即使组织具有高生理吸收，也可以推断完全代谢应答。在一些方面，使用CT在淋巴结中评估应答，其中CR被描述为没有患病的淋巴外部位，并且靶淋巴结/淋巴结肿块的病变的最长横径(LDi)必须恢复至≤1.5cm。其他评估部位包括骨髓，其中基于PET-CT的评估应表明在骨髓中缺乏嗜FDG疾病的证据，并且基于CT的评估应表明正常形态，如果不确定则应该是IHC阴性。其他部位可能包括器官增大的评估，其应该恢复正常。在一些方面，评估未测量的病变和新病变，其在CR的情况下应该不存在。(Cheson等人,JCO September 20,2014vol.32no.273059-3067；Johnson等人,(2015)Imaging for staging and response assessment in lymphoma.Radiology 2:323–338)。
C.毒性
在一些实施方案中，该方法不引起或降低毒性或毒性结果(如细胞因子释放综合征(CRS)、严重CRS(sCRS)、巨噬细胞激活综合征、肿瘤溶解综合征、至少38摄氏度或至少约38摄氏度发热三天或更多天以及至少20mg/dL或至少约20mg/dL的血浆CRP水平)、神经毒性和/或神经毒性的可能性。在一些实施方案中，根据该方法治疗的受试者中的至少50％(例如，所治疗的受试者中的至少60％、至少70％、至少80％、至少90％或更多)没有展现出毒性结果(例如CRS或神经毒性)或未展现出严重的毒性结果(例如严重的CRS或严重的神经毒性)。在一些实施方案中，受试者未展现出3级或更高的神经毒性和/或未展现出严重的CRS，或者在治疗后的一定时间段内(如在给予细胞的一周、两周或一个月内)没有展现出上述情况。
给予过继T细胞疗法如用表达嵌合抗原受体的T细胞进行的治疗可以诱导毒性作用或结果，如细胞因子释放综合征和神经毒性。在一些例子中，这种效果或结果与高水平的循环细胞因子并行，高水平的循环细胞因子可能是观察到的毒性的基础。
在一些方面，毒性结果是细胞因子释放综合征(CRS)或严重CRS(sCRS)或与细胞因子释放综合征(CRS)或严重CRS(sCRS)相关或指示细胞因子释放综合征(CRS)或严重CRS(sCRS)。通常，CRS由例如通过T细胞、B细胞、NK细胞、单核细胞和/或巨噬细胞介导的过度的全身免疫应答引起。这种细胞可释放大量炎症介质如细胞因子和趋化因子。细胞因子可引发急性炎症应答和/或诱导内皮器官损伤，该内皮器官损伤可能导致微血管渗漏、心力衰竭或死亡。严重危及生命的CRS可导致肺浸润和肺损伤、肾衰竭或弥散性血管内凝血。其他严重危及生命的毒性可包括心脏毒性、呼吸窘迫、神经毒性和/或肝衰竭。
可以使用抗炎疗法如抗IL-6疗法(例如抗IL-6抗体，例如托珠单抗或抗生素)治疗CRS。CRS的结果、体征和症状是已知的，并且包括本文所述的那些。在一些实施方案 中，当特定剂量方案或给药影响或不影响给定的CRS相关的结果、体征或症状时，可指定特定结果、体征和症状和/或量或程度。
在给予CAR表达细胞的情况下，CRS通常在输注表达该CAR的细胞后6-20天发生。在一些情况下，CRS在CAR T细胞输注后少于6天或超过20天发生。CRS的发生率和时间可能与输注时的基线细胞因子水平或肿瘤负荷有关。通常，CRS包括干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α和/或白细胞介素(IL)-2的血清水平升高。可在CRS中快速诱导的其他细胞因子是IL-1β、IL-6、IL-8和IL-10。
与CRS相关的示例性结果包括发热、寒颤、发冷、低血压、呼吸困难、急性呼吸窘迫综合征(ARDS)、脑病、ALT/AST升高、肾衰竭、心脏病、缺氧、神经紊乱和死亡。神经系统并发症包括谵妄、癫痫样活动、意识模糊、找词困难、失语和/或变得迟钝。与CRS相关的其他结果包括疲劳、恶心、头痛、癫痫、心跳过速、肌痛、皮疹、急性血管渗漏综合征、肝功能损害和肾衰竭。在一些方面，CRS与一种或多种因子(如血清铁蛋白、d-二聚体、氨基转移酶、乳酸脱氢酶和甘油三酯)的增加相关，或与低纤维蛋白原血或肝脾肿大相关。
在一些实施方案中，与CRS相关的结果包括以下一种或多种：持续发热例如指定温度的发热(例如大于38摄氏度或大于约38摄氏度)两天或更多天，例如三天或更多天，例如四天或更多天或至少连续三天；大于38摄氏度或大于约38摄氏度的发热；与至少两种细胞因子(例如，下组中的至少两种，该组由以下各项组成：干扰素γ(IFNγ)、GM-CSF、IL-6、IL-10、Flt-3L、fracktalkine和IL-5和/或肿瘤坏死因子α(TNFα))的治疗前水平相比细胞因子升高，如最大倍数变化例如至少75倍或至少约75倍，或至少一种这样的细胞因子的最大倍数变化例如至少250倍或至少约250倍；和/或至少一种毒性临床体征如低血压(例如，如通过至少一种静脉内血管活性加压器测量的)；缺氧(例如，血浆氧(PO2)水平低于90％或低于约90％)；和/或一种或多种神经病症(包括精神状态改变、迟钝和癫痫)。
示例性CRS相关结果包括一种或多种因子的增加的或高血清水平，该一种或多种因子包括细胞因子和趋化因子以及与CRS相关的其他因子。示例性结果进一步包括一种或多种此类因子的合成或分泌的增加。这种合成或分泌可以通过T细胞或与T细胞相互作用的细胞(如先天免疫细胞或B细胞)。
在一些实施方案中，CRS相关的血清因子或CRS相关的结果包括炎性细胞因子和/或趋化因子，该炎性细胞因子和/或趋化因子包括干扰素γ(IFN-γ)、TNF-a、IL-1β、IL-2、IL-6、IL-7、IL-8、IL-10、IL-12、sIL-2Ra、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞炎性蛋白(MIP)-1、肿瘤坏死因子α(TNFα)、IL-6和IL-10、IL-1β、IL-8、IL-2、MIP-1、Flt-3L、fracktalkine和/或IL-5。在一些实施方案中，该因子或结果包括C反应蛋白(CRP)。除了作为CRS的早期且易于测量的风险因子外，CRP也是细胞扩增的标志物。在一些实施方案中，测量具有高水平CRP(如≥15mg/dL)的受试者患有CRS。在一些实施方案中，测量具有高水平CRP的受试者不具有CRS。在一些实施方案中，CRS的量度包括CRP 的量度和指示CRS的另一因子。
在一些实施方案中，在CAR治疗之前、期间或之后监测一种或多种炎性细胞因子或趋化因子。在一些方面，一种或多种细胞因子或趋化因子包括IFN-γ、TNF-α、IL-2、IL-1β、IL-6、IL-7、IL-8、IL-10、IL-12、sIL-2Rα、粒细胞巨噬细胞集落刺激因子(GM-CSF)或巨噬细胞炎性蛋白(MIP)。在一些实施方案中，监测IFN-γ、TNF-α和IL-6。
关于CRS的诊断和管理的其他指南是已知的(参见例如Lee等人,Blood.2014；124(2):188-95)。如本文所用，受试者被认为响应于或继发于细胞疗法或其一剂细胞的给予而产生“严重CRS”(“sCRS”)，条件是给药后该受试者显示：(1)至少38摄氏度的发热至少三天；(2)细胞因子升高，其包括(a)与紧跟着给药后的水平相比，下列七种细胞因子中至少两种的最大倍数变化为至少75倍：干扰素γ(IFNγ)、GM-CSF、IL-6、IL-10、Flt-3L、fracktalkine和IL-5和/或(b)与紧跟着给药后的水平相比，下列七种细胞因子中的至少一种的最大倍数变化为至少250倍：干扰素γ(IFNγ)、GM-CSF、IL-6、IL-10、Flt-3L、fracktalkine和IL-5；(c)至少一种毒性临床症状如低血压(需要至少一次静脉血管活性加压药)或缺氧(PO2<90％)或一种或多种神经病症(包括精神状态改变、迟钝和/或或癫痫)。在一些实施方案中，严重CRS包括3级或更高等级的CRS。
在一些实施方案中，CRS包括以下项的组合：(1)持续发热(至少38摄氏度的发热至少三天)和(2)CRP的血清水平为至少20mg/dL或至少约20mg/dL。
在一些实施方案中，CRS包括需要使用两种或更多种血管加压药的低血压或需要机械通气的呼吸衰竭。
在一些方面，毒性结果是神经毒性或与神经毒性相关。在一些实施方案中，与神经毒性的临床风险相关的症状包括意识模糊、谵妄、表达性失语、迟钝、肌阵挛、嗜睡、精神状态改变、惊厥、癫痫样活动、癫痫(任选地通过脑电图[EEG]证实)、升高的β淀粉样蛋白(Aβ)水平，升高的谷氨酸水平和升高的氧自由基水平。在一些实施方案中，基于严重程度对神经毒性进行分级(例如，使用1-5级量表(参见例如Guido Cavaletti&Paola MarmiroliNature Reviews Neurology 6,657-666(December 2010)；美国国家癌症研究所—常见毒性标准第4.03版(NCI-CTCAE v4.03))。
在一些实施方案中，与其他方法相比，该方法减少了与神经毒性相关的症状。例如，与通过其他方法治疗的受试者相比，根据本方法治疗的受试者可能具有减轻的神经毒性症状如四肢无力或麻木、丧失记忆、视力和/或智力、无法控制的强迫性和/或强制性行为、妄想、头痛、认知和行为问题(包括丧失运动控制、认知恶化和自主神经系统功能障碍以及性功能障碍)。在一些实施方案中，根据本发明方法治疗的受试者可具有与外周运动神经病、外周感觉神经病、感觉迟钝、神经痛或感觉异常相关的症状减轻。
在一些实施方案中，该方法减轻与神经毒性相关的结果，包括对神经系统和/或脑的损害，如神经元的死亡。在一些方面，该方法降低与神经毒性相关的因子的水平，如β淀粉样蛋白(Aβ)、谷氨酸和氧自由基。
II.细胞表达的重组抗原受体
在一些实施方案中，用于所提供的方法或与所提供的方法一起给予的细胞含有或经工程化以含有工程化受体例如工程化抗原受体如嵌合抗原受体(CAR)或T细胞受体(TCR)。还提供了此类细胞群、含有此类细胞和/或富含此类细胞的组合物，如其中富集或选择某种类型的细胞如T细胞或CD8+或CD4+细胞。在这些组合物中有用于给予(如过继细胞疗法)的药物组合物和制剂。还提供了根据所提供的方法将该细胞和组合物给予受试者例如患者的治疗方法。
在一些实施方案中，该细胞包括经由基因工程引入的一种或多种核酸，从而表达此类核酸的重组或基因工程化产物。在一些实施方案中，通过首先刺激细胞来完成基因转移，如通过将细胞与诱导例如通过细胞因子或激活标志物的表达所测量的应答(如增殖、存活和/或激活)的刺激组合，随后转导激活的细胞，并在培养物中扩增至足以用于临床应用的数量。
细胞通常表达重组受体如抗原受体(包括功能性非TCR抗原受体，例如嵌合抗原受体(CAR))和其他抗原结合受体如转基因T细胞受体(TCR)。受体中还有其他嵌合受体。
A.嵌合抗原受体(CAR)
所述的免疫效应细胞表达有识别CD19或其变体的嵌合抗原受体，嵌合抗原受体(CAR)通常包括细胞外抗原结合结构域，例如抗体分子的一部分，通常是抗体的可变重(VH)链区和/或可变轻(VL)链区，例如，scFv抗体片段。
术语“CD19”包括但不限于人CD19的变体、同种型和物种同源物。在某些情况下，本发明的CD19-CAR可识别人以外的物种的CD19。示例性的人CD19的完整氨基酸序列具有SwissPort登录号P15391(SEQ ID NO：31)。CD19也被称为B细胞表面抗原B4，B细胞抗原CD19、CD19抗原或Leu-12。人CD19被Entrez Gene称为Gene ID：930，被HGNC称为HGNC：1633。CD19可以被称为CD19的基因编码。本文中“人CD19”的使用涵盖了人CD19的所有已知的或仍未被发现的等位基因和多态形式。
在一些实施方式中，所述抗体的重链可变区和轻链可变区具有：SEQ ID NO:5所示的HCDR1、SEQ ID NO:6所示的HCDR2、SEQ ID NO:7所示的HCDR3，以及SEQ ID NO:2所示的LCDR1、SEQ ID NO:3所示的LCDR2、SEQ ID NO:4所示的LCDR3，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列；在一优选方案中，所述抗体的重链可变区和轻链可变区具有SEQ ID NO：8以及SEQ ID NO:9所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列；在另一优选方案中，所述抗体的重链可变区和轻链可变区具有SEQ ID NO：11以及SEQ ID NO:12所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、 92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。
在一些实施方式中，所述抗体具有SEQ ID NO：1或10所示的scFv的序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。
在一些实施方式中，所述的嵌合抗原受体具有SEQ ID NO：13、14、15、16、17、18、19或20所示氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。
在一些实施方式中，嵌合抗原受体(例如CAR)的抗体部分还包括连接序列，其可以是或包括免疫球蛋白恒定区或其变体或其修饰形式的至少一部分，例如铰链区，例如，IgG4铰链区和/或CH1/CL和/或Fc区和/或CD8铰链区。在一些实施方式中，所述恒定区或部分是人IgG的，例如IgG4或IgG1的。在一些实施例中给，所述CD8铰链区具有SEQ ID NO：21所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。
该抗原识别结构域一般连接至一个或多个胞内信号转导部分，例如在CAR的情况中，通过抗原受体复合物(例如TCR复合物)模拟活化的信号转导部分，和/或通过另一细胞表面受体的信号。因此，在一些实施方式中，抗原结合组分(例如，抗体)与一个或多个跨膜和细胞内信号转导结构域连接。在一些实施方式中，所述跨膜结构域融合至所述胞外结构域。
在一些实施方式中，所述跨膜结构域源自天然或合成来源。当所述来源是天然来源时，在一些方面中，所述结构域源自任何膜结合或跨膜蛋白。跨膜区包括源自T-细胞受体的α、β或ζ链，CD28，CD3ε，CD45，CD4，CD5，CD8，CD9，CD 16，CD22，CD33，CD37，CD64，CD80，CD86，CD 134，CD137，CD 154，和/或跨膜区包含其功能变体(例如基本保留其结构部分(例如，跨膜结构部分)、性质的那些)的那些(即包含至少它们的跨膜区域)。在一些实施方式中，所述跨膜结构域是源自CD4、CD28或CD8的跨膜结构域，例如，CD8α或其功能变体。或者，在一些实施方式中，所述跨膜结构域是合成的。在一些方面中，所述合成跨膜结构域主要包含疏水残基例如亮氨酸和缬氨酸。在一些实施例中，所述CD8跨膜结构域具有SEQ ID NO：22所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。在一些实施例中，所述CD28跨膜结构域具有SEQ ID NO：23所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。
所述受体，例如，CAR，一般包括至少一种的一个或多个胞内信号转导部分。在 一些实施方式中，所述受体包括TCR复合物的胞内组分，例如介导T-细胞活化和细胞毒性的TCRCD3+链，例如，CD3ζ链。因此，在一些方面，抗原结合部分连接到一个或多个细胞信号转导模块。在一些实施方式中，细胞信号转导模块包括CD3跨膜结构域、CD3胞内信号转导结构域，和/或其它CD跨膜结构域。在一些实施方式中，所述受体，例如，CAR，还包括一种或多种其它分子的部分，例如Fc受体γ、CD8、CD4、CD25或CD16。例如，在一些方面，CAR或其它嵌合抗原受体包括CD3ζ(CD3-ζ)或Fc受体γ与CD8、CD4、CD25或CD16之间的嵌合分子。
在一些实施方式中，在CAR或其它嵌合抗原受体结合时，受体的细胞质结构域或细胞内信号转导结构域激活免疫细胞的正常效应功能或应答中的至少一种，例如经工程改造以表达CAR的T细胞。例如，在一些情况中，CAR诱导T细胞的功能，例如溶细胞活性或辅助性T细胞活性，例如细胞因子或其它因子的分泌。在一些实施方式中，采用抗原受体部分或共刺激分子的胞内信号转导结构域的截短部分来替代完整免疫刺激链，例如，如果其转导效应物功能信号的话。在一些实施方式中，细胞内信号转导结构域包括T细胞受体(TCR)的胞质序列，并且在一些方面，还包括天然情况下存在的共受体的那些与这些受体一致作用以在抗原受体接合后启动信号转导。
在其他实施方式中，CAR不包括用于生成共刺激信号的组分。在一些方面中，其他CAR在同一细胞中表达并且提供用于生成第二或共刺激信号的组分。
在一些方面中，T细胞活化描述为通过两类胞质信号转导序列介导：通过TCR起始抗原依赖性首活化的那些(首胞质信号转导序列)，和以抗原非依赖性方式作用以提供第二或共刺激信号的那些(第二胞质信号转导序列)。在一些方面中，所述CAR包括此类信号转导组分之一或两者。
在一些实施方式中，嵌合抗原受体(例如CAR)的抗体部分还包括信号肽，例如包括CD8或其变体的信号肽，例如与SEQ ID NO:27显示至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列相同性的氨基酸序列。
在一些方面中，所述CAR包括主要胞质信号转导序列，其调节TCR复合物的初始活化。以刺激方式作用的首胞质信号转导序列可包含信号转导基序，其已知是免疫受体基于酪氨酸的活化基序或ITAM。ITAM的实例包括细胞质信号转导序列，其包括源自如下的那些：TCRζ，FcRγ，FcRβ，CD3γ，CD3δ，CD3ε，CDS，CD22，CD79a，CD79b，和CD66d。在一些实施方式中，CAR中的胞质信号转导分子包含胞质信号转导结构域，其部分或源自CD3ζ的序列。
在一些实施方式中，所述CAR包括共刺激受体的跨膜部分和/或信号转导结构域，例如CD28，CD137，OX40，DAP10，和ICOS。在一些方面中，同一CAR同时包括活化和共刺激部分。
在一些实施方式中，激活结构域包括在一个CAR内，而共刺激组分由识别另一 种抗原的另一CAR提供。在一些实施方式中，所述CAR包括活化或刺激CAR、共刺激CAR，其均表达在同一细胞上(参见WO2014/055668)。在一些方面，细胞包括一种或多种刺激或活化CAR和/或共刺激C A R。在一些实施方式中，细胞还包括抑制性CAR(iCAR，参见Fedorov等，Sci.Transl.Medicine,5(215)(2013年12月))，例如识别除了与疾病或病症相关和/或特异性的CAR，由此通过所述疾病靶向CAR递送的激活信号通过抑制性CAR与其配体结合而减少或抑制，例如减少脱靶效应。
在一些实施方式中，所述嵌合抗原受体的胞内信号转导部分，例如CAR，包含CD3ζ胞内结构域和共刺激信号转导区。在某些实施方式中，所述胞内信号转导结构域包含CD28跨膜和信号转导结构域，其连接至CD3(例如，CD3-ζ)胞内结构域。在一些实施方式中，所述胞内信号转导结构域包含嵌合CD28和/或CD137(4-1BB、TNFRSF9)共刺激结构域，其连接至CD3ζ胞内结构域。在一些实施例中，所述CD28共刺激结构域具有SEQ ID NO：24所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。在一些实施例中，所述CD137共刺激结构域具有SEQ ID NO：25所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。在一些实施例中，所述CD3ζ胞内结构域具有SEQ ID NO：26所示的氨基酸序列，或者与上述序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同性的氨基酸序列。
在一些实施方式中，所述CAR涵盖一个或多个，例如，两个或更多个，共刺激结构域和活化结构域，例如，胞质部分中的初始活化结构域。示例性的CAR包括抗CD19的scfv、CD28跨膜区、CD137和CD3ζ的胞内结构域。
在一些情况中，CAR被称为第一、第二和/或第三代CAR。在一些方面，第一代CAR是在抗原结合时仅提供CD3链诱导信号的CAR；在一些方面，第二代CAR是提供这样的信号和共刺激信号的CAR，例如包括来自共刺激受体(例如CD28或CD137)的细胞内信号转导结构域的CAR；在一些方面，第三代CAR是包括不同共刺激受体的多个共刺激结构域的CAR。
在一些实施方式中，嵌合抗原受体包括含有抗体或抗体片段的细胞外部分。在一些方面，嵌合抗原受体包括含有抗体或片段的胞外部分和细胞内信号转导结构域。在一些实施方式中，所述抗体或片段包括scFv，且所述胞内结构域包含ITAM。在一些方面中，所述胞内信号转导结构域包括CD3-ζ链的ζ链的信号转导结构域。在一些实施方式中，所述嵌合抗原受体包括跨膜结构域，其连接胞外结构域和胞内信号转导结构域。在一些方面中，所述跨膜结构域包含CD28的跨膜部分。在一些实施方式中，嵌合抗原受体含有T细胞共刺激分子的细胞内结构域。胞外结构域和跨膜结构域可直接或间接相连。在一些实施方式中，所述胞外结构域和跨膜通过连接序列。在一些实 施方式中，所述受体包含作为跨膜结构域的衍生来源的分子的胞外部分，例如CD28胞外部分。在一些实施方式中，所述嵌合抗原受体包含源自T细胞共刺激分子或其功能变体的胞内结构域，例如在跨膜结构域和胞内信号转导结构域之间。在一些方面中，T细胞共刺激分子是CD28或41BB。例如，在一些实施方式中，CAR含有抗体，例如抗体片段，含有CD28的跨膜部分或其功能变体的跨膜结构域，以及含有CD28的信号转导部分或功能性变体的细胞内信号转导结构域，和CD3ζ的信号转导部分或其功能变体。在一些实施方式中，CAR含有抗体，例如抗体片段，含有CD28的跨膜部分或其功能变体的跨膜结构域，以及含有CD137的信号转导部分或功能变体的细胞内信号转导结构域，以及CD3ζ的信号转导部分或其功能变体。在一些所述实施方式中，所述受体还包括连接序列，其包含Ig分子(例如人Ig分子)的部分，例如Ig铰链，例如IgG4铰链，例如仅铰链连接序列。在一些实施方式中，所述的嵌合抗原受体具有：(i)特异性识别肿瘤抗原的抗体、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；或(ii)特异性识别肿瘤抗原的抗体、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；或(iii)特异性识别肿瘤抗原的抗体、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
例如，在一些实施方式中，CAR包括抗体，例如抗体片段，包括scFv，连接序列，例如含有免疫球蛋白分子的一部分的连接序列，例如铰链区和/或一个或多个重链分子恒定区，例如含有Ig-铰链的连接序列，含有全部或部分CD28衍生的跨膜结构域的跨膜结构域，CD28衍生的细胞内信号结构域和CD3ζ信号转导结构域。在一些实施方式中，CAR包括抗体或片段，例如scFv，连接序列，例如任何含有Ig-铰链的连接序列，CD28衍生的跨膜结构域，CD137衍生的细胞内信号转导结构域和CD3ζ衍生信号转导结构域。
在一些实施方式中，本文所述的方法包括向所述受试者给予一种过继细胞或者免疫效应细胞。在一些实施方式中，本文所述的方法包括在不同的疗程中向所述受试者给予针对相同的肿瘤抗原的两种或者更多种过继细胞或者免疫效应细胞。在一些实施方式中，本文所述的方法包括在不同的疗程中向所述受试者给予针对不同的肿瘤抗原的两种或者更多种过继细胞或者免疫效应细胞。在一些实施方式中，本文所述的方法包括在不同的疗程中向所述受试者给予针对相同的肿瘤抗原的相同表位的两种或者更多种过继细胞或者免疫效应细胞。在一些实施方式中，本文所述的方法包括在不同的疗程中向所述受试者给予针对相同的肿瘤抗原的不同表位的两种或者更多种过继细胞或者免疫效应细胞。在一些实施方式中，本文所述的方法包括在不同的疗程中向所述受试者给予两种或者更多种过继细胞或者免疫效应细胞以治疗相同部位的肿瘤。在一些实施方式中，本文所述的方法包括在不同的疗程中向所述受试者给予两种或者更多种过继细胞或者免疫效应细胞以治疗不同部位的肿瘤。在一些实施方式中，本文所述的方法所采用的过继细胞或者免疫效应细胞中的至少一种靶向CD19的CAR-T 细胞。在一些实施方式中，本文所述的方法所采用的过继细胞或者免疫效应细胞中的至少一种是本文所述的靶向CD19的CAR-T细胞。在上述情况下，本领域技术人员，例如临床医师可以根据在先治疗的情况决定本发明的CD19-CAR-T细胞的给予次数以及剂量。
B.TCR
在一些实施方案中，基因工程化的抗原受体包括从天然存在的T细胞克隆的重组T细胞受体(TCR)和/或TCR。在一些实施方案中，鉴定针对靶抗原(例如癌抗原)的高亲和力T细胞克隆，从患者分离并引入细胞中。在一些实施方案中，已经在用人免疫系统基因(例如人白细胞抗原系统或HLA)工程化的转基因小鼠中生成了针对靶抗原的TCR克隆。
III.免疫效应细胞
通过本文所述的方法提供的细胞是表达嵌合抗原受体的免疫效应细胞。所述细胞一般是哺乳动物细胞，并且通常是人细胞。在一些实施方式中，细胞衍生自血液、骨髓、淋巴或淋巴器官，是免疫系统的细胞，如先天或适应性免疫的细胞，例如，骨髓或淋巴样细胞，包括淋巴细胞，一般是T细胞和/或NK细胞。其他示例性的细胞包括干细胞，如多能性(multipotent)和亚全能性(pluripotent)干细胞，包括诱导性多能干细胞(iPSC)。细胞一般是原代细胞，如直接从受试者分离和/或从受试者分离并冷冻的那些细胞。在一些实施方式中，细胞包括T细胞或其他细胞类型的一个或多个亚组，如全T细胞群、CD4+细胞、CD8+细胞及其亚群，如由功能、活化状态、成熟、分化潜力、扩增、再循环、定位，和/或持久能力、抗原特异性、抗原受体类型、特定器官或隔室中的存在、标志物或细胞因子分泌概况，和/或分化程度限定的那些。关于待治疗的受试者，细胞可以是同种异体和/或自体的。方法包括现成的方法。在一些方面中，如对于现成的技术，细胞是多能和/或专能的，如干细胞，如诱导性多能干细胞(iPSC)。在一些实施方式中，该方法包括从受试者分离细胞，对其进行制备、处理、培养和/或工程改造，并且在冷冻保存前或后将其再导入同一患者。
T细胞和/或CD4+和/或CD8+T细胞的亚型和亚群包括：原初T(TN)细胞、效应T细胞(TEFF)、记忆T细胞及其亚型，例如干细胞记忆T(TSCM)、中心记忆T(TCM)、效应记忆T(TEM)、或最终分化的效应记忆T细胞、肿瘤-浸润性淋巴细胞(TIL)、不成熟T细胞、成熟T细胞、辅助性T细胞、细胞毒性T细胞、粘膜相关的非变体T(MAIT)细胞、天然产生的和过继性调节T(Treg)细胞、辅助性T细胞，例如TH1细胞、TH2细胞、TH3细胞、TH17细胞、TH9细胞、TH22细胞、滤泡辅助性T细胞、α/βT细胞，和δ/γT细胞。
在一些实施方式中，所述细胞是自然杀伤(NK)细胞。在一些实施方式中，所述细胞是单核细胞或粒细胞，例如，骨髓细胞、巨噬细胞、嗜中性粒细胞、树突细胞、肥大细胞、嗜酸性粒细胞，和/或嗜碱性粒细胞。
在一些实施方式中，所述细胞包括通过遗传工程改造导入的一种或多种核酸，并 由此表达所述核酸的重组或经遗传工程改造的产物。在一些实施方式中，核酸是异源性的，即，通常不存在于细胞或从该细胞获得的样品中，如从另一个生物体或细胞获得的样品，其例如通常不在工程改造中的细胞和/或这类细胞来源的生物体中发现。在一些实施方式中，核酸不是天然存在的，如核酸不是自然中发现的，包括包含编码来自多种不同细胞类型的各种结构域的核酸的嵌合组合。
IV.用于遗传工程改造的方法和载体
本发明还提供了用于产生表达嵌合抗原受体的遗传工程改造的细胞的方法、组合物和试剂盒。遗传工程改造一般涉及将编码所述重组或经工程改造的部分的核酸导入细胞，如通过病毒转导、电转等导入细胞。
在一些实施方式中，基因转移通过如下方式进行：首先，刺激细胞，例如，通过将其与刺激物合并，所述刺激物诱导响应，例如增殖、存活和/或活化，例如，通过细胞因子或活化标志物的表达来检测，然后转导该活化的细胞，并且在培养物中扩增至足以供于临床应用的数量。
在一些方面中，细胞还经工程改造以促进细胞因子或其他因子的表达。用于引入遗传工程改造的组分，例如，抗原受体(例如，CAR)的各种方法是熟知的，并可采用本文提供的方法和组合物。示例性方法包括用于转移编码受体的核酸的那些，包括通过病毒，例如，逆转录病毒或慢病毒，转导，转座子，和电穿孔。
在一些实施方式中，采用重组感染性病毒颗粒将重组核酸转移进入细胞，例如，源自猿病毒40(SV40)、腺病毒、腺相关病毒(AAV)的载体。在一些实施方式中，采用重组慢病毒载体或逆转录病毒载体，例如γ-逆转录病毒载体，将重组核酸转移进入T细胞。
A.免疫效应细胞的制备
在一些实施方式中，经工程改造的细胞的制备包括一种或多种培养和/或一个或多个制备步骤。用于导入编码转基因受体(例如，CAR)的核酸的细胞可从样品(例如生物样品，例如获自或源自受试者的样品)分离。在一些实施方式中，从中分离细胞的受试者是患有一定疾病或病症或需要细胞治疗或将给予细胞治疗的受试者。在一些实施方式中，受试者是需要特定治疗性介入的人，例如，需要过继细胞疗法或效应细胞疗法的人，用于该疗法的细胞是经分离、加工和/或经工程改造的。在一些实施方式中，所述细胞是原代细胞，例如，原代人细胞。所述样品包括直接取自受试者的组织、体液和其它样品，以及获自一个或多个处理步骤，例如分离、离心、遗传工程改造(例如采用病毒载体的转导)、清洗和/或孵育的样品。所述生物样品可以是直接获自生物来源的样品或经处理的样品。生物样品包括但不限于，体液，例如血液、血浆、血清、脑脊髓液、滑膜液、尿液和汗液，组织和器官样品，包括源自它们的经处理的样品。
示例性样品包括全血、外周血单核细胞(PBMC)、白细胞、骨髓、胸腺、组织活检物、肿瘤、白血病、淋巴瘤、淋巴结、肠相关的淋巴样组织、粘膜相关的淋巴样组 织、脾、其它淋巴样组织、肝、肺、胃、肠、结肠、肾、胰腺、乳腺、骨、前列腺、子宫颈、睾丸、卵巢、扁桃体或其它器官，和/或源自其中的细胞。在细胞治疗(例如，过继细胞治疗或免疫效应细胞治疗)的情况中，样品包括来自自体和同种异体来源的样品。
在一些实施方式中，细胞的分离包括一个或多个制备和/或不基于亲和性的细胞分离步骤。在一些实例中，细胞在一种或多种物质的存在下经清洗、离心和/或孵育，例如，以移除不需要的组分，富集所需组分，裂解或移除对具体物质敏感的细胞。在一些实例中，细胞基于一种或多种性质，例如密度、粘附性质、尺寸、对具体组分的敏感性和/或抗性被分离。在一些实例中，例如，通过单采或白细胞去除术，获得来自受试者循环血液的细胞。在一些方面中，所述样品包括淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其它有核血液白细胞、血红细胞，和/或血小板。
在一些实施方式中，从所述受试者收集的血液细胞经清洗，例如，以移除血浆部分，并将该细胞置于合适的缓冲液或培养基中以供后续处理步骤。在一些实施方式中，所述细胞用磷酸盐缓冲盐水(PBS)清洗。在一些实施方式中，清洗溶液缺乏钙和/或镁和/或许多或全部二价阳离子。在一些方面中，清洗步骤按照生产商说明，通过半自动化的“流通”离心法(例如，COBE 2991细胞处理器，百特公司(BaXter))完成。在一些方面中，清洗步骤按照生产商说明，通过内切流过滤(TFF)完成。在一些实施方式中，在清洗后，所述细胞在多种生物相容缓冲液中重悬，例如，无Ca++/Mg++PBS。在某些实施方式中，移除血液细胞样品组分，并将细胞直接重悬于培养基中。
在一些实施方式中，所述方法包括基于密度的细胞分离方法，例如，通过裂解血红细胞或者不裂解红细胞并通过Percoll或Ficoll梯度离心外周血或者单采样品或白细胞去除术样品制备获得外周血单个核细胞(PBMC)。
在一些实施方式中，所述分离方法包括，基于细胞中一种或多种特定分子，例如表面标志物，例如，表面蛋白质、胞内标志物或核酸的表达或存在来分离不同的细胞类型。在一些实施方式中，可采用基于此类标志物进行分离的任何已知方法。在一些实施方式中，所述分离是基于亲和性或基于免疫亲和性的分离。例如，在一些方面中，所述分离包括基于细胞的一种或多种标志物(通常是细胞表面标志物)的表达或表达水平来分离细胞和细胞群，例如，通过与特异性地结合至此类标志物的抗体或结合伴侣孵育，随后通常是清洗步骤，和从尚未结合至所述抗体或结合伴侣的那些细胞分离已结合所述抗体或结合伴侣的细胞。
此类分离步骤可基于正选择，其中已结合所述试剂的细胞被保留用于进一步应用，和/或，负选择，其中尚未结合至所述抗体或结合伴侣的细胞被保留。
例如，在一些方面中，T细胞的特定亚群，例如一种或多种表面标志物阳性细胞或表达高水平的一种或多种表面标志物的细胞，例如，CD3+、CD28+、CD62L+、CCR7+、CD27+、CD127+、CD4+、CD8+、CD45RA+和/或CD45RO+T细胞，通过 正或负选择技术分离。
例如，CD3+、CD28+T细胞可以采用CD3/CD28连接的磁珠(例如，DYNA珠M-450CD3/CD28T细胞扩增器)来正选择。
在一些实施方式中，T细胞通过对在非T细胞(例如B细胞、单核细胞或其它血液白细胞)上表达的标志物(例如CD14)进行负选择来从PBMC样品分离分离。在一些方面中，使用CD4+或CD8+选择步骤来分离辅助性CD4+和CD8+细胞毒性T细胞。通过针对一种或多种原初、记忆和/或效应T细胞亚群上表达或以相对较高的程度表达的标志物进行正或负选择，可将此类CD4+和CD8+群进一步分选成亚群。
在一个实例中，为了通过负选择对CD4+细胞进行富集，单克隆抗体混合物通常包括针对CD14、CD20、CD11b、CD16、HLA-DR，和CD8的抗体。在一些实施方式中，使所述抗体或结合伴侣结合至固体支持物或基质，例如磁珠或顺磁珠，以允许分离用于正和/或负选择的细胞。
在一些实施方式中，制备方法包括：冷冻步骤，例如，在分离、孵育和/或工程改造之前或之后，冻存所述细胞。在一些实施方式中，所述冷冻和后续融化步骤移除粒细胞，并且，在某种程度上，移除细胞群中的单核细胞。在一些实施方式中，例如，在清洗步骤以移除血浆和血小板之后，将所述细胞悬浮于冷冻溶液中。在一些方面中，可采用任何各种已知冷冻溶液和参数。一个实例涉及采用包含20％DMSO和8％人血清白蛋白(HAS)的PBS或其它合适的细胞冷冻培养基。然后，其用培养基1:1稀释，从而DMSO和HSA的终浓度分别是10％和4％。然后，一般以程控降温装置按照既定的程序或者原理如1°/分钟的速率将细胞冷冻至-80℃或者-90℃，并贮存在液氮储罐的汽相中。
B.组合物和制剂
本发明还提供了包括用于给药的细胞的组合物，包括药物组合物和制剂，如来自包含给定剂量或其部分的用于给药的细胞数量的单位剂型组合物。所述药物组合物和制剂一般包括一种或多种任选的药学上可接受的运载体或赋形剂。在一些实施方式中，所述组合物包括至少一种其它治疗剂。
术语“药物制剂”指此类形式的制剂：所述制剂允许其中所含活性成分的生物学活性有效，并且不含具有待给予该制剂的受试者不可接受的毒性的额外成分。
“药学上可接受的运载体”指，药物制剂中的一种成分，其不是活性成分，其对于受试者无毒性。药学上可接受的运载体包括但是不限于，缓冲剂、赋形剂、稳定剂或防腐剂。
在一些方面中，运载体的选择部分由特定细胞和/或给药方法来确定。因此，存在多种合适的配方。例如，所述药物组合物可包含防腐剂。合适的防腐剂可包括，例如，对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸钠和苯扎氯铵。在一些方面中，采用两种或更多种防腐剂的混合物。防腐剂或其混合物的存在量通常是约0.0001％ 至约2％(以组合物总重量计)。运载体描述于，例如，《雷明顿药物科学》(Remington's Pharmaceutical Sciences)，第16版，Osol,A.编(1980)。在所用剂量和浓度下，药学上可接受的运载体通常是对受者无毒的，包括但不限于：缓冲剂如磷酸盐、柠檬酸盐和其它有机酸缓冲剂；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(如十八烷基二甲基苄基氯化铵；氯化六烃季铵；苯扎氯铵、苄索氯铵；苯酚、丁基或苄基醇；对羟基苯甲酸烷酯，如对羟基苯甲酸甲酯或丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(小于约10个残基)的多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷胺酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其它糖，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖，如蔗糖、甘露醇、海藻糖或山梨糖醇；形成盐的抗衡离子，如钠；金属络合物(如Zn-蛋白质络合物)；和/或非离子型表面活性剂，例如聚乙二醇(PEG)。
在一些方面中，所述组合物包含缓冲剂。合适的缓冲剂包括，例如，柠檬酸、柠檬酸钠、磷酸、磷酸钾和多种其他酸和盐。在一些方面中，采用两种或更多种缓冲剂的混合物。缓冲剂或其混合物的存在量通常是约0.001％至约4％(以组合物总重量计)。用于制备可给予的药物组合物的方法是已知的。
该制剂可包含水溶液。所述制剂或组合物还可包含多于一种活性成分，该活性成分可用于待用所述细胞治疗的特定适应症、疾病或病症，优选对所述细胞具有补充活性的那些，其中相应活性剂彼此不产生负面影响。这类活性成分适合以有效用于所需目的的用量联合存在。因此，在一些实施方式中，所述药物组合物还包含其它药学活性物质或药物，例如化疗剂，例如，天冬酰胺酶，白消安，卡铂，顺铂，柔红霉素，多柔比星，氟尿嘧啶，吉西他滨，羟基脲，甲氨蝶呤，紫杉醇，利妥昔单抗，长春碱，和/或长春新碱。
在一些实施方式中，药物组合物包含有效治疗或预防疾病或病症的量，如治疗有效或预防有效量的细胞。在一些实施方式中，通过定期评估治疗的受试者来监测治疗或预防性功效。所需剂量可以通过单药丸给予所述细胞，通过多药丸给予所述细胞或通过连续输注给予细胞来递送。
在一些实施方式中，该组合物包含有效降低疾病或病症负荷的量，和/或在受试者中不导致CRS或严重CRS的量和/或实现本文所述的方法的任何其他结果的量的细胞。
该细胞和组合物可采用标准给予技术、制剂和/或装置给予。所述细胞的给予可以是自体同源或异源的。例如，免疫抑制细胞或祖细胞可获自一个受试者，并给予相同受试者或不同的相容受试者。外周血衍生的免疫抑制细胞或其后代(例如，体内、离体或体外衍生的)可通过局部注射给予，包括导管给药、全身注射、局部注射、静脉注射、或胃肠外给药。当给予治疗性组合物(例如，含有遗传修饰的免疫抑制细胞 的药物组合物)时，其通常被配制成单位剂型的可注射形式(溶液、悬液、乳液)。
制剂包括用于口服、静脉内、腹膜内、皮下、肺、透皮、肌肉内、鼻内、粘膜、舌下或栓剂给药的那些。在一些实施方式中，所述细胞群胃肠外给予。本文所用的术语“胃肠外”，包括静脉内、肌内、皮下、直肠、阴道和腹膜内给予。在一些实施方式中，所述细胞通过静脉内，腹膜内或皮下注射采用外围全身递送给予受试者。
在一些实施方式中，组合物以无菌液体制剂的形式提供，例如，等渗水性溶液、悬液、乳液、分散体或粘性组合物，其在一些方面中可缓冲至选择的pH。液体制剂通常比凝胶、其他粘性组合物和固体组合物更容易制备。另外，液体组合物多少更便于给药，尤其是通过注射。在另一方面，粘性组合物可在合适的粘度范围内配制以提供与特定组织更长的接触时间。液体或粘性组合物可包括运载体，其可以是溶剂或分散介质，其含有，例如，水、盐水、磷酸盐缓冲盐水、多羟基化合物(例如，甘油、丙二醇、液体聚乙二醇)及其合适混合物。可通过将所述细胞纳入溶剂中，如与合适运载体、稀释剂、或赋形剂如无菌水、生理盐水、葡萄糖、右旋糖等来制备无菌可注射溶液。组合物可含有辅助性物质，如润湿、分散、或乳化剂(例如，甲基纤维素)、pH缓冲剂、胶凝或粘度增强添加剂、防腐剂、风味剂、和/或色素，取决于所需的给药和制备途径。
C.制品
本发明还提供了制品，如试剂盒和装置，用于按照提供的用于过继细胞治疗或免疫效应细胞治疗的方法向受试者给予细胞，并用于储存和给予该细胞和组合物。
制品包括一个或多个容器，一般是多个容器，包装材料，和与一个或多个容器和/或包装结合或其上的标签或包装插页，一般包括向受试者给予细胞的说明。
容器一般含有待给予的细胞，例如，其一个或多个单位剂量。制品一般包括多个容器，各自含有单个单位剂量的细胞。单位剂量可以是以在先疗程的免疫效应细胞的待给予受试者的细胞的量或数量或两倍(或更多)于待以首或在后疗程的免疫效应细胞给予的细胞的数量。其可以是与给药方法相关的给予受试者的细胞的最低剂量或最低可能剂量。在一些实施方式中，单位剂量是将按照本发明的方法以单位剂量给予具有特定疾病或病症的任何受试者或任何受试者的细胞数量或细胞最小数量。例如，在一些方面，单位剂量可包括将给予较低体重和/或较低疾病负荷的受试者的细胞的最小量，如以在先疗程的免疫效应细胞向给定受试者给予一个或在一些情况中超过一个单位剂量并且在一个或多个在后疗程的免疫效应细胞中向给定受试者给予一个或超过一个单位剂量，例如，按照提供的方法。在一些实施方式中，单位剂量中的细胞数是需要以在先疗程的免疫效应细胞给予特定受试者，如细胞衍生的受试者的嵌合抗原受体-表达或CAR-表达的数量或细胞数。在一些实施方式中，细胞衍生自待通过本文提供的方法治疗的受试者。
在一些实施方式中，各容器单独包含单位剂量的细胞，例如，包括相同或基本相 同数量的细胞。因此，在一些实施方式中，各容器包含相同或大约或基本相同数量的细胞或嵌合抗原受体-表达细胞。在一些实施方式中，单位剂量包括小于约1x10 10、小于约1x10 9、小于约1x10 8或小于约1x10 7个工程改造的细胞、总细胞、T细胞或PBMC/千克待治疗和/或细胞衍生的受试者。
合适的容器包括，例如，瓶、小瓶、注射器、和柔性袋如冷冻袋。在特定实施方式中，容器是袋，例如，柔性袋，如适于向受试者输注细胞的那些，例如，柔性塑料或PVC袋或者EVA或者ULPDE，和/或IV溶液袋。在一些实施方式中，袋是可密封和/或能够灭菌的，以提供无菌溶液以及细胞和组合物的递送。在一些实施方式中，容器，例如，袋的容积等于或约或至少或约10、20、30、40、50、60、70、80、90、100、200、300、400、500、或1000ml容积，如等于或约10至等于或约100mL或者等于或约10至等于或约500mL容积。在一些实施方式中，容器，例如，袋是和/或由在一个或多个不同温度下稳定和/或提供细胞的稳定储存和/或维持的材料制成，如在低温下，例如，低于或约或等于或约-20℃、-80℃、-120℃、135℃、-196℃和/或适于冷冻保存的温度，和/或其他温度，如适于冻融细胞的温度和体温，如等于或约37℃或-38℃、或-39℃、或-40℃，以允许在治疗前冻融，例如，在受试者的地点或治疗地点。
制品还可包括包装插页或标签，其一片或多片显示使用信息和/或说明。在一些实施方式中，信息或说明显示可以或应该用于治疗特定疾病或病症的内容，和/或提供其说明。标签或包装插页可显示待用于治疗疾病或病症的制品的内容。在一些实施方式中，标签或包装插页提供了治疗受试者的说明，例如，细胞已衍生的受试者，通过包括给予首和一个或多个在后疗程的免疫效应细胞的细胞，例如，按照提供的方法的实施方式中的任一个的方法。在一些实施方式中，说明指定了在在先疗程的免疫效应细胞中，给予一个单位剂量，例如，制品的单个单独容器的内容物，之后在指定时间点或指定时间窗内和/或检测到受试者中的一个或多个因子或结果的存在或缺失或量或程度之后给予一个或多个在后疗程的免疫效应细胞。
在一些实施方式中，制品包括一个或多个，通常为含有包含细胞的组合物的多个容器，例如其单独的单位剂量形式，并且还包括其中包含组合物的一个或多个其他容器，其该组合物包含其他试剂，例如细胞毒性或其它治疗剂，其例如将与细胞组合，例如，同时或以任何顺序一次给予。或者或此外，所述制品还可包括包含药学上可接受的缓冲剂的另一或相同容器。其还可包括其它材料，例如其它缓冲剂、稀释剂、滤器、管、针头，和/或注射器。
术语“包装插页”指治疗性产品的商品包装中常包括的说明书，所述说明书包含关于这类治疗性产品使用的说明、用法、剂量、给药、联合治疗、禁忌症和/或警告的信息。
本发明的方法可以总结为：
通过基于“去单个核细胞分离术”从患有肿瘤的人类受试者的外周血单个核细胞(PBMC)或者T细胞，并且用编码嵌合抗原受体(CAR)的病毒载体培养和转导细胞，所述嵌合抗原受体(CAR)特异性结合受试者中由肿瘤表达的抗原，其是肿瘤相关或肿瘤特异性抗原，例如CD19。细胞在单独的柔性冷冻袋中的输注介质中冷冻保存，每个包含单个单位剂量的细胞，其为约1×10 5个细胞至1×10 9个细胞。在输注之前，将细胞保持在约低于-130℃或约低于-175℃的温度下。
在开始细胞治疗之前，从受试者获得血液，并且任选地通过ELISA和/或MSD和/或CBA的方法评估血清中指示细胞因子释放综合征(CRS)的一种或多种血清因子的水平，例如肿瘤坏死因子α(TNFα)、干扰素γ(IFNγ)、IL-10和IL-6。在治疗开始之前，可以通过例如通过PET或CT扫描测量实体肿瘤的大小或质量来任选地评估肿瘤负荷。
通过升温至约38℃进行复苏，并且受试者通过多次输注给予本发明免疫效应细胞的细胞。输注为约1-20ml/min内连续输注静脉内(IV)给予。
在给予一定剂量免疫效应细胞后，受试者接受身体检查，并监测任何毒性或毒性结果的症状，例如发热、低血压、缺氧、神经障碍或炎性细胞因子或C反应蛋白(CRP)的血清水平升高。任选地，在给予免疫效应细胞后，在一次或多次的情况下，从患者获得血液，并通过ELISA和/或MSD和/或CBA的方法评估指示CRS的血清因子的水平。将血清因子的水平与刚好给予在先疗程的免疫效应细胞之前获得的血清因子的水平进行比较。如有必要，给予抗IL6或其他CRS治疗以减少CRS的症状。
在给予免疫效应细胞治疗后，例如在给药开始后1、2、3和/或4周，任选地检测受试者中抗CAR免疫应答的存在或不存在，例如，通过qPCR、ELISA、ELISPOT、基于细胞的抗体测定和/或混合淋巴细胞反应。
通过输注本发明的免疫效应细胞实现的肿瘤负荷减少百分比可任选地在通过扫描(例如PET和CT扫描)在实体瘤患者中给予在先疗程的免疫效应细胞后一次或多次进行测量，和/或通过量化在血液或肿瘤部位疾病阳性细胞。
从第一疗程给予免疫效应细胞开始并持续长达数年，定期监测受试者。在随访期间，测量肿瘤负荷、和/或通过流式细胞术和定量聚合酶链反应(qPCR)检测CAR-表达细胞，以测量所给予的细胞的体内增殖和持久性、和/或评估抗-CAR免疫应答的发展。
本发明的优点：
本发明的目的在于提供一种工程化细胞(例如，T细胞)及其组合物用于治疗患有或可疑患有CD19阳性的血液肿瘤的受试者的方法和用途，通常包括白血病或淋巴瘤，尤其是急性、慢性性淋巴细胞白血病和/或非霍奇金淋巴瘤(NHL)。在一些方面，与现有技术相比，该方法和用途提供或实现改善的或更持久的应答或功效和/或降低的毒性风险或其他副作用。本发明采用不同剂量(剂量范围为1.5×10 5至4.9×10 6CAR-T细胞/kg、或总剂量为1×10 7至3×10 8CAR-T细胞)的抗CD19 CAR-T细胞治疗CD19阳性的血液肿瘤， 受试者的ORR达到67％(8/12受试者)，CR达到50％(6/12受试者)；12例受试者中6例受试者的PFS超过6个月，其中3例疗效为CRS的受试者的PFS超过1年最长达到近3年且随访至今，1例疗效为PR的受试者的PFS超过500天且随访至今；并且安全性高，12例受试者均没有出现3级或更高级的CRS及神经毒性，最常见的与本发明抗CD19CAR-T细胞相关的不良事件是发热、其次是血小板下降和白细胞下降，经过对症、支持治疗均得到缓解或在缓解中。其中，最高输注剂量达到4.9×10 6CAR-T细胞/kg或输注总剂量达到3×10 8CAR-T细胞的受试者在细胞输注后疗效评估达到CR，且随访至今约19个月数次疗效评估均为CR，目前仍在随访中；受试者在CAR-T细胞输注后仅出现淋巴细胞计数降低、白细胞计数降低、乳酸脱氢酶升高等不良反应且自行缓解。本发明抗CD19 CAR能很好的植入CD19阳性的血液肿瘤受试者体内且在体内扩增能力强且持续数月。在输注剂量高达到4.9×10 6CAR-T细胞/kg或输注总剂量高达到3×10 8CAR-T细胞也是安全且有显著疗效的。
本发明采用不同剂量(剂量范围为1.5×10 5至4.9×10 6CAR-T细胞/kg、或总剂量为1×10 7至3×10 8CAR-T细胞)的抗CD19 CAR-T细胞治疗CD19阳性的NHL，受试者的ORR达到56％(5/9受试者)，CR达到33％(3/9受试者)；9例受试者中5例受试者的PFS超过6个月，其中3例疗效为CRS的受试者的PFS超过1年最长达到近3年且随访至今，1例疗效为PR的受试者的PFS超过500天且随访至今；并且安全性高，9例NHL受试者均没有出现3级或更高级的CRS及神经毒性，最常见的与本发明抗CD19 CAR-T细胞相关的不良事件是发热、其次是血小板下降和白细胞下降，经过对症、支持治疗均得到缓解或在缓解中。其中，最高输注剂量达到4.9×10 6CAR-T细胞/kg或输注总剂量达到3×10 8CAR-T细胞的受试者在细胞输注后疗效评估达到CR，且随访至今约19个月数次疗效评估均为CR，目前仍在随访中；受试者在CAR-T细胞输注后仅出现淋巴细胞计数降低、白细胞计数降低、乳酸脱氢酶升高等不良反应且自行缓解。本发明抗CD19 CAR能很好的植入CD19阳性的NHL受试者体内且在体内扩增能力强能持续数月。在输注剂量高达到4.9×10 6CAR-T细胞/kg或输注总剂量高达到3×10 8CAR-T也是安全且有显著疗效的。
本发明采用不同剂量(剂量范围为1.8×10 6至2.6×10 6CAR-T细胞/kg、或总剂量为1×10 8至1.5×10 8CAR-T细胞)的抗CD19 CAR-T细胞治疗CD19阳性的ALL，3例ALL受试者最佳疗效评价均为CR，PFS为120-432天；均没有出现3级或更高级的CRS及神经毒性，最常见的与本发明抗CD19 CAR-T细胞相关的不良事件是发热、其次是血小板下降和白细胞下降，经过对症、支持治疗均得到缓解或在缓解中。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料与方法：
本发明采用的各种材料，包括试剂均可自商业渠道购得。
示例性抗原受体，包括CAR，以及用于将这样的受体工程化并将这样的受体引入到细胞中的方法，例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN 108884459 A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN 109908176 A、CN109880803A、CN 110055275 A、CN110123837A、CN 110438082 A、CN 110468105 A国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306 A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018/219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029中公开的全文内容。
作为示例性的，本发明下述实施例中，嵌合抗原受体的scFv部分的氨基酸序列如SEQ ID NO:10所示，scFv具有SEQ ID NO：11所示的重链可变区以及SEQ ID NO：12所示的轻链可变区，嵌合抗原受体具有SEQ ID NO：19所示的氨基酸序列。应理解，包含有上述scFv的CAR还可以具有其他的胞内域，因此，CAR的序列还可以为SEQ ID NO：17、18或20所示的序列。
SEQ ID NO:10所示的scFv具有SEQ ID NO：5所示的HCDR1、SEQ ID NO：6所示的HCDR2、SEQ ID NO：7所示的HCDR3、及SEQ ID NO：2所示的LCDR1、SEQ ID NO：3所示的LCDR2、SEQ ID NO：4所示的LCDR3。
实施例1.用CD19 CAR治疗患有复发或难治性CD19阳性的血液肿瘤的受试者
给予复发性或难治性CD19阳性的血液肿瘤的12名受试者输注表达抗CD19嵌合抗原受体(CAR)的自体T细胞。受试者的年龄范围为22至62岁，平均年龄为43岁。受试者展现出多种疾病类型，包括6例弥漫性大B细胞淋巴瘤(DLBCL)、1例套细胞淋巴瘤(MCL)、1例滤泡性淋巴瘤、1例B淋巴母细胞淋巴瘤/急性淋巴细胞白血病(ALL)和3例急性淋巴细胞白血病(ALL)。所有受试者先前均已进行治疗，其中平均数为4次治疗且范围为1至7次现有治疗，其中2名受试者采用依鲁替尼治疗过。在所有受试者中，6名受试者已用大于或等于4次现有疗法进行治疗。1名已经接受了自体造血干细胞移植。
在治疗前，来自供者的PBMC制备而成的抗CD19 CAR-T细胞(按中国专利申请CN201711330443.7方法制备)。在抗CD19 CAR T细胞输注前至少2天至最长6天开始， 受试者接受淋巴细胞清除疗法，输注约17-27mg/m 2/d的氟达拉滨3或4次、和21-59mg/kg的环磷酰胺(分1次或2次或3次给予)。
CAR-T细胞输注：分别以剂量1.5×10 5、5.8×10 5、1.1×10 6、1.7×10 6、1.8×10 6、1.9×10 6、2.0×10 6、2.1×10 6、2.6×10 6或4.9×10 6CAR-T细胞/kg受试者体重，向受试者输注，其中CAR-T细胞输注剂量为1.8×10 6、1.9×10 6CAR-T细胞/kg各有2例受试者、其余剂量均为1例受试者；或以总剂量为1×10 7、或3×10 7、或6×10 7、或8×10 7、或9×10 7、或1×10 8、或1.5×10 8或3×10 8CAR-T细胞向受试者输注，其中CAR-T细胞输注总剂量为1×10 8CAR-T细胞为2例受试者、1.5×10 8CAR-T细胞为4例受试者、其余剂量均为1例受试者。CAR-T细胞剂量可以为1次性输注、也可以分2或3次输注。
在给予抗CD19 CAR-T细胞之前和之后4周，患者进行PET-CT全身成像以及诊断质量CT扫描或骨髓细胞形态学和活检组织病理学来评估治疗应答情况。对骨髓进行形态学分析，在骨髓中具有至少5％的原始及幼稚细胞的对象被认为具有形态学疾病(MD)。完全缓解(CR)的受试者骨髓涂片：骨髓中具有小于5％的原始及幼稚淋巴细胞，但在骨髓中显示分子可检测的残留疾病(通过流式细胞术)被认为具有最小的残留疾病(MRD)。将淋巴结肿瘤体积评估为在诊断质量CT扫描中鉴定的6个最大指数淋巴结的横截面积的总和。淋巴瘤负荷按照Lugano标准(Cheson等人,JCO September 20,2014vol.32no.273059-3067)以靶病灶的SPD值定量检测。根据影像学检查及骨髓穿刺/活检结果以Lugano2014标准评估疗效。根据美国国立癌症研究所(NCI)不良事件通用术语标准(CTCAE)4.03版本，对不良事件的严重程度进行分级。细胞因子释放综合征(CRS)如2014 Lee DW和2019 ASTCT中所述进行分级。
通过测量总体缓解率(ORR)来监测对象的治疗功效。根据影像学检查及骨髓穿刺/活检结果以lugano标准或NCCN临床实践指南来评估疗效，ORR被确定为具有完全缓解(CR)或部分缓解(PR)的对象的比例。
在12例受试者中，ORR为67％(8/12受试者)，CR为50％(6/12受试者)。CR组的PFS有高于非CR组的趋势。
为了进一步评估给予抗CD19 CAR-T细胞的功效，通过检测抗CD19 CAR-T细胞在体内持续存活期，即CAR-T细胞“植入”体内持续存活的期间。从初次输注(为第0天)结束后起每个访视点采用Q-PCR的方法，所用探针为Probe(核苷酸序列见SEQ ID NO:28CGTGGAGGCCAACGACACCG)；上游引物序列为：Primer(核苷酸序列见SEQ ID NO:29TTCACCCTGACCATCAACCC)；下游引物序列为：Primer(核苷酸序列见SEQ ID NO:30CTGCTGGCAGTAGTAGTTGG)，检测外周血中含有抗CD19 CAR DNA的拷贝数，直到任何2次连续的检测为阴性，记录为抗CD19 CAR-T细胞持续存活期。检测结果显示，抗CD19 CAR-T细胞在所有受试者中增殖，约在细胞输注后第1-7天检测到CAR拷贝数，约在第9-22天达到峰值，约至第5至6个月 检测不到。
给予抗CD19 CAR-T细胞后评估受试者的疾病状态以评估对治疗的反应。治疗后，对受试者进行评估并监测神经毒性(神经系统并发症，包括混乱症状，失语症，癫痫发作，抽搐，嗜睡和/或改变的精神状态)，根据严重程度分级(使用1-5级量表例如，Guido Cavaletti和Paola Marmiroli Nature Reviews Neurology 6，657-666(2010年12月)，其中3级(严重症状)，4(危及生命的症状)或5(死亡)被认为是严重的神经毒性。
(一)确定并监测细胞因子释放综合征(CRS)：
1级(轻微)-不威胁生命，仅需要全身治疗如退热剂和止吐剂(例如，发热、恶心、疲劳、头痛、肌痛、不适)；
2级(中等)-需要并响应中等程度干预：
氧气需求<40％，或相应体液或低剂量单一血管升压类药物的低血压，或2级器官毒性(通过CTCAE v4.0)；
3级(严重)-需要并响应积极介入：
氧气需求≥40％，或需要高剂量单一血管升压类药物(例如，去甲肾上腺素≥20ug/kg/分钟，多巴胺≥10ug/kg/分钟，苯肾上腺素≥200ug/kg/分钟，或肾上腺素≥10ug/kg/分钟)的低血压，或需要多种血管升压类药物(例如，抗利尿素+上述试剂之一，或等于≥20ug/kg/分钟去甲肾上腺素的血管升压类药物的组合)的低血压，或3级器官毒性，或4级转氨酶升高(通过CTCAE v4.0)；
4级(威胁生命)-需要通气机支持，或4级器官毒性(排除转氨酶升高)；
5级(致命)-死亡。
(二)神经毒性的示例性分级标准：
1级(无临床症状或轻微)-轻微或无临床症状；
2级(中等)-存在限制日常积极活动(ADL)的症状，如做饭、买菜或买衣服、使用电话、管理钱；
3级(严重)-存在限制性自我管理ADL,如洗澡、穿衣或脱衣、进食、使用厕所、服用药物的症状；
4级(威胁生命)-威胁生命，需要紧急介入的症状；
5级(致命)-死亡。
本发明中12名受试者中没有出现3级或更高级的细胞因子释放综合征(CRS)及神经毒性，见表1。
表1不良事件统计
Figure PCTCN2021076518-appb-000001
Figure PCTCN2021076518-appb-000002
本发明采用不同剂量(剂量范围为1.5×10 5至4.9×10 6CAR-T细胞/kg、或总剂量为1×10 7至3×10 8CAR-T细胞)的抗CD19 CAR-T细胞治疗CD19阳性的血液肿瘤，受试者的ORR达到67％(8/12受试者)，CR达到50％(6/12受试者)；12例受试者中6例受试者的PFS超过6个月，其中3例疗效为CRS的受试者的PFS超过1年最长达到近3年且随访至今，1例疗效为PR的受试者的PFS超过500天且随访至今；并且安全性高，12例受试者均没有出现3级或更高级的CRS及神经毒性，最常见的与本发明抗CD19CAR-T细胞相关的不良事件是发热、其次是血小板下降和白细胞下降，经过对症、支持治疗均得到缓解或在缓解中。
12例CD19阳性的血液肿瘤受试者中，最高输注剂量达到4.9×10 6CAR-T细胞/kg或输注总剂量达到3×10 8CAR-T细胞的受试者在细胞输注后疗效评估达到CR，且随访至今约19个月数次疗效评估均为CR，目前仍在随访中；受试者在CAR-T细胞输注后仅出现淋巴细胞计数降低、白细胞计数降低、乳酸脱氢酶升高等不良反应且自行缓解。
结论：本发明抗CD19 CAR能很好的植入CD19阳性的血液肿瘤受试者体内且在体内扩增能力强且持续数月。在输注剂量高达到4.9×10 6CAR-T细胞/kg或输注总剂量高达到3×10 8CAR-T细胞也是安全且有显著疗效的。
实施例2.非霍奇金淋巴瘤(NHL)的抗CD19 CAR-T细胞治疗
给予复发性或难治性CD19阳性的非霍奇金淋巴瘤(NHL)的9名受试者输注表达抗CD19嵌合抗原受体(CAR)的自体T细胞。受试者的年龄范围为25至62岁，平均年龄为45岁。该组受试者展现出多种疾病类型，包括6例弥漫性大B细胞淋巴瘤(DLBCL)、1例套细胞淋巴瘤(MCL)、1例滤泡性淋巴瘤、1例B淋巴母细胞淋巴瘤/急性淋巴细胞白血病(ALL)。所有受试者先前均已进行治疗，其中平均数为4次治疗且范围为1至7次现有治疗，在所有受试者中，3名受试者已用大于或等于4次现有疗法进行治疗，其中2名受试者采用依鲁替尼治疗过。1名已经接受了自体造血干细胞移植。
在抗CD19 CAR T细胞输注前至少2天至最长6天开始，受试者接受淋巴细胞清除化疗，输注约17-24mg/m 2/d的氟达拉滨3或4次、和21-59mg/kg的环磷酰胺(分1次或2次或3次给予)。
采用实施例1所述的抗CD19 CAR-T细胞进行输注：分别以剂量1.5×10 5、5.8×10 5、1.1×10 6、1.7×10 6、1.8×10 6、1.9×10 6、2.0×10 6、2.1×10 6或4.9×10 6CAR-T细胞/kg受试者体重，向受试者输注；或以总剂量为1×10 7、或3×10 7、或6×10 7、或8×10 7、或1×10 8、或1.5×10 8或3×10 8CAR-T细胞向受试者输注，其中CAR-T细胞输注总剂量为1.5×10 8CAR-T细胞为3例受试者、其余剂量均为1例受试者。CAR-T细胞输注可以为1次性输注、也可以分2或3次输注。
在给予抗CD19 CAR-T细胞之前和之后4周，患者进行PET-CT全身成像以及诊断质量CT扫描或骨髓细胞形态学和活检组织病理学来评估治疗应答情况。根据影像学检查及骨髓穿刺/活检结果以Lugano2014标准评估疗效。根据美国国立癌症研究所(NCI)不良事件通用术语标准(CTCAE)4.03版本，对不良事件的严重程度进行分级。细胞因子释放综合征(CRS)如2014Lee DW和2019ASTCT中所述进行分级。
通过测量ORR来监测对象的治疗功效。在9例NHL受试者中，ORR为56％(5/9受试者)，CR为33％(3/9受试者)。CR组的PFS有高于非CR组的趋势。
本发明采用不同剂量(剂量范围为1.5×10 5至4.9×10 6CAR-T细胞/kg、或总剂量为1×10 7至3×10 8CAR-T细胞)的抗CD19 CAR-T细胞治疗CD19阳性的NHL，受试者的ORR达到56％(5/9受试者)，CR达到33％(3/9受试者)；9例受试者中5例受试者的PFS超过6个月，其中3例疗效为CRS的受试者的PFS超过1年最长达到近3年且随访至今，1例疗效为PR的受试者的PFS超过500天且随访至今；并且安全性高，9例NHL受试者均没有出现3级或更高级的CRS及神经毒性，最常见的与本发明抗CD19 CAR-T细胞相关的不良事件是发热、其次是血小板下降和白细胞下降，经过对症、支持治疗均 得到缓解或在缓解中。
9例NHL受试者中，最高输注剂量达到4.9×10 6CAR-T细胞/kg或输注总剂量达到3×10 8CAR-T细胞的受试者在细胞输注后疗效评估达到CR，且随访至今约19个月数次疗效评估均为CR，目前仍在随访中；受试者在CAR-T细胞输注后仅出现淋巴细胞计数降低、白细胞计数降低、乳酸脱氢酶升高等不良反应且自行缓解。
结论：本发明抗CD19 CAR能很好的植入CD19阳性的NHL受试者体内且在体内扩增能力强能持续数月。在输注剂量高达到4.9×10 6CAR-T细胞/kg或输注总剂量高达到3×10 8CAR-T也是安全且有显著疗效的。
实施例3.非霍奇金淋巴瘤(NHL)的高剂量的抗CD19 CAR-T细胞治疗
给予复发性或难治性CD19阳性的弥漫大B细胞淋巴瘤高剂量组4名受试者输注表达抗CD19嵌合抗原受体(CAR)的自体T细胞。受试者的年龄范围为30至63岁。所有受试者先前均已进行治疗，范围为2至5次现有治疗
在抗CD19 CAR T细胞输注前至少5天始，受试者接受淋巴细胞清除化疗，输注约21-27mg/m2/d的氟达拉滨3或4次、和28-31mg/kg的环磷酰胺(分2次给予)。
采用实施例1所述的抗CD19 CAR-T细胞进行输注：均已总剂量为3×10 8CAR-T细胞向受试者输注；CAR-T细胞输注为1次性或分2次输注。
给予抗CD19 CAR-T细胞之前和之后4周，患者进行PET-CT全身成像以及诊断质量CT扫描或骨髓细胞形态学和活检组织病理学来评估治疗应答情况。根据影像学检查及骨髓穿刺/活检结果以Lugano2014标准评估疗效。根据美国国立癌症研究所(NCI)不良事件通用术语标准(CTCAE)4.03版本，对不良事件的严重程度进行分级。细胞因子释放综合征(CRS)如2014Lee DW和2019ASTCT中所述进行分级。
在4例NHL受试者中，其中获得CR患者2例，PR1例，SD1例。4例受试者均没有出现3级或更高级的CRS及神经毒性，最常见的与本发明抗CD19 CAR-T细胞相关的不良事件是发热、其次是血小板下降和白细胞下降，经过对症、支持治疗均得到缓解或在缓解中。
实施例4.急性淋巴细胞性白血病(ALL)的抗CD19 CAR-T细胞治疗
给予复发性或难治性CD19阳性的急性淋巴细胞性白血病(ALL)的3名受试者输注表达抗CD19嵌合抗原受体(CAR)的自体T细胞。受试者的年龄范围为22至57岁，平均年龄为38岁。所有受试者先前均已进行治疗，其中平均数为4次治疗且范围为4至5次现有治疗。
在抗CD19 CAR T细胞输注前至少2天至最长6天开始，受试者接受淋巴细胞清除化疗，输注约21-27mg/m 2/d的氟达拉滨3或4次、和28-31mg/kg的环磷酰胺(分2次给予)。
采用实施例1所述的抗CD19 CAR-T细胞进行输注：分别以剂量1.8×10 6、1.9×10 6、2.6×10 6CAR-T细胞/kg受试者体重，向受试者输注；或以总剂量为9×10 7、 或1×10 8或1.5×10 8CAR-T细胞向受试者输注；CAR-T细胞输注为1次性输注。
在给予抗CD19 CAR-T细胞之前和之后4周，患者进行骨髓细胞形态学和活检组织病理来评估治疗应答情况。根据骨髓穿刺/活检结果以NCCN临床实践指南标准评估疗效。根据美国国立癌症研究所(NCI)不良事件通用术语标准(CTCAE)4.03版本，对不良事件的严重程度进行分级。
本发明采用不同剂量(剂量范围为1.8×10 6至2.6×10 6CAR-T细胞/kg、或总剂量为1×10 8至1.5×10 8CAR-T细胞)的抗CD19 CAR-T细胞治疗CD19阳性的ALL，3例ALL受试者最佳疗效评价均为CR，PFS为120-432天；均没有出现3级或更高级的CRS及神经毒性，最常见的与本发明抗CD19 CAR-T细胞相关的不良事件是发热、其次是血小板下降和白细胞下降，经过对症、支持治疗均得到缓解或在缓解中。
在上述实施例中，示例性的，选择了SEQ ID NO：19所示CAR，然而，应理解，其他靶向CD19的CAR，也可以应用于本申请所述的技术方案。如SEQ ID NO：13、14、15、16、17、18或20所示的CAR。
其中，SEQ ID NO：13、14、15及16所示的CAR的scFv具有SEQ ID NO：8所示的重链可变区的氨基酸序列，和SEQ ID NO：9所示的轻链可变区的氨基酸序列，CDR区分别为：SEQ ID NO：5所示的HCDR1、SEQ ID NO：6所示的HCDR2、SEQ ID NO：7所示的HCDR3、及SEQ ID NO：2所示的LCDR1、SEQ ID NO：3所示的LCDR2、SEQ ID NO：4所示的LCDR3。
其中，SEQ ID NO：17、18及20所示的CAR的scFv具有SEQ ID NO：11所示的重链可变区的氨基酸序列，和SEQ ID NO：12所示的轻链可变区的氨基酸序列，CDR区分别为：SEQ ID NO：5所示的HCDR1、SEQ ID NO：6所示的HCDR2、SEQ ID NO：7所示的HCDR3、及SEQ ID NO：2所示的LCDR1、SEQ ID NO：3所示的LCDR2、SEQ ID NO：4所示的LCDR3。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明涉及的序列
Figure PCTCN2021076518-appb-000003
Figure PCTCN2021076518-appb-000004
Figure PCTCN2021076518-appb-000005

Claims (24)

  1. 一种治疗患有或怀疑患CD19阳性血液肿瘤的方法，其特征在于，包括向所述受试者给予表达抗CD19嵌合抗原受体(CAR)的细胞，所给予细胞的剂量如下：
    以每公斤受试者体重给予的表达抗CD19嵌合抗原受体(CAR)的细胞的个数来计或者以每个受试者给予的表达抗CD19嵌合抗原受体(CAR)的细胞的总个数来计，所述剂量选自以下中的任意：
    (a)约4.9x10 6个细胞/kg或以下，
    (b)约3x10 8个总细胞或以下，
    (c)约1x10 7个细胞/kg或以下，
    (d)约6x10 8个总细胞或以下，
    (e)约1.5x10 5个细胞/kg～约4.9x10 6个细胞/kg，
    (f)约1.5x10 5个细胞/kg～约1x10 7个细胞/kg，
    (g)约1x10 7个总细胞～约3x10 8个总细胞，以及
    (h)约1x10 7个总细胞～约6x10 8个总细胞；
    所述细胞能结合肿瘤细胞上表达的CD19，
    进一步优选CD19是如SEQ ID NO：31所示的人来源的CD19。
  2. 如权利要求1所述的方法，其特征在于，
    以每公斤受试者体重给予的表达抗CD19嵌合抗原受体(CAR)的细胞的个数来计，所给予细胞的剂量选自以下中的任意：
    (a)约5.8x10 5个细胞/kg，(b)约1.1x10 6个细胞/kg，(c)约1.7x10 6个细胞/kg，(d)约1.8x10 6个细胞/kg，(e)约1.9x10 6个细胞/kg，(f)约2.0x10 6个细胞/kg，(g)约2.1x10 6个细胞/kg，以及(h)约2.6x10 6个细胞/kg，或者；
    以每个受试者给予的表达抗CD19嵌合抗原受体(CAR)的细胞的总个数来计，所给予细胞的剂量选自以下中的任意：
    (h)约3x10 7个总细胞，(i)约6x10 7个总细胞，(j)约8x10 7个总细胞，(k)约9x10 7个总细胞，(l)约1x10 8个总细胞，(m)约1.5x10 8个总细胞，或者：
    以每公斤受试者体重给予的表达抗CD19嵌合抗原受体(CAR)的细胞的个数来计，所述剂量选自以下中的任意：
    (n)约5.8x10 5个细胞/kg～约4.9x10 6个细胞/kg，(o)约1.1x10 6个细胞/kg～约4.9x10 6个细胞/kg，(p)约1.7x10 6个细胞/kg～约4.9x10 6个细胞/kg，(q)约1.8x10 6个细胞/kg～约4.9x10 6个细胞/kg，(r)约1.9x10 6个细胞/kg～约4.9x10 6个细胞/kg，(s)约2x10 6个细胞/kg～约4.9x10 6个细胞/kg，(t)约2.6x10 6个细胞/kg～约4.9x10 6个细胞/kg，
    以每个受试者给予的表达抗CD19嵌合抗原受体(CAR)的细胞的总个数来计，所给 予细胞的剂量选自以下中的任意：
    (u)约3x10 7个总细胞～约3x10 8个总细胞，(v)约6x10 7个总细胞～约3x10 8个总细胞，(w)约8x10 7个总细胞～约3x10 8个总细胞，(x)约1x10 8个总细胞～约3x10 8个总细胞，和/或(y)约1.5x10 8个总细胞～约3x10 8个总细胞；
    其中在细胞给予之前所述受试者接受淋巴细胞清除疗法。
  3. 如权利要求1或2所述的方法，其特征在于，所述受试者在给予所述细胞时或给予之前：
    所述受试者被鉴定出患有B细胞恶性肿瘤；
    优选所述B细胞恶性肿瘤选自：急性淋巴细胞白血病(ALL)、成人ALL、慢性淋巴细胞白血病(CLL)、和/或非霍奇金淋巴瘤(NHL)；和/或
    所述受试者被鉴定出或已经被鉴定出具有一种或多种细胞遗传学异常，任选地与高风险NHL和/或白血病相关；
    优选所述受试者被鉴定出或已经被鉴定出患有高风险NHL；
    进一步优选所述NHL选自下组：侵袭性NHL、弥漫性大B细胞淋巴瘤(DLBCL)、原发性纵隔大B细胞淋巴瘤(PMBCL)、富含T细胞/组织细胞的大B细胞淋巴瘤(TCHRBCL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)和/或滤泡性淋巴瘤(FL)；和/或
    所述受试者是成年人和/或年龄超过20岁、30岁、40岁、50岁、60岁或70岁或者超过约20岁、30岁、40岁、50岁、60岁或70岁。
  4. 如权利要求1-3任一所述的方法，其特征在于，在给予所述细胞之前所述受试者已经接受表达CD19CAR细胞以外的其他两种或三种及以上针对所述血液肿瘤的先前疗法，任选2、3或4种或5种以上，任选地，先前疗法包括激酶抑制剂治疗任选依鲁替尼，或单克隆抗体治疗任选利妥昔单抗，或CHOP疗法，和/或造血干细胞移植(HSCT)治疗；所述先前疗法不耐受、无效、或治疗后出现缓解后复发难治。
  5. 如权利要求2-4任一项所述的方法，其特征在于，所述淋巴细胞清除疗法包括：
    (i)给予氟达拉滨，任选地为还包括给予环磷酰胺；
    (ii)在给予细胞前至少48小时或至少约48小时或在48小时、或约48小时和144小时或约144小时之间的时间开始；并且(iii)以约21-59mg/kg给予环磷酰胺，任选每天1次持续1天或2天或3天，或以约250mg/m 2～500mg/m 2/日给予环磷酰胺，任选2天或3天或4天，和/或以约17-27mg/m 2/d给予氟达拉滨，持续3-4天。
  6. 如权利要求1-5中任一项所述的方法，其特征在于，所述细胞输注和/或淋巴细胞清除疗法施与由门诊递送。
  7. 如权利要求1-6中任一项所述的方法，其特征在于，所述细胞剂量分1、2、3、4或以上次在3或4天内经由胃肠外给予，任选静脉内给予。
  8. 如权利要求1-7中任一项所述的方法，根据所述方法治疗的受试者中的至少30％实现完全缓解(CR)和/或总体缓解(OR)。
  9. 如权利要求1-8中任一项所述的方法，其特征在于：
    根据所述方法治疗并实现完全缓解(CR)的受试者中的至少50％展现出超过12个月的无进展生存期(PFS)和/或总生存期(OS)；根据该方法治疗的受试者展现出超过6个月、12个月或18个月或者超过约6个月、12个月或18个月的平均PFS或OS；和/或该受试者展现出至少6、12、18或更多个月或者至少约6、12、18或更多个月的治疗后PFS或OS。
  10. 如权利要求1-9中任一项所述的方法，其特征在于：根据所述方法治疗的受试者均没有出现3级或更高级的CRS及神经毒性。
  11. 如权利要求1-10任一项所述的方法，其特征在于，所述CAR包含结合CD19的scFv、跨膜结构域、4-1BB或CD28的共刺激结构域、以及CD3ζ胞内结构域；所述scFv包含SEQ ID NO：2所示的LCDR1、SEQ ID NO：3所示的LCDR2、SEQ ID NO：4所示的LCDR3、及SEQ ID NO：5所示的HCDR1、SEQ ID NO：6所示的HCDR2、SEQ ID NO：7所示的HCDR3；或
    所述scFv包含SEQ ID NO：8或11所示的重链可变区以及SEQ ID NO：9或12所示的轻链可变区；或
    所述scFv具有SEQ ID NO：1或10所示序列；
    所述CAR包含SEQ ID NO：13、14、15、16、17、18、19或20所示的氨基酸序列；和/或
    所述共刺激结构域包含SEQ ID NO:24或25、或其变体，该变体与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性；和/或
    所述CD3ζ胞内结构域包含SEQ ID NO:26，它们具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性。
  12. 如权利要求1-11任一所述的方法，其特征在于，所述细胞是免疫效应细胞，优选地为T细胞，更优选地为自体同源或同种异体T细胞，最优选地为从受试者获得的原代T 细胞。
  13. 一种包含表达特异性地结合至CD19阳性血液肿瘤的嵌合抗原受体(CAR)的细胞的组合物，其特征在于，所述组合物用于治疗患有或怀疑患有CD19阳性血液肿瘤的受试者，所述组合物包括能够给予受试者给定剂量的所述CAR的细胞，
    以每公斤受试者体重给予的表达抗CD19嵌合抗原受体(CAR)的细胞的个数来计或者以每个受试者给予的表达抗CD19嵌合抗原受体(CAR)的细胞的总个数来计，所述给定剂量选自以下中的任意：
    (a)约4.9x10 6个细胞/kg或以下，或者
    (b)约3x10 8个总细胞或以下，
    (c)约1x10 7个细胞/kg或以下，
    (d)约6x10 8个总细胞或以下，
    (e)约1.5x10 5个细胞/kg～约4.9x10 6个细胞/kg，
    (f)约1.5x10 5个细胞/kg～约1x10 7个细胞/kg，
    (g)约1x10 7个总细胞～约3x10 8个总细胞，以及
    (h)约1x10 7个总细胞～约6x10 8个总细胞；
    所述细胞能结合肿瘤细胞上表达的CD19，
    进一步优选CD19是如SEQ ID NO：31所示的人来源的CD19。
  14. 如权利要求13所述的组合物，其特征在于，
    以每公斤受试者体重给予的表达抗CD19嵌合抗原受体(CAR)的细胞的个数来计，所给予细胞的剂量选自以下中的任意：
    (a)约5.8x10 5个细胞/kg，(b)约1.1x10 6个细胞/kg，(c)约1.7x10 6个细胞/kg，(d)约1.8x10 6个细胞/kg，(e)约1.9x10 6个细胞/kg，(f)约2.0x10 6个细胞/kg，(g)约2.1x10 6个细胞/kg，以及(h)约2.6x10 6个细胞/kg，或者
    以每个受试者给予的表达抗CD19嵌合抗原受体(CAR)的细胞的总个数来计，所给予细胞的剂量选自以下中的任意：
    (h)约3x10 7个总细胞，(i)约6x10 7个总细胞，，(j)约8x10 7个总细胞，(k)约9x10 7个总细胞，(l)约1x10 8个总细胞，(m)约1.5x10 8个总细胞，或者：
    以每公斤受试者体重给予的表达抗CD19嵌合抗原受体(CAR)的细胞的个数来计，所述剂量选自以下中的任意：
    (n)约5.8x10 5个细胞/kg～约4.9x10 6个细胞/kg，(o)约1.1x10 6个细胞/kg～约4.9x10 6个细胞/kg，(p)约1.7x10 6个细胞/kg～约4.9x10 6个细胞/kg，(q)约1.8x10 6个细胞/kg～约4.9x10 6个细胞/kg，(r)约1.9x10 6个细胞/kg～约4.9x10 6个细胞/kg，(s)约2x10 6个细胞/kg～约4.9x10 6个细胞/kg，(t)约2.6x10 6个细胞/kg～约4.9x10 6个细胞/kg，
    以每个受试者给予的表达抗CD19嵌合抗原受体(CAR)的细胞的总个数来计，所给予细胞的剂量选自以下中的任意：
    (u)约3x10 7个总细胞～约3x10 8个总细胞，(v)约6x10 7个总细胞～约3x10 8个总细胞，(w)约8x10 7个总细胞～约3x10 8个总细胞，(x)约1x10 8个总细胞～约3x10 8个总细胞，和/或(y)约1.5x10 8个总细胞～约3x10 8个总细胞；
    其中在组合物给予之前所述受试者接受淋巴细胞清除疗法。
  15. 如权利要求13或14所述的组合物，其特征在于，所述组合物还包含用于淋巴细胞清除疗法的药物，任选地，所述药物包括氟达拉滨和环磷酰胺。
  16. 如权利要求13-15任一所述的组合物，其特征在于，所述受试者在给予所述组合物时或给予之前：
    所述受试者被鉴定出患有B细胞恶性肿瘤；
    优选所述B细胞恶性肿瘤选自：急性淋巴细胞白血病(ALL)、成人ALL、慢性淋巴细胞白血病(CLL)、和/或非霍奇金淋巴瘤(NHL)；和/或
    所述受试者被鉴定出或已经被鉴定出具有一种或多种细胞遗传学异常，任选地与高风险NHL和/或白血病相关；
    优选所述受试者被鉴定出或已经被鉴定出患有高风险NHL；
    进一步优选所述NHL选自下组：侵袭性NHL、弥漫性大B细胞淋巴瘤(DLBCL)、原发性纵隔大B细胞淋巴瘤(PMBCL)、富含T细胞/组织细胞的大B细胞淋巴瘤(TCHRBCL)、伯基特淋巴瘤、套细胞淋巴瘤(MCL)和/或滤泡性淋巴瘤(FL)；和/或
    所述受试者是成年人和/或年龄超过20岁、30岁、40岁、50岁、60岁或70岁或者超过约20岁、30岁、40岁、50岁、60岁或70岁。
  17. 如权利要求13-16任一所述的组合物，其特征在于，所述受试者在给予所述组合物之前所述受试者已经接受表达CD19CAR细胞以外的其他两种或三种以上针对所述血液肿瘤的先前疗法，任选2、3或4种或5种以上，任选地，先前疗法包括激酶抑制剂治疗任选依鲁替尼，或单克隆抗体治疗任选利妥昔单抗，或CHOP疗法，和/或造血干细胞移植(HSCT)治疗；所述先前疗法不耐受、无效、或治疗后出现缓解后复发难治。
  18. 如权利要求14-17任一所述的组合物，其特征在于，所述淋巴细胞清除疗法：
    (i)在给予细胞前至少48小时或至少约48小时或在48小时、或约48小时和144小时或约144小时之间的时间开始；和/或
    (ii)以约21-59mg/kg给予环磷酰胺，任选每天1次持续1天或2天或3天，或以约250mg/m 2～500mg/m 2/日给予环磷酰胺，任选2天或3天或4天，和/或以约17-27mg/m 2/d 给予氟达拉滨，持续3-4天。
  19. 如权利要求13-18中任一所述的组合物，其特征在于，所述组合物中的细胞分1、2、3、4或以上次在3或4天内，通过肠胃外给药，任选静脉内给药。
  20. 如权利要求13-19中任一所述的组合物，其特征在于，在门诊环境中、和/或在所述受试者无需住院过夜或连续多天的情况下、和/或所述受试者无需住院一天或多天的情况下给予或将给予所述组合物中的细胞。
  21. 如权利要求13-20中任一所述的组合物，其特征在于，所述细胞是免疫效应细胞，优选地为T细胞，更优选地为自体同源或同种异体T细胞，最优选地为从受试者获得的原代T细胞。
  22. 如权利要求13-21任一项所述的组合物，其特征在于，所述CAR包含结合CD19的scFv、跨膜结构域、4-1BB或CD28的共刺激结构域、以及CD3ζ胞内结构域；所述scFv包含SEQ ID NO：2所示的LCDR1、SEQ ID NO：3所示的LCDR2、SEQ ID NO：4所示的LCDR3、及SEQ ID NO：5所示的HCDR1、SEQ ID NO：6所示的HCDR2、SEQ ID NO：7所示的HCDR3；或
    所述scFv包含SEQ ID NO：8或11所示的重链可变区以及SEQ ID NO：9或12所示的轻链可变区；或
    所述scFv具有SEQ ID NO：1或10所示序列；或
    所述CAR包含SEQ ID NO：13、14、15、16、17、18、19或20所示的氨基酸序列；和/或
    所述共刺激结构域包含SEQ ID NO:24或25、或其变体，该变体与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性；和/或
    所述CD3ζ胞内结构域包含SEQ ID NO:26，它们具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列一致性。
  23. 一种用于治疗患有或怀疑患有CD19阳性血液肿瘤的制品，其特征在于，所述制品包含：
    1)如权利要求13-22任一所述的组合物；
    2)利用所述试剂盒治疗患有或怀疑患有CD19阳性血液肿瘤受试者的给药说明书。
  24. 如权利要求23所述的制品，其特征在于，每个剂量包含表达抗CD19嵌合抗原受体(CAR)的细胞，所述说明书规定了向患有CD19阳性血液肿瘤的受试者给予的细胞的剂量；并且该说明书规定了给予一定数量的CD19 CAR表达细胞，或规定了给予对应于所述指定数量的细胞或含有所述指定数量的细胞的一定量或体积的一种或多种制剂。
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