WO2020020210A1 - 免疫效应细胞治疗肿瘤的方法 - Google Patents

免疫效应细胞治疗肿瘤的方法 Download PDF

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WO2020020210A1
WO2020020210A1 PCT/CN2019/097453 CN2019097453W WO2020020210A1 WO 2020020210 A1 WO2020020210 A1 WO 2020020210A1 CN 2019097453 W CN2019097453 W CN 2019097453W WO 2020020210 A1 WO2020020210 A1 WO 2020020210A1
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cells
seq
immune effector
subject
course
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PCT/CN2019/097453
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English (en)
French (fr)
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李宗海
王华茂
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科济生物医药（上海）有限公司
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Priority to US17/262,443 priority Critical patent/US20210292427A1/en
Priority to CN201980049601.2A priority patent/CN112930199A/zh
Priority to JP2021503898A priority patent/JP7262568B2/ja
Priority to EP19841265.2A priority patent/EP3834849A4/en
Priority to CA3107515A priority patent/CA3107515A1/en
Priority to KR1020217003555A priority patent/KR20210055034A/ko
Priority to AU2019310855A priority patent/AU2019310855A1/en
Publication of WO2020020210A1 publication Critical patent/WO2020020210A1/zh

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    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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Definitions

  • the invention belongs to the field of immunotherapy; in particular, it relates to immune cell therapy that targets and recognizes tumor antigens, triggers activation of immune effector cells, and exerts antitumor effects.
  • MM Multiple myeloma
  • the main condition is the infinite expansion and enrichment of plasma cells in the bone marrow, leading to osteonecrosis.
  • the main treatment options are chemotherapy and stem cell transplantation.
  • the chemotherapy drugs are mainly steroids, thalidomide, lenalidomide, bortezomib, and so on.
  • BCMA B-cell maturation antigen
  • B-cell mature antigen a type III transmembrane protein composed of 185 amino acid residues, belonging to the TNF receptor superfamily, and its ligand belongs to the TNF superfamily, such as the proliferation-inducing ligand (APRIL ), B lymphocyte stimulating factor (BAFF), BCMA can activate B cell proliferation and survival after binding to its ligand.
  • APRIL proliferation-inducing ligand
  • BAFF B lymphocyte stimulating factor
  • BCMA can activate B cell proliferation and survival after binding to its ligand.
  • BCMA is specifically overexpressed in plasma cells and multiple myeloma cells, but not in hematopoietic stem cells and other normal tissue cells. Therefore, BCMA can be an ideal target for targeted treatment of MM.
  • An object of the present invention is to provide a technical means capable of having excellent killing effect on BCMA-positive tumors.
  • a method for treating a BCMA-positive tumor comprising administering to a subject at least one course of an immune effector cell expressing a chimeric antigen receptor (CAR), the immune effector cell Specific recognition of BCMA.
  • CAR chimeric antigen receptor
  • the dose of immune effector cells per course of treatment does not exceed about 1 ⁇ 10 9 cells / kg of the subject's body weight or the total amount does not exceed about 1 ⁇ 10 10 .
  • the dose of immune effector cells per course does not exceed about 1 ⁇ 10 8 cells / kg of the subject's body weight or the total amount of said cells does not exceed about 1 ⁇ 10 9 .
  • the dose of immune effector cells per course does not exceed about 1 ⁇ 10 7 cells / kg of the subject's body weight or the total amount of said cells does not exceed about 5 ⁇ 10 8 .
  • the total dose of immune effector cells in each course is not less than 1 ⁇ 10 5 .
  • the total dose of immune effector cells in each course is not less than 1 ⁇ 10 6 .
  • the total dose of immune effector cells in each course is not less than 1 ⁇ 10 7 .
  • the subject is administered the immune effector cells for 2-5 courses.
  • the immune effector cells are administered in a later course of treatment.
  • the immune effector cells in the subsequent course are administered at a time point of about 4 to 24 weeks after the administration in the previous course.
  • the dose of immune effector cells administered in a later course is lower than, equal to, or higher than the immune effector cells administered in a previous course.
  • the dose of immune effector cells given in a later course is higher than the immune effector cells given in a previous course.
  • the dose of the immune effector cells administered in the subsequent course is 2 times, 5 times, 7 times, or 10 times the dose of the previously administered immune effector cells.
  • the immune effector cells of each course are divided into N administrations within 15 days, N is a natural number not less than 1, and in a preferred embodiment, N is 1, 2, 3, or 4.
  • the subject when the immune effector cells are administered in a later course of treatment, the subject has any of the following characteristics:
  • Factors indicative of cytokine release syndrome have a serum level fold in the subject that is about 10 times smaller and about 25 times smaller than the level in the subject immediately before the administration of immune effector cells in the previous course of treatment , And / or about 50 times smaller;
  • neurotoxicity or CRS levels are reduced compared to peak levels of neurotoxicity or CRS levels after administration of immune effector cells during the previous course of treatment;
  • the subject does not show a detectable humoral or cell-mediated immune response against CAR expressed by cells of the previous course of treatment.
  • the CRS level is compared with the peak level of CRS after administration of immune effector cells in a previous course of treatment, The reduction is at least 50%, preferably, at least 20%, more preferably, at least 5%, or the CRS level is comparable to the CRS level prior to the administration of immune effector cells in the previous course of treatment.
  • the method further comprises pre-treating the immune effector cells, the pre-treating comprising administering a chemotherapeutic agent or radiation therapy to the subject, or a combination thereof.
  • said pretreatment is performed 2-12 days before the administration of immune effector cells. In a preferred embodiment, it is performed 2-7 days before the administration of immune effector cells.
  • the chemotherapeutic agent is selected from any one or a combination of the following: cyclophosphamide, fludarabine.
  • the fludarabine is administered an amount of about 10-50mg / m 2 / day, or about 15-40mg / m 2 / day, or about 15-35mg / m 2 / day, or 15 -30 mg / m 2 / day, or about 20-36 mg / m 2 / day, or about 20-30 mg / m 2 / day.
  • the amount of fludarabine administered is about 20-30 mg / m 2 / day.
  • the amount of fludarabine administered is about 20-26 mg / m 2 / day.
  • the cyclophosphamide is administered is about 100-700mg / m 2 / day, or about 150-600mg / m 2 / day, or about 190-600mg / m 2 / day, or about 190 -560mg / m 2 / day.
  • the cyclophosphamide is administered in an amount of about 150-400 mg / m 2 / day, preferably about 190-350 mg / m 2 / day.
  • the administration of cyclophosphamide is about 400-600mg / m 2 / day, preferably about 450-600mg / m 2 / day, more preferably about 450-560mg / m 2 / day.
  • fludarabine and cyclophosphamide are administered simultaneously, the respective doses of fludarabine and cyclophosphamide are also as described above.
  • the chemotherapeutic agent is used continuously for no more than 6 days.
  • the cyclophosphamide is used continuously for 1-5 days.
  • the fludarabine is used continuously for 2-4 days.
  • the tumor is multiple myeloma.
  • the chimeric antigen receptor includes an antibody that specifically binds BCMA, a transmembrane domain, and an intracellular domain.
  • the heavy chain and light chain variable regions of the antibody have:
  • HCDR1 shown in SEQ ID NO: 1 HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, and LCDR1 shown in SEQ ID NO: 6 and LCDR2 shown in SEQ ID NO: 7 LCDR3 shown in SEQ ID NO: 8; or
  • HCDR1 shown in SEQ ID NO: 1 HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 4, and LCDR1 shown in SEQ ID NO: 6 and LCDR2 shown in SEQ ID NO: 7 LCDR3 shown in SEQ ID NO: 9; or
  • HCDR1 shown in SEQ ID NO: 1 HCDR2 shown in SEQ ID NO: 2
  • HCDR3 shown in SEQ ID NO: 5 LCDR1 shown in SEQ ID NO: 6
  • LCDR2 shown in SEQ ID NO: 7 LCDR3 shown in SEQ ID NO: 10; or
  • HCDR1 shown in SEQ ID NO: 11 HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7 LCDR3 shown in SEQ ID NO: 10; or
  • HCDR1 shown in SEQ ID NO: 13 HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6 and LCDR2 shown in SEQ ID NO: 7 LCDR3 shown in SEQ ID NO: 10.
  • the heavy chain variable region and light chain variable region of the antibody have HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12, and HCDR2 shown in SEQ ID NO: 12.
  • the chimeric antigen receptor includes an antibody that specifically binds BCMA, a transmembrane domain, and an intracellular domain.
  • the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 6. LCDR1, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 10.
  • the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20.
  • the heavy chain variable region of the antibody has HCDR3 as shown in SEQ ID NO: 5.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 16; or
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 17 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 18; or
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 19 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20; or
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20; or
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20.
  • the antibody has the sequence of the scFv shown in SEQ ID NO: 25, 27, or 29.
  • the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41.
  • the chimeric antigen receptor has the amino acid sequence shown in any one of SEQ ID NO: 36, 37, and 38.
  • the chimeric antigen receptor has an amino acid sequence represented by SEQ ID NO: 36.
  • the immune effector cells are T cells, NK cells or NKT cells; in a preferred embodiment, the immune effector cells are T cells.
  • the immune effector cell is from the subject's own body.
  • said immune effector cell is from said allogeneic.
  • the dosing interval of the immune effector cells is 4 to 24 weeks per course.
  • the number of immune cells in each course of treatment is substantially the same.
  • the number of immune effector cells administered in a later course is higher than the number of immune cells administered in a later course.
  • the number of immune effector cells administered in a later course is lower than the number of immune cells administered in a later course.
  • the subject prior to administering the immune effector cells, the subject has not been treated with immune cells that target a BCMA-expressing chimeric antigen receptor.
  • the subject prior to administering the immune effector cell therapy, has undergone surgical treatment, chemotherapy, or immunotherapy different from a BCMA-targeting immune cell expressing a chimeric antigen receptor.
  • the subject prior to administering immune effector cells for each course of treatment, the subject is indicated with a factor indicating CRS, a factor indicating neurotoxicity, a factor indicating tumor burden, and / or a host anti-CAR immunity
  • a factor indicating CRS a factor indicating CRS
  • a factor indicating neurotoxicity a factor indicating tumor burden
  • a host anti-CAR immunity The serum levels of responding factors were evaluated.
  • the factor indicating the tumor burden is: the total number of tumor cells in the subject, or the total number of tumor cells in the organ of the subject, or the tumor in the tissue of the subject The total number of cells, or the mass or volume of the tumor, or the degree of tumor metastasis, or the number of tumors.
  • the factors indicating tumor burden include:
  • the dose of the immune effector cells is about 0.1 ⁇ 10 6 cells / kg subject weight to 5 ⁇ 10 7 cells / kg subject weight, or the total dose of the immune effector cells is About 0.1x10 7 cells to 1x10 10 cells.
  • the total dose of the immune effector cells is about 0.1 ⁇ 10 8 cells to 1 ⁇ 10 9 cells.
  • the total dose of the immune effector cells is about 0.1 ⁇ 10 8 cells to 9 ⁇ 10 8 cells.
  • the BCMA expression rate of the subject is greater than 50%, preferably, greater than 70%, or greater than 80%. More preferably, it is greater than 85%. More preferably, it is greater than 90%.
  • the disease classification of the subject is IgG ⁇ type, or IgG ⁇ type, or IgA ⁇ type, or IgA ⁇ type, or ⁇ light chain type.
  • Figure 1 shows the efficacy results for different groups of patients.
  • Figure 2 shows the proliferation of BCMA CAR-T cells in a subject.
  • CAR chimeric antigen receptor
  • dose interval refers to the time elapsed between the administration of multiple courses of immune effector cell treatment to a subject and the administration of a pretreatment drug. Therefore, the dosing interval can be indicated as a range.
  • the "dose” described herein may be expressed as a dose calculated on a weight basis or a dose calculated on a body surface area (BSA) basis.
  • the dose calculated on the basis of the weight is the dose given to the patient calculated based on the weight of the patient, such as mg / kg, the number of immune effector cells / kg, and the like.
  • the dose calculated based on BSA is the dose given to the patient based on the patient's surface area, such as mg / m2 and the number of immune effector cells / m2.
  • number of administrations refers to the frequency with which a dose of immune effector cells or pretreatment drugs is administered within a given period of time.
  • the number of dosing can be indicated as the number of doses per given time.
  • fludarabine can be administered as follows: once daily for 4 consecutive days, once daily for 3 consecutive days, once daily for 2 consecutive days, or once daily.
  • Cyclophosphamide can be administered as follows: once daily for 4 consecutive days, once daily for 3 consecutive days, once daily for 2 consecutive days, or once daily.
  • This application relates to adoptive cells or immune effector cells for the treatment of tumors, including the administration of cells for one or more courses, and methods, compositions and articles of use thereof.
  • Cells typically express a chimeric antigen receptor, for example, a chimeric antigen receptor (CAR) or other transgenic receptor such as a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the present invention provides methods and compositions for treating diseases (eg, tumors) associated with BCMA expression.
  • the present invention provides a method for treating a tumor in a subject using adoptive cells or immune effector cells expressing a genetically engineered (recombinant) chimeric receptor.
  • the method includes single-course infusion of adoptive cells or immune effector cells, or multi-course infusion.
  • dose refers to the total number of adoptive cells or immune effector cells administered or returned within a course of treatment. In some embodiments, where multiple courses are included according to the methods described herein, the dose for each course is the same. In some embodiments, where a plurality of courses is included according to the methods described herein, the dose of each course is different.
  • “Divided dose” refers to a single administration dose in the case where the dose of the entire treatment course is divided into multiple administrations to a subject within a course of treatment. In some embodiments, where the dose within a course of treatment is divided into multiple administrations to a subject, the divided dose for each administration is the same. In some embodiments, where the dose within a course of treatment is divided into multiple administrations to a subject, the divided dose for each administration is different. For the purposes of this document, doses refer to the total number of adoptive cells or immune effector cells administered or returned within a course of treatment, unless otherwise specified.
  • the methods described herein include returning the adoptive cells or immune effector cells in a single course of treatment.
  • a single course of treatment refers to returning a certain amount of adoptive cells or immune effector cells within a certain period of time.
  • a certain amount of adoptive cells or immune effector cells are reinfused at one time during the course of treatment.
  • a certain amount of adoptive cells or immune effector cells are infused back in two or more times.
  • a certain amount of adoptive cells or immune effector cells are infused three or more times.
  • each adoptive adoptive cell or immune effector cell is an aliquot of the adoptive cell or immune effector cell to be returned.
  • each adoptive adoptive cell or immune effector cell is a non-equivalent portion of the adoptive cell or immune effector cell to be returned.
  • the amount of adoptive cells or immune effector cells per infusion is determined by the physician based on the specific circumstances of the subject.
  • the specific condition of the subject can be, for example, the overall health of the subject, the severity of the disease, the response to the previous dose of the same course of treatment, the response to the previous course of treatment, the combination of the subject, the degree of toxic reaction, or Likelihood, complications, and any other factors that the physician believes will affect the amount of adoptive or immune effector cells that the subject is suitable for infusion.
  • the amount of adoptive cells or immune effector cells that are infused each time is increasing. In some embodiments, among the number of adoptive cells or immune effector cells that are infused a plurality of times, the amount of adoptive cells or immune effector cells that are infused each time decreases. In some embodiments, among the number of adoptive cells or immune effector cells that are infused a plurality of times, the number of adoptive cells or immune effector cells that are infused each time increases first and then decreases. In some embodiments, among the number of adoptive cells or immune effector cells that are infused back a plurality of times, the amount of adoptive cells or immune effector cells that are infused each time decreases first and then increases.
  • Multi-course infusion refers to having a plurality of said time periods, and a certain amount of adoptive cells or immune effector cells are infused in each time period.
  • the lengths of the plurality of time periods are uniform.
  • the lengths of the plurality of time periods are unequal.
  • the multiple courses refers to having at least two of the time periods. In some embodiments, the multiple courses refers to having at least three or more of the time periods.
  • a certain amount of adoptive cells or immune effector cells are returned at a time within one of the plurality of treatment courses. In some embodiments, a certain amount of adoptive cells or immune effector cells are reinfused two or more times during one of the plurality of treatment courses. In some embodiments, a certain amount of adoptive cells or immune effector cells are returned in three or more times within one of the plurality of treatment courses. In some embodiments, within one of the multiple courses of treatment, the adoptive cells or immune effector cells returned each time are equal parts of the adoptive cells or immune effector cells to be returned.
  • the adoptive cells or immune effector cells returned each time are non-equivalent parts of the adoptive cells or immune effector cells to be returned.
  • the amount of adoptive cells or immune effector cells infused back within each course of the plurality of courses is determined by the physician according to the specific conditions of the subject.
  • the specific condition of the subject can be, for example, the overall health of the subject, the severity of the disease, the response to the previous dose of the same course of treatment, the response to the previous course of treatment, the combination of the subject, the degree of toxic reaction Likelihood, complications, cancer metastasis, and any other factors that the physician believes will affect the amount of adoptive or immune effector cells that the subject is suitable for infusion.
  • a certain amount of adoptive cells or immune effector cells are infused back in the same number of times in each of the plurality of treatment courses. In some embodiments, a certain amount of adoptive cells or immune effector cells are infused back at different times in each of the plurality of treatment courses. In some embodiments, a certain amount of adoptive cells or immune effector cells are infused back in the same number of times in each of the plurality of treatment courses. In some embodiments, the same amount of adoptive cells or immune effector cells are returned in each of the plurality of treatment courses. In some embodiments, a different amount of adoptive cells or immune effector cells are returned in each of the plurality of treatment courses.
  • the total number of adoptive cells or immune effector cells in each of the plurality of treatment courses is increasing. In some embodiments, the total number of adoptive cells or immune effector cells in each of the plurality of treatment courses is decreasing. In some embodiments, the total number of the adoptive cells or immune effector cells in each of the plurality of treatment courses has a tendency of increasing first and then decreasing. The total amount of the adoptive cells or immune effector cells in each of the plurality of treatment courses has a tendency of decreasing first and then increasing.
  • the methods described herein include monitoring a subject's degree of exposure to the adoptive cells or immune effector cells, and determining the dose and interval of subsequent divided doses or subsequent courses of treatment based on the degree of exposure time. In some embodiments, the methods described herein include monitoring a subject's degree of exposure to the adoptive cells or immune effector cells, and maintaining or reducing subsequent divided doses based on the degree of exposure reaching or exceeding a certain level or Dosing doses for subsequent courses and / or maintaining or extending the interval between subsequent divided doses or subsequent courses.
  • the methods described herein include monitoring a subject's degree of exposure to the adoptive cells or immune effector cells, and maintaining or increasing subsequent fractional administration or follow-up based on the degree of exposure being below a certain level The administered dose of a course of treatment and / or maintaining or shortening the interval between subsequent divided doses or subsequent courses of treatment.
  • the methods described herein include monitoring a subject's degree of toxicity or risk to the adoptive cells or immune effector cells, and determining subsequent fractional administration or subsequent treatment based on the degree of toxicity or risk The dose and interval of administration.
  • the degree or risk of toxic reactions includes, but is not limited to, for example, CRS, neurotoxicity, macrophage activation syndrome, tumor lysis syndrome, and the like.
  • immune effector cells are administered in a later course of treatment when a host-adaptive immune response to the cell has not been detected, has not been established, and / or has not reached a certain level or degree or stage.
  • the so-called tumor burden includes typing, staging, the proportion of abnormal plasma cells in the bone marrow at the time of onset, cytogenetic changes, the presence of extramedullary infiltration, and the response to treatment.
  • the tumor burden passes serum, Urine protein electrophoresis, bone marrow smear, bone marrow biopsy, MRD, MRI, and / or CT were evaluated.
  • the immune effector cell is administered in an amount that includes a cell dose in an amount sufficient to reduce the tumor burden in the subject.
  • the serum level of a factor indicative of cytokine-release syndrome (CRS) in a subject does not exceed 10 or 25 times the serum level of the subject prior to the administration of immune effector cells when cells are administered in a later course of treatment And / or the peak level of CRS-related results in the subject and began to decline after the administration of immune effector cells, and the subject did not have a chimeric antigen receptor specifically expressed by the cells of the immune effector cells in the previous course of treatment. Detection of adaptive host immune response.
  • the immune effector cell is administered at a dose of not less than about 0.5 ⁇ 10 5 cells / kg of the subject's body weight or a total dose of not less than 1 ⁇ 10 5 cells; preferably, not less than about 0.1 ⁇ 10 6 cells / kg body weight or total dose is not less than 1x10 6 cells; preferably, not less than about 0.5x10 6 cells / kg subject weight or total dose is not less than 1x10 7 cells; preferably, not less than about 0.9x10 6 cells / kg body weight of the subject or the total dose of not less than 1x10 7 cells.
  • the immune effector cells are administered at a dose of no more than about 1 ⁇ 10 9 cells / kg of the subject ’s weight or total amount not more than about 1 ⁇ 10 10 cells; preferably, no more than about 1 ⁇ 10 8 cells or the total number of cells
  • the amount does not exceed about 1x10 9 cells; preferably, not more than about 1x10 7 cells or the total number of cells does not exceed about 1x10 9 cells, preferably, not more than about 5x10 6 cells or the total number of cells does not exceed about 9x10 8 cell.
  • the dose of the immune effector cells is about 0.1 ⁇ 10 6 cells / kg subject weight to 5 ⁇ 10 7 cells / kg subject weight, or the total dose of the immune effector cells is About 0.1x10 7 cells to 1x10 10 cells.
  • it is about 0.5 ⁇ 10 6 cells / kg subject weight to 1 ⁇ 10 7 cells / kg subject weight, or the total dose of the immune effector cells is about 0.1 ⁇ 10 8 cells to 1 ⁇ 10 9 cells. More preferably, it is about 0.9 ⁇ 10 6 cells / kg subject weight to 5 ⁇ 10 6 cells / kg subject weight, or the total dose of the immune effector cells is about 0.1 ⁇ 10 8 cells to 9 ⁇ 10 8 cells.
  • the method provided by the present invention comprises administering at least one course of immune effector cells expressing a chimeric antigen receptor (such as CAR, TCR, TFP, TAC) that recognizes BCMA to a tumor subject expressing BCMA.
  • a chimeric antigen receptor such as CAR, TCR, TFP, TAC
  • a "subject" is a mammal, such as a human or other animal, usually a human.
  • the subject has been treated with chemotherapy or radiation prior to the administration of immune effector cells in a previous course and / or the administration of immune effector cells in a subsequent course.
  • the subject is refractory or non-responsive to other therapeutic agents.
  • the tumor is persistent or relapsed, for example, after intervention with another treatment (including chemotherapy, radiation), the tumor still progresses or relapses after control. .
  • the subject is responsive to other therapeutic agents, reducing tumor burden.
  • the subject is initially responsive to the therapeutic agent, but shows recurrence of the tumor over time.
  • the subject is determined to have a risk of recurrence, for example at a high risk of recurrence, and thus the CAR T cells of the invention are prophylactically administered in order to reduce the likelihood of recurrence or prevent recurrence.
  • the size of the dose and the time of infusion are determined by the subject's initial tumor burden. For example, in some cases, the number of cells of immune effector cells that are generally given to the subject during the previous course of treatment is lower, and in the case of lower tumor burden, such as solid tumors, tumor tumor size can be assessed by tumor marker detection and / Or small residual lesions, the initial dose may be higher. In other cases, in subjects with higher tumor burden, continuous cell infusions can be used for each course, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
  • the single administration is preferably 1-5 administrations, and more preferably, 2-3 administrations. Each administration is separated by 1, 2, 3, 4, 5, 6 days or more.
  • treatment refers to the complete or partial reduction or reduction of a tumor or a symptom associated therewith, and the improvement of an objective indicator related to the assessment. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a tumor, reducing symptoms, reducing any direct or indirect pathological results of the tumor, preventing extramedullary disease, slowing the rate of tumor progression, improving or reducing the state of the tumor, and reducing or Improve prognosis.
  • inhibiting a function or activity means a reduction in function or activity when compared to the same condition in other circumstances or compared to another condition.
  • the cell therapy can be treated by autologous infusion.
  • the cells are derived from a subject in need of treatment and are administered to the same subject after isolation and processing.
  • the cell therapy is treated by allogeneic infusion, wherein the cells are isolated and / or otherwise extracted and prepared from the donor, and the donor and the subject returning the cells different.
  • the donor and recipient are genetically the same.
  • the donor and recipient are genetically similar.
  • the recipient and the donor are of the same HLA category or supertype.
  • the cells can be administered by any suitable means, for example, by injection, such as intravenous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, reverse interval injection, subscleral injection, intrachoroidal injection, anterior chamber Injection, subconjectval injection, subconjuntival injection, suprascleral injection, post-ball injection, periocular injection, or peri-ball delivery.
  • injection such as intravenous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, reverse interval injection, subscleral injection, intrachoroidal injection, anterior chamber Injection, subconjectval injection, subconjuntival injection, suprascleral injection, post-ball injection, periocular injection, or peri-ball delivery.
  • the cells are administered by drip.
  • the cells are administered by multiple instillations, for example, multiple administrations over no more than 30 days, or the cells are administered by continuous infusion.
  • the dose administered may depend on the type of disease, the chimeric antigen receptor or cell type, the severity and duration of the disease, the subject's previous treatment history, and the response of the administered cells, and the treating physician Judge's judgment.
  • the immune effector cells are used as part of a combination therapy, for example, in combination with another interventional therapy, such as antibodies, engineered immune effector cells, receptors or agents, cytotoxic drugs, or other treatment methods , Given simultaneously or sequentially, in any order.
  • immune effector cells are used in combination with one or more other treatments, or in combination with another interventional treatment, simultaneously or sequentially in any order.
  • the immune effector cells are co-administered with another treatment at a time close enough to produce a therapeutic effect greater than the aforementioned immune effector cell population or one or more other therapeutic drugs or methods, and vice versa.
  • the immune effector cell is administered before one or more other therapeutic agents.
  • the immune effector cell is administered after one or more other therapeutic agents.
  • one or more other therapeutic agents include cytokines, such as IL-2, IL-12, to enhance persistence.
  • the method includes administering a pretreatment prior to administering immune effector cells, such as, for example, a chemotherapeutic drug (chemotherapeutic agent), systemic radiation, local radiation therapy, etc., or a combination thereof.
  • a chemotherapeutic drug chemotherapeutic agent
  • the method includes pretreating the subject with one or more chemotherapeutic agents.
  • the method comprises pretreating a subject with a tubulin inhibitor and one or more other chemotherapeutic agents.
  • the effects of pretreatment are considered to include, but not limited to, lymphocyte clearance, reducing tumor burden, and the like.
  • the chemotherapeutic agent refers to a drug used in chemotherapy, for example, cyclophosphamide, fludarabine, a protease inhibitor (such as bortezomib, carfilzomib, etc.), an immunomodulator (such as thalidomide, Lenalidomide, pomalidomide, etc.), melphalan, doxorubicin, dexamethasone, prednisone, etc.
  • a drug used in chemotherapy for example, cyclophosphamide, fludarabine, a protease inhibitor (such as bortezomib, carfilzomib, etc.), an immunomodulator (such as thalidomide, Lenalidomide, pomalidomide, etc.), melphalan, doxorubicin, dexamethasone, prednisone, etc.
  • Pretreatment can improve the effect of immune effector cell therapy.
  • the first day of infusion of immune effector cells e.g., CAR T cells
  • Pretreatment can be given at any time prior to the administration of immune effector cells.
  • fludarabine or cyclophosphamide is used alone or in combination as a pretreatment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 9 before CAR-T cell infusion. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days, preferably at least 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, more preferably, at least 2, 3, 4, 5 Subjects were administered on, 6, 7, or 8 days.
  • pretreatment includes administering fludarabine and cyclophosphamide 2-12 days before CAR-T cell infusion. In some embodiments, pretreatment includes administering fludarabine and cyclophosphamide 7 days before CAR-T cell infusion.
  • the timing of the pretreatment component can be adjusted so that the CAR T treatment achieves the maximum effect.
  • fludarabine and cyclophosphamide can be given daily.
  • fludarabine is administered daily
  • cyclophosphamide is administered for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.
  • fludarabine is administered daily for 4 consecutive days
  • cyclophosphamide is administered daily for 2 consecutive days.
  • fludarabine is administered daily for 2 consecutive days
  • cyclophosphamide is administered daily for 2 consecutive days.
  • fludarabine is administered daily for 3 consecutive days
  • cyclophosphamide is administered once, or cyclophosphamide is administered continuously for 2 or 3 or 5 days.
  • the day when the patients were given CAR T cell therapy in each round was designated as the 0th day.
  • the patient is given fludarabine on the 6th (i.e., -6th), -5th, and -4th days before the 0th day.
  • the patient is administered fludarabine on days -5, -4, and -3.
  • the patient is administered fludarabine on days -6, -5, -4, and -3.
  • the patient is administered fludarabine on days -5, -4, -3, and -2.
  • the patient is administered fludarabine on days -4, -3, and -2.
  • the patient is administered fludarabine on days -3 and -2. In some embodiments, the patient is administered fludarabine on days -3, -2, and -1. In some embodiments, the patient is administered cyclophosphamide on days -6, -5, -4, -3, and -2. In some embodiments, the patient is administered cyclophosphamide on days -6 and -5. In some embodiments, the patient is administered cyclophosphamide on days -4, -3, and -2. In some embodiments, the patient is administered cyclophosphamide on days -4 and -3. In some embodiments, the patient is administered cyclophosphamide on days -5 and -4.
  • the patient is administered cyclophosphamide on day -5. In some embodiments, the patient is administered cyclophosphamide on days -3 and -2. In some embodiments, the patient is administered cyclophosphamide on days -3, -2, and -1.
  • Fludarabine and cyclophosphamide can be given on the same day or on different days.
  • fludarabine and cyclophosphamide may be administered simultaneously or sequentially.
  • Fludarabine and cyclophosphamide can be administered by any route, including intravenous drip (I.V.). In some embodiments, fludarabine and cyclophosphamide can be administered according to the drug instructions for fludarabine and cyclophosphamide.
  • I.V. intravenous drip
  • fludarabine and cyclophosphamide can be administered according to the drug instructions for fludarabine and cyclophosphamide.
  • adoptive cell therapy or immune effector cell therapy is selected from the group consisting of tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.
  • TIL tumor infiltrating lymphocyte
  • eACT engineered autologous cell therapy
  • allogeneic T cell transplantation is selected from the group consisting of tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.
  • the immune effector cell therapy is the administration of a chimeric antigen receptor modified T cell that targets a tumor antigen (such as BCMA).
  • pretreatment includes administering fludarabine no higher than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mg / m 2 / day; and / or given cyclophosphamide not higher than about 700, 690, 680, 670, 660, 650, 640, 630, 620, 610, 600, 595, 590, 585, 580, 579, 578, 576, 575, 574, 573, 572, 571, 570, 569, 568, 567, 566, 565, 564, 563, 562, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545,
  • the fludarabine is administered an amount of about 10-50mg / m 2 / day, or about 15-40mg / m 2 / day, or about 15-35mg / m 2 / day, 15-30mg or / m 2 / day, or about 20-30 mg / m 2 / day.
  • the cyclophosphamide is administered is about 100-700mg / m 2 / day, or about 150-600mg / m 2 / day,, or from about 190-600mg / m 2 / day, or about 190 to 560 mg / m 2 / day.
  • the cyclophosphamide is administered in an amount of about 150-400 mg / m 2 / day, preferably about 190-350 mg / m 2 / day.
  • the cyclophosphamide is administered is about 400-600mg / m 2 / day, preferably about 450-600mg / m 2 / day, more preferably about 450-560mg / m 2 / day.
  • cyclophosphamide and fludarabine may cause adverse reactions in patients after administration.
  • the scope of the invention includes administering a composition to a patient to reduce some of these adverse events.
  • the method comprises administering physiological saline to the patient.
  • the patient may be given saline before or after administration of cyclophosphamide and / or fludarabine, or before and after administration of cyclophosphamide and / or fludarabine.
  • the patient is given saline before cyclophosphamide and / or fludarabine is administered on each infusion day, and after cyclophosphamide and / or fludarabine is administered.
  • adjuvants and excipients can be administered to patients.
  • mesna sodium 2-mercaptoethanesulfonate
  • patients can be administered exogenous cytokines.
  • pretreatment prior to infusion of immune effector cells in a previous course or immune effector cells in a subsequent course improves the outcome of the treatment. For example, in some aspects, pretreatment improves the efficacy of treatment with immune effector cells in a previous course or immune effector cells in a later course, or increases the persistence of immune effector cells (such as CAR-T cells) in a subject . In some embodiments, pretreatment treatment increases the stable phase of the disease.
  • the sustained survival of the immune effector cell population in vivo and its biological activity are measured by any of a number of known methods.
  • the continuous survival period of CAR-T cells in vivo is the continuous survival period of CAR-T cells in vivo after the "implantation" of CAR-T cells.
  • the number of copies of CAR DNA contained in peripheral blood was detected by Q-PCR at the visiting points until any two consecutive tests were negative, and the CAR-T cell survival period was recorded.
  • the biological activity of immune effector cells can be assessed by the specific binding of engineered or natural T cells or other immune cells to the antigen, such as by ELISA or flow cytometry.
  • the cell killing capacity of engineered immune effector cells can be detected using any suitable method known in the art, such as by measuring certain cytokines such as CD107a, IFN ⁇ , IL-2 and TNF Expression and / or secretion to measure the biological activity of immune effector cells.
  • biological activity is assessed by clinical outcomes, such as tumor burden or reduction in burden.
  • the cells are evaluated for toxicity, persistence and / or proliferation, and / or the presence or absence of a host immune response.
  • the administration of a given "dose” includes a single composition and / or a single uninterrupted administration, such as a single injection or continuous infusion to a given amount or number of immune effector cells, and Also included in no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 20, 19, 18, 17, 16, 15, 15, 14, 13, 12, 11, 10, 9, 8, 7
  • a given period of time of 6, 6, 5, 4, 3 or 2 days divided doses are provided in multiple separate compositions or infusions to give a given amount or number of immune effector cells. Therefore, each course of immune effector cells is a single or continuous administration of a specified number of immune effector cells given or started at a single time point.
  • the immune effector cells in each course do not exceed 30, 29, 28, 27, 26, 25, 24, 23, 22, 20, 19, 18, 17, 16, 15, 14, 14, 13 Multiple injections or infusions over a period of 1, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, such as once a day for three or two days, or by one day Multiple infusions within.
  • the immune effector cells of the previous course of immune effector cells are administered as a single pharmaceutical composition.
  • the term "after the course of treatment” refers to the dose given to the subject without any intervening doses being given to the subject during the period after the first course of treatment. Nonetheless, the term does not include the second, third, and / or other injections or infusions in a series of infusions or injections contained in a single divided dose. Therefore, unless otherwise stated, a second infusion within one, two, or three days is not considered a "post-treatment course” as used herein. Similarly, the second, third, and other doses in a series of multiple doses within a divided dose are not considered to be “intervening" doses in the meaning of the "after course” dose.
  • the dose given over a period of more than 30 days after the first course or after the start of the first course The dose considered to be "after the course".
  • multiple administrations of the same immune effector cells are considered as a single dose over a period of up to 30 days, and 30 days after the initial administration (the first infusion of each course (i.e. the initial Administration of immune effector cells on the first day of the day) is not considered to be administered after the course of treatment and is not considered to determine whether the second dose is "continuous" with the immune effector cells of the previous course of treatment. Insert dose.
  • first course is used to describe the total dose administered during the first course of treatment performed according to the methods described herein. This dose is equal to the total dose given in a single course of administration, or the total dose given in the first course of multiple courses.
  • the term does not necessarily mean that the subject has never been treated with immune effector cells before, or that the subject has not previously been treated with immune effector cells that target the same antigen.
  • the size of the dose of the immune effector cells of the previous course and / or of the immune effector cells of the subsequent course is usually designed to provide improved efficacy and / or reduced risk of toxicity.
  • the immune cell dose per course is higher than, lower than, or equal to about 0.5 ⁇ 10 6 cells / kg subject weight to 1 ⁇ 10 7 cells / kg subject weight, such as higher, lower, or Equal to about 0.6 ⁇ 10 6 , 0.7 ⁇ 10 6 , 0.8 ⁇ 10 6 , 0.9 ⁇ 10 6 , 1.0 ⁇ 10 6 , 1.1 ⁇ 10 6 , 1.2 ⁇ 10 6 , 1.3 ⁇ 10 6 , 1.4 ⁇ 10 6 , 1.5 ⁇ 10 6 , 1.6 ⁇ 10 6 , 1.7 ⁇ 10 6 , 1.8 ⁇ 10 6 , 1.9 ⁇ 10 6 , 2.0 ⁇ 10 6 , 2.1 ⁇ 10 6 , 2.2 ⁇ 10 6 , 2.3 ⁇ 10 6 , 2.4 ⁇ 10 6 ,
  • the total dose of immune cells administered per course is higher than, lower than, or equal to about 0.1 ⁇ 10 8 cells to 1 ⁇ 10 10 cells.
  • it is higher than, lower than or equal to about 1 ⁇ 10 6 cells / kg subject weight to 1 ⁇ 10 7 cells / kg subject weight, or the total dose of the immune effector cells is higher, lower or Equal to about 0.5x10 8 cells to 1x10 9 cells.
  • it is higher than, lower than or equal to about 1.5x10 6 cells / kg subject weight to 3x10 6 cells / kg subject weight, or the total dose of the immune effector cells is about 0.5x10 8 cells to 5x10 8 cells.
  • the number of immune effector cells refers to the number of immune effector cells expressing the chimeric antigen receptor, such as the number of CAR-T cells. In other embodiments, the number of immune effector cells may also refer to the number of T cells or PBMCs or total cells administered.
  • the dose after the course of treatment is sufficient to reduce tumor burden or an indicator thereof, and / or one or more symptoms of the disease or disorder.
  • the tumor burden such as the proportion of plasma cells in the bone marrow, blood / urinary M protein, and extramedullary soft tissue plasma cell tumors, is reduced by about 1 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%.
  • M protein is a large number of abnormal immunoglobulins produced by the malignant proliferation of plasma cells or B lymphocytes.
  • the dose specifically disclosed in the present invention is a safe and effective dose obtained by the inventor after research.
  • Those skilled in the art may The tumor load, the patient's own physical condition, and other factors determine the dose of immune effector cells in the previous course of treatment; if further administration of immune effector cells in the later course of treatment is needed, those skilled in the art can, for example, change the tumor load after giving immune effector cells To determine the dose of immune effector cells in the later course of treatment.
  • the dose of immune effector cells for each course includes an amount that does not cause or reduce toxicity, and the toxicity may be cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation Syndrome, tumor lysis syndrome, fever of at least 38 degrees Celsius or about 38 degrees Celsius for three or more days, CRP plasma levels at least equal to about 20 mg / dL, and / or neurotoxicity.
  • the number of cells administered in an immune effector cell after a course of treatment is determined based on the likelihood that the subject will show toxicity or toxic results after the cells are administered. For example, in some embodiments, the likelihood of developing a toxic outcome in a subject is predicted based on tumor burden.
  • the method includes detecting or assessing toxicological results and / or tumor burden before administering immune effector cells.
  • the dosing time in the subsequent course is calculated from the completion of the previous course (the total dose infusion completion date of the previous course is set to 0 day).
  • the serum level of a factor indicative of CRS in a subject is no more than about 10-fold, 25-fold, 50-fold, or 50-fold greater than the serum level of the indicator in the subject after the administration of immune effector cells in the previous course At 100 times, immune effector cells were administered during the subsequent course.
  • a CRS-related result e.g., a serum factor associated with or indicative of CRS
  • a clinical sign or symptom such as fever, hypoxia, hypotension, or neurological disorder in a subject
  • the immune effector cells are given in the later course of treatment.
  • the immune effector cells are administered during a later course of treatment when a decrease is observed after administration compared to the highest level of such results, or when the maximum or level of results is reached after administration.
  • an indicator of a toxic effect eg, a serum indicator of CRS
  • the Immune effector cells are administered after a course of treatment when the subject does not show CRS or does not show severe CRS.
  • the immune effector cells in the subsequent course are administered at a time point when the tumor load in the patient is reduced compared to the tumor load before the immune effector cells administered in the previous course.
  • the immune effector cells of the subsequent course of treatment are given the immune effector cells of the previous course of treatment, when the tumor burden such as the proportion of plasma cells in the bone marrow, blood / urine M protein, extramedullary soft tissue plasmacytoma has been reduced 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more.
  • the immune effector cell in a subsequent course is administered when the subject's disease or condition does not recur after a decrease in response to the previous course or in response to the previous dose.
  • reducing tumor burden is indicated by a reduction in one or more indicators, such as typing, staging, the proportion of abnormal plasma cells in the bone marrow at the time of onset, cytogenetic changes, the presence of extramedullary infiltration, and Response after treatment.
  • immune effector cells are administered in a later course of treatment when the host adaptive immune response has not been detected, has not been established, or has not reached a certain level, degree, or stage. In some aspects, immune effector cells are administered in a later course of treatment before the subject's memory immune response develops.
  • the time between the administration of immune effector cells in a previous course and the administration of immune effector cells in a subsequent course is from about 4 weeks to about 24 weeks.
  • additional or further immune effector cells are administered after the subsequent course of immune effector cells, for example, a second immune effector cell (the third course dose), a third Immune effector cells in the later course (dose of the fourth course), and so on.
  • the tumor burden is reduced by at least about 25%, 30%, after administration of the immune effector cells in the subsequent course, compared to the time immediately before or after the immune effector cells in the subsequent course, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more.
  • tumor burden can be assessed by serum, urine protein electrophoresis, bone marrow smear, bone marrow biopsy, MRD, MRI, and / or CT.
  • one or more doses does not exist in the subject's immune response (eg, an adaptive or specific immune response to a transgenic receptor or cell), and is not detectable, Or when it can not be detected above a certain level.
  • the presence or extent of a specific immune response to a transgene is often related to the immunogenicity of the recipient (e.g., a CAR expressed by a cell or a transgenic TCR) and / or the time the subject is exposed to the cell.
  • an immune response, an adaptive or specific immune response, a detectable immune response, and / or a memory response to a chimeric antigen receptor or cell has been developed in a subject. Effector cells.
  • the decision of when and / or whether to give immune effector cells at a later course of treatment depends on whether the subject exhibits such an immune response or a detectable reading thereof, such as for a cell or a chimeric antigen receptor Specific detectable specific or adaptive host immune responses, such as CAR expressed by cells of immune effector cells in a previous course of treatment, and / or whether such a response was detected at a certain level.
  • the subject is not administered an immune effector cell at a later course of treatment.
  • a specific or adaptive (e.g., humoral or cell-mediated) immune response to the receptor e.g., CAR expressed by cells of an immune effector cell in the previous course
  • a detectable level or an acceptable level of such a response or indicator is administered to immune effector cells during a later course of treatment.
  • the subject exhibits reduced humoral or cell-mediated CAR against cells expressed by immune effector cells of the previous course of treatment when administered to immune effector cells of the later course of treatment compared to when the initial dose is greater. Immune response.
  • provided methods increase the persistence of a subject to an administered immune effector cell, e.g., an increase in the number or duration of cells over time, and / or improve efficacy and treatment outcomes in immune cell therapy .
  • this method has the advantage that compared to other methods, cells (such as CAR-T cells) that express the chimeric antigen receptor can improve treatment outcomes to a greater extent and / or longer. These results can include patient survival and remission, even in subjects with severe tumor burden.
  • the presence of cells expressing a chimeric antigen receptor e.g., CAR-expressing cells
  • a chimeric antigen receptor e.g., CAR-expressing cells
  • quantitative PCR is used to assess the amount of cells (e.g., CAR-T) that express a chimeric antigen receptor in the subject's blood or serum or organ or tissue (e.g., a disease site).
  • persistence is quantified as a copy of DNA or plasmid encoding a receptor per microgram of DNA, such as CAR, or as receptor expression per microliter of sample, such as blood or serum, such as the number of CAR-expressing cells, or Total number of peripheral blood mononuclear cells (PBMC) or white blood cells or T cells per microliter of sample.
  • a receptor per microgram of DNA such as CAR
  • receptor expression per microliter of sample such as blood or serum, such as the number of CAR-expressing cells, or Total number of peripheral blood mononuclear cells (PBMC) or white blood cells or T cells per microliter of sample.
  • PBMC peripheral blood mononuclear cells
  • the administration of immune effector cells in a previous course or at least on the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, Cells were detected in the subject at 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • at least at least 2, 4, or 6 weeks after the first or subsequent course of immune effector cells are administered, or at 3, 6 or 12, 18 or 24, or 30 or 36 months, or 1, 2, 3 Cells were detected at 4, 4, 5 or more years.
  • the method results in at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, Maximum concentration of 1500, 2000, 5000, 10,000, or 15,000 copies or receptor-encoding nucleic acids, such as CAR per microgram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 Somatic expression, such as the total number of CAR-expressed cells / peripheral blood mononuclear cells (PBMC), total monocytes, total T cells, or total microliters.
  • PBMC peripheral blood mononuclear cells
  • the receptor-expressing cells are detected as at least 10%, 20%, 30%, 40%, 50%, or 60% of the total PBMCs in the subject's blood, and / or at this level upon first administration Drug or subsequent administration for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks, or for 1 week after such administration , 2, 3, 4 or 5 years or more.
  • the method results in, for example, a copy of a nucleic acid encoding a chimeric antigen receptor, such as CAR, in a subject's serum increased by at least 2-fold, at least 4-fold, at least 10-fold, or at least 20-fold per microgram of DNA.
  • a chimeric antigen receptor such as CAR
  • the receptor-expressing cells can be detected in the subject's blood or serum, for example, by a specified method, such as qPCR or a flow cytometry-based detection method, the immune effector cells given to the previous course of treatment At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19 after administration of immune effector cells , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 or more days, or after the administration of immune effector cells in a previous course or later
  • the course of immune effector cells continues for at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 or more weeks.
  • the number of copies of a nucleic acid encoding a chimeric antigen receptor per 100 cells such as a vector copy number, such as in peripheral blood or bone marrow or In the chamber, at least 0.01, at least 0.1, at least 1, or at least 10, the cells are administered, for example, about 1 week, about 2 weeks, about 3 weeks after the immune effector cells in the previous course or the immune effector cells in the subsequent course Week, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 2 or 3 years .
  • the immune effector cells express a chimeric antigen receptor that recognizes BCMA or a variant thereof.
  • 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 antibody. Heavy (VH) chain regions and / or variable light (VL) chain regions, such as scFv antibody fragments.
  • the heavy chain and light chain variable regions of the antibody have: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR2 shown in SEQ IDNO: 2 HCDR3, and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 8, or at least 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences; or HCDR1 shown in SEQ ID NO: 1 , HCDR3 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 4, and LCDR1 shown in SEQ IDNO: 6, LCDR2 shown in SEQ IDNO: 7, LCDR2 shown in SEQ IDNO: 7, and LCDR3, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%
  • the heavy chain variable region and light chain variable region of the antibody have HCDR1 shown in SEQ ID NO: 11, HCDR2 shown in SEQ ID NO: 12, and SEQ ID NO: 5 HCDR3 and LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 10, or at least 85%, 86%, 87%, 88% of the above sequence , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity amino acid sequences.
  • the light chain variable region of the antibody has LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 10, or the same sequence as above Amino acids with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity sequence.
  • the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20, or has at least 85%, 86%, 87%, 88%, 89%, 90% of the above sequence. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity amino acid sequences.
  • variable region of the heavy chain of the antibody has HCDR3 shown in SEQ ID NO: 5
  • variable region of the light chain has the amino acid sequence shown in SEQ ID NO: 20, or has at least 85 with the above sequence. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity amino acid sequences.
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 15 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 16, or Identical to the above sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
  • the variable region of the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO: 17 and the variable region of the light chain of the antibody has the amino acid sequence shown in SEQ ID NO: 18, or Identical to the above sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
  • the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 19 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20, or With at least 85%, 8
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 21 and the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 20, or Identical to the above sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Sexual amino acid sequence.
  • the antibody has the sequence of the scFv shown in SEQ ID NO: 25, 27, or 29, or has at least 85%, 86%, 87%, 88%, 89%, 90% of the sequence described above. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity amino acid sequences.
  • the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41, or The above sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity Amino acid sequence.
  • the chimeric antigen receptor has the amino acid sequence shown in any one of SEQ ID NO: 36, 37, 38, or has at least 85%, 86%, 87%, 88%, Amino acid sequences with 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 36, or has at least 85%, 86%, 87%, 88%, 89%, 90%, Amino acid sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the antibody portion of a chimeric antigen receptor further includes a linker 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.
  • a linker 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.
  • the constant region or portion is of human IgG, such as IgG4 or IgG1.
  • the antigen recognition domain is typically linked to one or more intracellular signal transduction moieties, such as in the case of CAR, mimicking an activated signal transduction moiety through an antigen receptor complex (eg, a TCR complex), and / or via Signal from another cell surface receptor.
  • an antigen-binding component eg, an antibody
  • the transmembrane domain is fused to the extracellular domain.
  • a transmembrane domain that is one of the domains in a naturally associated receptor (eg, CAR) is used.
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding the domain to the transmembrane domain of the same or different surface membrane protein, so that Minimize interaction.
  • the transmembrane domain is derived from a natural or synthetic source.
  • the domain is derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions include ⁇ -, ⁇ -, or ⁇ chains derived from T-cell receptors, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 CD137, CD154, and / or transmembrane regions contain those functional variants (such as those that substantially retain their structural parts (eg, transmembrane structural parts), properties) (ie, include at least their transmembrane regions).
  • the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, such as CD8 ⁇ or a functional variant thereof.
  • the transmembrane domain is synthetic.
  • the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine.
  • trimers of phenylalanine, tryptophan, and valine will occur at each end of a synthetic transmembrane domain.
  • the linking occurs through a linker, a spacer, and / or a transmembrane domain.
  • the intracellular signal transduction domain includes those that mimic or approximate signals through natural antigen receptors, signals that co-stimulate receptors through such receptors, and / or signals that co-stimulate receptors alone.
  • a short oligopeptide or polypeptide linker is present, for example, a linker that is 2-10 amino acids in length, such as a linker comprising glycine and serine, for example, a glycine-serine duplex, and in the cytoplasm of the CAR A connection is formed between the signal transduction domain and the transmembrane domain.
  • the receptor for example, CAR, generally includes at least one type of one or more intracellular signal transduction moieties.
  • the receptor includes an intracellular component of a TCR complex, such as a TCRCD3 + chain, such as a CD3 ⁇ chain, that mediates T-cell activation and cytotoxicity.
  • the antigen binding moiety is connected to one or more cellular 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 eg, CAR
  • the receptor further comprises a portion of one or more other molecules, such as an Fc receptor gamma, CD8, CD4, CD25, or CD16.
  • a CAR or other chimeric antigen receptor includes a chimeric molecule between CD3 [zeta] (CD3- [zeta]) or an Fc receptor [gamma] 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 functions or responses of immune cells, such as engineered T cells engineered to express CAR.
  • CAR induces the function of T cells, such as cytolytic activity or helper T cell activity, such as secretion of cytokines or other factors.
  • a truncated portion of an intracellular signal transduction domain of an antigen receptor moiety or a costimulatory molecule is used in place of a complete immunostimulatory chain, for example, if it transduces effector function signals.
  • the intracellular signal transduction domain includes the cytoplasmic sequence of T cell receptors (TCRs), and in some aspects, also those co-receptors that occur in nature acting in concert with these receptors to Signal transduction is initiated after antigen receptor binding.
  • TCRs T cell receptors
  • the CAR does not include components for generating a co-stimulatory signal.
  • other CARs are expressed in the same cell and provide components for generating a second or costimulatory signal.
  • T cell activation is described as mediated by two types of cytoplasmic signal transduction sequences: those that initiate antigen-dependent primary activation by TCR (primary cytoplasmic signal transduction sequences), and in an antigen-independent manner 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 (e.g., CAR) further includes a signal peptide, such as a signal peptide including CD8 or a variant thereof, such as comprising the amino acid sequence shown in SEQ ID NO: 35, or IDNO: 35 shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or more sequence identity amino acid sequences.
  • the CAR includes a major cytoplasmic signal transduction sequence that modulates the initial activation of the TCR complex.
  • the first cytoplasmic signal transduction sequence acting in a stimulating manner may comprise a signal transduction motif, which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM include first cytoplasmic signal transduction sequences, including those derived from: TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b, and CD66d.
  • the cytoplasmic signal transduction molecule in the CAR comprises a cytoplasmic signal transduction domain, which is partially or derived from a sequence of CD3 ⁇ .
  • the CAR includes a transmembrane portion of a costimulatory receptor and / or a signal transduction domain, such as CD28, CD137, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both activating and co-stimulating portions.
  • the activation domain is included in one CAR and the co-stimulatory component is provided by another CAR that recognizes another antigen.
  • the CAR comprises an activated or stimulated CAR, a co-stimulated CAR, all of which are expressed on the same cell (see WO2014 / 055668).
  • the cells include one or more of a stimulating or activating CAR and / or a co-stimulating CAR.
  • the cell further comprises an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl.
  • identifying cells other than those associated with and / or specific to a disease or disorder CAR, thereby activating signals delivered by the disease-targeting CAR, are reduced or inhibited by inhibiting the binding of CAR to its ligand, such as reducing off-target effects.
  • the intracellular signal transduction portion of the chimeric antigen receptor, such as CAR comprises a CD3 ⁇ intracellular domain and a co-stimulatory signal transduction region.
  • the intracellular signal transduction domain comprises a CD28 transmembrane and a signal transduction domain connected to a CD3 (eg, CD3- ⁇ ) intracellular domain.
  • the intracellular signal transduction domain comprises a chimeric CD28 and / or CD137 (4-1BB, TNFRSF9) co-stimulatory domain that is linked to a CD3 ⁇ intracellular domain.
  • the CAR encompasses one or more, for example, two or more, a co-stimulatory domain and an activation domain, for example, an initial activation domain in a cytoplasmic portion.
  • exemplary CARs include the intracellular portions of CD3- [zeta], CD28, and CD137.
  • the CAR is referred to as a first, second, and / or third generation CAR.
  • the first-generation CAR is a CAR that provides only the CD3 chain-inducing signal upon antigen binding;
  • the second-generation CAR is a CAR that provides such signals and co-stimulatory signals, including, for example, from co-stimulatory receptors ( (Eg, CD28 or CD137), a CAR of an intracellular signal transduction domain;
  • a third-generation CAR is a CAR that includes multiple co-stimulatory domains of different co-stimulatory receptors.
  • the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, a 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 comprises a scFv, and the intracellular domain comprises ITAM. In some aspects, the intracellular signal transduction domain comprises a signal transduction domain of the zeta chain of the CD3-zeta chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain that connects an extracellular domain and an intracellular signal transduction domain. In some aspects, the transmembrane domain comprises a transmembrane portion of CD28.
  • the chimeric antigen receptor contains an intracellular domain of a T-cell costimulatory molecule.
  • the extracellular domain and the transmembrane domain can be connected directly or indirectly.
  • the extracellular domain and the transmembrane pass a linker sequence.
  • the receptor comprises an extracellular portion of a molecule that is a derived source of a transmembrane domain, such as a CD28 extracellular portion.
  • the chimeric antigen receptor comprises an intracellular domain derived from a T cell co-stimulatory molecule or a functional variant thereof, such as between a transmembrane domain and an intracellular signal transduction domain.
  • the T-cell costimulatory molecule is CD28 or 41BB.
  • a CAR contains an antibody, such as an antibody fragment, is or contains a transmembrane domain of a CD28 or a functional variant thereof, and a cell containing a signal transduction moiety or a functional variant of CD28 Internal signal transduction domain, and the signal transduction part of CD3 ⁇ or a functional variant thereof.
  • the CAR contains an antibody, such as an antibody fragment, is a transmembrane domain comprising or containing a CD28 transmembrane portion or a functional variant thereof, and an intracellular signal comprising a signal transduction moiety or functional variant of CD137 The transduction domain, as well as the signal transduction part of CD3 ⁇ or a functional variant thereof.
  • the receptor further comprises a linker sequence comprising a portion of an Ig molecule (eg, a human Ig molecule), such as an Ig hinge, such as an IgG4 hinge, such as a hinge-only linker sequence.
  • the chimeric antigen receptor has: (i) an antibody that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signal domain of CD28, and CD3 ⁇ ; or (ii) specific Antibodies that sexually recognize tumor antigens, transmembrane regions of CD28 or CD8, co-stimulatory signal domains of CD137, and CD3 ⁇ ; or (iii) antibodies that specifically recognize tumor antigens, transmembrane regions of CD28 or CD8, and co-stimulus signals of CD28 Domain, CD137 co-stimulatory signal domain, and CD3 ⁇ .
  • the CAR includes an antibody, such as an antibody fragment, including scFv, a linker sequence, such as a linker sequence that contains a portion of an immunoglobulin molecule, such as a hinge region and / or one or more heavy chain molecule constant regions,
  • an Ig-hinge-containing linking sequence a transmembrane domain containing all or part of a CD28-derived transmembrane domain, a CD28-derived intracellular signal domain, and a CD3 ⁇ signal transduction domain.
  • the CAR includes an antibody or fragment, such as a scFv, a linker sequence, such as any Ig-hinge-containing linker sequence, a CD28-derived transmembrane domain, a CD137-derived intracellular signal transduction domain, and a CD3 ⁇ -derived signal Transduction domain.
  • a linker sequence such as any Ig-hinge-containing linker sequence
  • CD28-derived transmembrane domain such as any Ig-hinge-containing linker sequence
  • CD137-derived intracellular signal transduction domain such as CD137-derived intracellular signal transduction domain
  • CD3 ⁇ -derived signal Transduction domain such as any Ig-hinge-containing linker sequence
  • the methods described herein include administering to the subject an adoptive cell or an immune effector cell. In some embodiments, the methods described herein include administering to the subject two or more adoptive cells or immune effector cells against the same tumor antigen in different courses of treatment. In some embodiments, the methods described herein include administering to the subject two or more adoptive cells or immune effector cells to different tumor antigens in different courses of treatment. In some embodiments, the methods described herein include administering to the subject two or more adoptive cells or immune effector cells against the same epitope of the same tumor antigen in different courses of treatment.
  • the methods described herein include administering to the subject two or more adoptive cells or immune effector cells to different epitopes of the same tumor antigen in different courses of treatment. In some embodiments, the methods described herein include administering two or more adoptive cells or immune effector cells to the subject in different courses of treatment to treat a tumor at the same site. In some embodiments, the methods described herein include administering to the subject two or more adoptive cells or immune effector cells 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 employed by the methods described herein targets BCMA CAR-T cells.
  • At least one of the adoptive cells or immune effector cells employed in the methods described herein is a BCMA-targeting CAR-T cell described herein.
  • a person skilled in the art for example, a clinician, can decide the number of administrations and doses of the BCMA-CAR-T cells of the present invention according to the situation of the previous treatment.
  • the cells provided by the methods described herein are immune effector cells that express a chimeric antigen receptor.
  • the cells are generally mammalian cells, and are typically human cells.
  • the cells are derived from blood, bone marrow, lymph, or lymphoid organs and are cells of the immune system, such as innate or adaptive immune cells, such as bone marrow or lymphoid cells, including lymphocytes, typically 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).
  • 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 status, maturity, differentiation potential, Amplification, recycling, localization, and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or those defined by the degree of differentiation.
  • the cells may be allogeneic and / or autologous. Methods include ready-made methods.
  • cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSC).
  • the method includes isolating cells from a subject, preparing, processing, culturing, and / or engineering them, and reintroducing them into the same patient before or after cryopreservation.
  • Subtypes and subpopulations of T cells and / or CD4 + and / or CD8 + T cells include: primary 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 eventually differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxicity T cells, mucosa-associated non-variant T (MAIT) cells, naturally occurring and adoptively regulated 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 primary T
  • TEFF effector T cells
  • TCM stem cell memory T
  • TCM Central memory T
  • TEM
  • the cells are natural killer (NK) cells.
  • the cells are monocytes or granulocytes, for example, bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils granulocyte.
  • the cell comprises 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, typically not 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 engineered And / or such cell-derived organisms.
  • the nucleic acid is not naturally occurring, such as a nucleic acid is not found in nature, including a chimeric combination comprising nucleic acids encoding various domains from multiple different cell types.
  • the invention also provides methods, compositions and kits for generating genetically engineered cells expressing a chimeric antigen receptor.
  • Genetic engineering generally involves introducing a nucleic acid encoding the recombined or engineered portion into a cell, such as through viral transduction, electrotransduction, or the like.
  • gene transfer is performed by first stimulating a cell, for example, by combining it with a stimulus that induces a response, such as proliferation, survival, and / or activation, for example, by a cytokine or The expression of the activation marker is detected, and then the activated cells are transduced and expanded in culture to an amount sufficient for clinical use.
  • the cells are also engineered to promote the expression of cytokines or other factors.
  • cytokines e.g., CAR
  • Various methods for introducing genetically engineered components, such as antigen receptors (e.g., CAR) are well known and can employ the methods and compositions provided herein. Exemplary methods include those used to transfer a nucleic acid encoding a receptor, including by viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.
  • a recombinant infectious virus particle is used to transfer the recombinant nucleic acid into a cell, for example, a vector derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV).
  • SV40 simian virus 40
  • AAV adeno-associated virus
  • a recombinant lentiviral vector or a retroviral vector such as a gamma-retroviral vector, is used to transfer the recombinant nucleic acid into T cells.
  • the preparation of the engineered cells includes one or more culture and / or one or more preparation steps.
  • Cells used to introduce a nucleic acid encoding a transgenic receptor can be isolated from a sample (eg, a biological sample, such as a sample obtained from or derived from a subject).
  • a sample eg, a biological sample, such as a sample obtained from or derived from a subject.
  • the subject from which the cells are isolated is a subject who has a certain disease or disorder or who requires or will be administered a 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, such as a primary human cell.
  • the samples include tissue, body fluids, and other samples taken directly from the subject, as well as obtained from one or more processing steps, such as isolation, centrifugation, genetic engineering (e.g., transduction with viral vectors), washing, and / or Incubate the samples.
  • 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, bowel-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen , Other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organ, and / or cells derived from it.
  • samples include samples from autologous and allogeneic sources.
  • the isolation of the cells includes one or more steps of preparation and / or affinity-based cell isolation.
  • the cells are washed, centrifuged, and / or incubated in the presence of one or more substances, for example, to remove unwanted components, enrich the desired components, lyse or remove Sensitive cells.
  • cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to specific components. In some examples, for example, by apheresis or leukapheresis, cells from the subject's circulating blood are obtained.
  • the sample includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated blood leukocytes, red blood cells, and / or platelets.
  • blood cells collected from the subject are washed, for example, to remove a portion of plasma, 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 is deficient in calcium and / or magnesium and / or many or all divalent cations.
  • the washing step is performed by a semi-automatic "flow-through" centrifugation method (eg, COBE 2991 Cell Processor, BaXter) according to the manufacturer's instructions.
  • the washing step is performed by end-cut filtration (TFF) according to the manufacturer's instructions.
  • the cells after washing, the cells are resuspended in a variety of biocompatible buffers, for example, Ca ++ / Mg ++ PBS-free.
  • biocompatible buffers for example, Ca ++ / Mg ++ PBS-free.
  • blood cell sample components are removed and the cells are resuspended directly in the culture medium.
  • the method comprises a density-based cell separation method, for example, obtaining peripheral blood cells by lysing red blood cells or not lysing red blood cells and preparing peripheral blood or single sample or leukapheresis samples by Percoll or Ficoll gradient centrifugation. Nuclear cells (PBMC).
  • PBMC Nuclear cells
  • the method of isolation includes separating different cell types based on the expression or presence of one or more specific molecules in a cell, such as surface markers, such as surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is an affinity-based or an immunoaffinity-based separation.
  • the isolating comprises isolating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers) of the cell, for example, by specifically binding thereto
  • a marker-like antibody or binding partner is incubated, usually followed by a washing step, and cells that have bound the antibody or binding partner are isolated from those cells that have not yet bound to the antibody or binding partner.
  • isolation 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. In some examples, both parts are reserved for further applications. In some aspects, negative selection may be particularly useful when there are no antibodies available to specifically identify cell types in a heterogeneous population, so isolation is best based on markers expressed by cells different from the desired population .
  • the isolation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • a particular type of cell being selected or enriched refers to an increase in the number or percentage of the cells, but need not result in the complete absence of cells that do not express the marker.
  • negatively selecting, removing, or depleting specific types of cells, such as those expressing markers refers to reducing the number or percentage of the cells, but not necessarily causing complete removal of all such cells.
  • multiple rounds of separation steps are performed, where a portion of positive or negative selection from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single isolation step simultaneously consumes cells that express multiple markers, such as by incubating the cells with multiple antibodies or binding partners that are each specific for a negatively selected targeted marker.
  • multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on various cell types.
  • a specific subpopulation 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 are isolated by positive or negative selection techniques.
  • CD3 + and CD28 + T cells can be positively selected using CD3 / CD28-linked magnetic beads (eg, DYNA beads M-450CD3 / CD28T cell amplifier).
  • CD3 / CD28-linked magnetic beads eg, DYNA beads M-450CD3 / CD28T cell amplifier.
  • the isolation is performed by enriching a specific cell population by positive selection or depleting a specific cell population by negative selection.
  • positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers, the one or more surfaces Markers are expressed on positively or negatively selected cells (marker +) or at relatively high levels (marker high), respectively.
  • T cells are isolated from a PBMC sample by negative selection of a marker (eg, CD14) expressed on 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.
  • Such CD4 + and CD8 + populations can be further sorted into subpopulations by positive or negative selection of markers expressed on one or more primary, memory, and / or effector T cell subpopulations or expressed to a relatively high degree. group.
  • the CD8 + cells are further enriched or depleted for primary, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with corresponding subpopulations.
  • enrichment for central memory T (TCM) cells is performed to increase efficacy, such as to improve long-term survival, expansion, and / or implantation after administration. In some aspects, it is in such sub-types
  • the group is particularly strong. See Terakura et al. (2012) Blood. 1: 72-82; Wang et al. (2012) J Immunother. 35 (9): 689-701.
  • TCM-enriched CD8 + T cells are combined with CD4 + T cells to further enhance efficacy.
  • memory T cells are present in the CD62L + and CD62L- subsets of CD8 + peripheral blood lymphocytes.
  • PBMCs can be enriched or consumed against CD62L-CD8 + and / or CD62L + CD8 + portions, for example using anti-CD8 and anti-CD62L antibodies.
  • the enrichment for central memory T (TCM) cells is based on CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127 positive or high surface expression; in some aspects, it is based on CD45RA and / or granules Cells expressing Enzyme B or highly expressed were negatively selected.
  • isolation of the CD8 + population enriched for TCM cells is performed by depleting cells expressing CD4, CD14, CD45RA, and enriching for cells that are being selected or targeted for CD62L.
  • the enrichment for central memory T (TCM) cells is performed by starting with a negative portion of cells selected based on CD4 expression, which performs negative selection based on the expression of CD14 and CD45RA, and positive selection based on CD62L. In some aspects, such selections occur simultaneously, and in other aspects, such selections occur sequentially in a certain order. In some aspects, the same CD4 expression-based selection step is used to prepare a CD8 + cell population or subpopulation, and also to generate a CD4 + cell population or subpopulation, thereby retaining the positive and negative portions from the CD4-based separation, and optionally After one or more further positive or negative selection steps, for subsequent steps of the method.
  • CD4 + cell selection is performed on a PBMC sample or other blood leukocyte sample, with the negative and positive portions retained. Then, negative selection is performed on the negative part based on the expression of CD14 and CD45RA or CD19, and positive selection is performed based on a central memory T cell characteristic marker, such as CD62L or CCR7, wherein the positive and negative selection are performed in a certain order.
  • a central memory T cell characteristic marker such as CD62L or CCR7
  • helper CD4 + T cells were sorted into primary, central memory, and effector cells.
  • CD4 + lymphocytes can be obtained by standard methods.
  • the original CD4 + T lymphocytes are CD45RO-, CD45RA +, CD62L +, CD4 + T cells.
  • the central memory CD4 + cells are CD62L + and CD45RO +.
  • the effector CD4 + cells are CD62L- and CD45RO-.
  • monoclonal antibody mixtures typically include 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 or paramagnetic beads, to allow isolation of cells for positive and / or negative selection.
  • the method of preparation includes a freezing step, such as freezing the cells before or after isolation, incubation, and / or engineering.
  • the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes in the cell population.
  • the cells are suspended in a frozen solution after a washing step to remove plasma and platelets.
  • any of a variety of known frozen solutions and parameters can be used.
  • 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, by a program-controlled 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
  • provided methods include breeding, incubating, culturing, and / or genetic engineering steps.
  • methods are provided for incubating and / or engineering depleted cell populations and culture starting compositions.
  • the cell population is incubated in a culture initiation composition.
  • Incubation and / or engineering can be performed in culture vessels, such as units, chambers, wells, columns, tubes, tube sets, valves, vials, culture dishes, bags, or other containers used to culture or grow cells.
  • the cells are incubated and / or cultured prior to or with genetic engineering.
  • the incubation step may include culturing, incubating, stimulating, activating, and / or propagating.
  • the cells or compositions are incubated in the presence of a stimulating condition or stimulating agent.
  • a stimulating condition or stimulating agent include those designed to induce cell proliferation, reproduction, activation, and / or survival in a population to mimic antigen contact, and / or trigger cells for genetic engineering, such as those used to introduce recombinant antigen receptors.
  • the conditions may include one or more of the following: specific media, temperature, oxygen content, carbon dioxide content, time, reagents, such as nutrients, amino acids, antibiotics, ions, and / or stimulating factors such as cytokines, Chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other substances designed to maintain the state of activated cells.
  • the stimulating conditions or agents include one or more substances, eg, ligands, which are capable of activating the intracellular signal transduction domain of the TCR complex.
  • the substance turns on or initiates a TCR / CD3 intracellular signal transduction cascade in T cells.
  • substances may include antibodies, such as those specific for TCR components and / or costimulatory receptors, such as anti-CD3, anti-CD28, for example, which bind to a solid support, such as beads, and / or an Or multiple cytokines.
  • the amplification method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml).
  • the stimulant includes 1L-2 and / or IL-15 and / or IL-7 and / or IL-21, for example, IL-2 at a concentration of at least about 10 units / mL.
  • the incubation is in accordance with techniques such as US Patent No. 6,040,177 to Riddell et al., Klebanoff et al., (2012) J Immunother. 35 (9): 651-660, Terakura et al., (2012) Blood. 1: 72- 82 and / or those described in Wang et al. (2012) J Immunother. 35 (9): 689-701.
  • the T cell population is expanded by adding to feeder cells of the culture initiation composition, such as non-dividing peripheral blood mononuclear cells (PBMC), (eg, to target the initial population to be expanded).
  • PBMC peripheral blood mononuclear cells
  • Each T lymphocyte, the resulting cell population comprises at least about 5, 10, 20, or 40 or more PBMC feeder layer cells); and the culture is incubated (eg, a time sufficient to expand the number of T cells).
  • the non-dividing feeder layer cells can include gamma-irradiated PBMC feeder layer cells.
  • the PBMC is irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division.
  • the feeder layer cells are added to the culture medium before the population of T cells is added.
  • the stimulation conditions include a temperature suitable for human T lymphocyte growth, for example, at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, and generally or about 37 degrees Celsius.
  • the incubating may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells.
  • LCL can be irradiated with gamma rays in the range of about 6000-10000 rad.
  • the LCL feeder layer cells are provided in any suitable amount, such as a ratio of LCL feeder layer cells to initial T lymphocytes is at least about 10: 1.
  • antigen-specific T cells such as antigen-specific CD4 + and / or CD8 + T cells
  • antigen-specific T cell lines or clones can be produced against cytomegalovirus antigens by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.
  • compositions and preparations are provided.
  • compositions including cells for administration including pharmaceutical compositions and formulations, such as unit dosage form compositions from a number of cells for administration that contain a given dose or portion 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 formulation refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective, and contains no additional ingredients with unacceptable toxicity to the subject to be administered the formulation.
  • “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation that is not the active ingredient and is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
  • the selection of the carrier is determined in part by the particular cell and / or method of administration. Therefore, there are many 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. Preservatives or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% (based on the total weight of the composition). Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).
  • pharmaceutically acceptable carriers are generally non-toxic to the recipient, including but not limited to: buffers such as phosphates, citrates and other organic acid buffers; antioxidants, including ascorbic acid and Methionine; Preservatives (such as stearyl dimethyl benzyl ammonium chloride; hexahydrocarbon quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; p-hydroxyl Alkyl benzoates, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) Polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspara
  • the composition comprises a buffering agent.
  • Suitable buffering agents 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 buffer or mixture thereof is typically present in an amount of from about 0.001% to about 4% (based on the total weight of the composition).
  • the formulation may include an aqueous solution.
  • the formulation or composition may also contain more than one active ingredient, which may be used for a particular indication, disease or condition to be treated with the cells, preferably those having complementary activity to the cells, wherein the corresponding activity
  • the agents do not negatively affect each other.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further comprises other pharmaceutically active substances or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin Bixin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.
  • chemotherapeutic agents for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin Bixin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.
  • the pharmaceutical composition comprises an amount effective to treat or prevent a disease or disorder, such as a therapeutically effective or preventive effective amount of cells.
  • a disease or disorder such as a therapeutically effective or preventive effective amount of cells.
  • therapeutic or prophylactic efficacy is monitored by periodically assessing treated subjects. The required dose may be delivered to the cells by a single pill, the cells by multiple pills or by continuous infusion.
  • the composition comprises an amount effective to reduce the burden of a disease or disorder, and / or an amount that does not cause CRS or severe CRS in a subject and / or an amount that achieves any other result 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 may 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 eg, derived in vivo, ex vivo, or in vitro
  • a therapeutic composition e.g., a pharmaceutical composition containing genetically modified immunosuppressive cells
  • it is usually formulated as an injectable form (solution, suspension, emulsion) in a unit dosage form.
  • 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 a 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 longer contact time with a particular tissue.
  • Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium, containing, for example, water, saline, phosphate buffered saline, polyhydroxy compounds (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and Suitable mixture.
  • a carrier such as water, saline, phosphate buffered saline, polyhydroxy compounds (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and Suitable mixture.
  • Sterile injectable solutions can be prepared by incorporating the cells into a solvent, such as with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, and the like.
  • composition may contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methyl cellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or pigments, depending on Desired route of administration and preparation.
  • auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methyl cellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or pigments, depending on Desired route of administration and preparation.
  • emulsifying agents e.g., methyl cellulose
  • pH buffering agents e.g., methyl cellulose
  • gelling or viscosity enhancing additives e.g., preservatives, flavoring agents, and / or pigments, depending on Desired route of administration and preparation.
  • preservatives e.g., methyl cellulose
  • flavoring agents e.
  • the invention also provides articles of manufacture, such as kits and devices, for administering cells to a subject according to the provided methods for adoptive cell therapy or immune effector cell therapy, and for storing and administering the cells and compositions.
  • Articles of manufacture include one or more containers, typically multiple containers, packaging materials, and labels or packaging inserts in conjunction with or on one or more containers and / or packaging, and generally include instructions for administering cells to a subject.
  • a container typically 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 in the previous course of immune effector cells or twice (or more) the number of cells to be administered in the first or subsequent course of immune effector cells. It may be the lowest dose or the lowest possible dose to the cells associated with the method of administration to the subject.
  • the unit dose is the number of cells or the minimum number of cells that will be administered in a unit dose to any subject with a particular disease or disorder in accordance with the methods of the invention.
  • a unit dose may include a 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 In some cases more than one unit dose and one or more unit doses are given to a given subject in one or more immune effector cells during a later course of treatment, for example, as provided.
  • the number of cells in a unit dose is the number of chimeric antigen receptor-expressing or CAR-expressing cells that need to be administered to a particular subject, such as a cell-derived subject, with a prior course of immune effector cells or Number of cells.
  • the cells are derived from a 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.
  • each container contains the same or about or substantially the same number of cells or chimeric antigen receptor-expressing cells.
  • the unit dose comprises less than about 1x10 10 , less than about 1x10 9 , less than about 1x10 8 or less than about 1x10 7 engineered cells, total cells, T cells or PBMC / kg to be treated and / or cell derived Subject.
  • 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 to 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 volume of the container for example, a 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.
  • a container for example, a bag is and / or is made of a material that is stable and / or provides stable storage and / or maintenance of cells at one or more different temperatures, such as at low temperatures, for example, low 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 before treatment, for example, at the subject's site or treatment site.
  • temperatures such as at low temperatures, for example, low At or about or equal to or about -20 ° C, -80 ° C, -120 ° C, 135 ° C, -196 ° C and / or a temperature suitable for cry
  • the container can be made from various materials such as glass or plastic.
  • the container has one or more ports, for example, a sterile immersion port, for example, for connecting to one or more tubes through a tube or catheter, for example, for intravenous or other infusion and / or For connection for purposes of transfer from and to other containers, such as cell culture and / or storage bags or other containers.
  • exemplary containers include freezer bags, intravenous solution bags, vials, including those with stopper caps that are pierceable by injection needles.
  • the article of manufacture may also include a package insert or label, one or more of which display usage information and / or instructions.
  • the information or instructions show content that may or should be used to treat a particular disease or condition, and / or provide instructions thereon.
  • a label or package insert can show the contents of an article to be used to treat a disease or condition.
  • the label or package insert provides instructions for treating a subject, e.g., a subject in which the cells have been derived, by including cells administered to the first and one or more immune effector cells in a subsequent course, e.g., A method according to any one of the provided method embodiments.
  • the instructions specify that a unit dose is administered in the immune effector cells of the previous course, eg, the contents of a single individual container of the article, and then at a specified time point or within a specified time window and / or detected The presence or absence or amount or extent of one or more factors or results in the subject is followed by the administration of one or more immune effector cells at a later course of treatment.
  • the instructions specify the administration of multiple unit doses to a subject by performing a first dose and a continuous dose.
  • the first administration comprises delivering one of the unit doses to the subject and the subsequent administration comprises administering one or more of the unit doses to the subject.
  • the label or package insert or package includes an identifier to indicate the identity of the subject from which the cells were derived and / or the subject to be administered.
  • the cells are derived from a subject to be administered to the cells.
  • the identifying information may specify that the cells are to be administered to a particular patient, such information may be present in the packaging material and / or label in the form of a barcode or other coded identifier, or may indicate the subject's name and / or other identifying characteristics.
  • the article of manufacture comprises one or more, typically multiple containers containing a composition comprising cells, such as its individual unit dosage form, and also includes one or more other containers containing the composition therein,
  • the composition comprises other agents, such as cytotoxic or other therapeutic agents, which will be combined with the cells, for example, administered at the same time or in any order.
  • the preparation 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, which contain information on the use, use, dosage, administration, combination therapy, contraindications, and / or warnings of the use of such therapeutic products.
  • PBMCs Peripheral blood mononuclear cells
  • T cells from a human subject with cancer by "mononuclear cell removal-free” and cultured and transduced with a viral vector encoding a chimeric antigen receptor (CAR) Cell
  • the chimeric antigen receptor (CAR) specifically binds an antigen expressed by a cancer in a subject, which is a tumor-associated or tumor-specific antigen.
  • CAR chimeric antigen receptor
  • Cells cryopreserved in freezing infusion medium separate flexible bags, each cell contains a single unit dose, which is about 1 ⁇ 10 5 cells to 1 ⁇ 10 9 cells. Prior to infusion, the cells are maintained at a temperature below about -130 ° C or about below -175 ° C.
  • tumor burden can optionally be assessed by measuring the size or quality of a solid tumor, such as by PET or CT scans.
  • Resuscitation was performed by warming to about 38 ° C, and the subject was given cells of the immune effector cells of the previous course by multiple infusions.
  • the infusion is given intravenously (IV) as a continuous infusion over about 1-20 ml / min.
  • the subject undergoes a physical examination and monitors for signs of any toxic or toxic outcome, such as fever, hypotension, hypoxia, neurological disorders or inflammatory cytokines or C-reactive protein (CRP ) Increased serum levels.
  • any toxic or toxic outcome such as fever, hypotension, hypoxia, neurological disorders or inflammatory cytokines or C-reactive protein (CRP ) Increased serum levels.
  • blood is obtained from the patient in one or more cases, and the level of the serum factor indicating CRS is assessed by ELISA and / or MSD and / or CBA methods .
  • the levels of serum factors were compared with the levels of serum factors obtained just prior to the administration of immune effector cells in the previous course. If necessary, give anti-IL6 or other CRS treatments to reduce the symptoms of CRS.
  • the presence or absence of an anti-CAR immune response is optionally detected in a subject after administration of a previous course of immune effector cells, such as 1, 2, 3, and / or 4 weeks after the start of administration, for example, by qPCR , ELISA, ELISPOT, cell-based antibody assays and / or mixed lymphocyte reactions.
  • the percentage reduction in tumor burden achieved by the previous course of immune effector cells may optionally be measured one or more times after the administration of immune effector cells of the previous course in patients with solid tumors by scanning (such as PET and CT scans), and / Or by quantifying disease-positive cells in the blood or tumor site.
  • Subjects are regularly monitored starting with the administration of immune effector cells for the first course of treatment and for several years. During follow-up, measure tumor load, and / or detect CAR-expressing cells by flow cytometry and quantitative polymerase chain reaction (qPCR) to measure the in vivo proliferation and persistence of the administered cells, and / or assess anti- Development of the CAR immune response.
  • qPCR quantitative polymerase chain reaction
  • a method of preparing an immune effector cell expressing a chimeric antigen receptor refer to, for example, Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, International Patent Application Publication No. WO2018006882A1, The full content disclosed in WO2015172339A8.
  • the amino acid sequence of the scFv portion of the chimeric antigen receptor is shown in SEQ ID NO: 27, the nucleotide sequence is shown in SEQ ID NO: 26, and the scFv has SEQ ID The heavy chain variable region shown by NO: 21 and the light chain variable region shown by SEQ ID NO: 20, and the chimeric antigen receptor has the amino acid sequence shown by SEQ ID NO: 36.
  • the CAR containing the scFv may also have other intracellular domains. Therefore, the sequence of the CAR may also be the sequence shown in SEQ ID NO: 37 or 38.
  • the scFv shown in SEQ ID NO: 27 has HCDR1 shown in SEQ ID NO: 11, HCDR1 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO: 6. LCDR shown in SEQ ID NO: 7, LCDR3 shown in SEQ ID NO: 10.
  • BCMA-positive multiple myeloma are given autologous T cells expressing anti-BCMA chimeric antigen receptor (CAR).
  • CAR anti-BCMA chimeric antigen receptor
  • T cells were obtained by separating PBMCs from the peripheral blood of the subject, transduced by a viral vector encoding a BCMA-targeting CAR, and performing a large number of amplifications to obtain a BCMA-targeting CAR from a frozen medium preparation -T cells, which were then aliquoted into frozen bags and stored under liquid nitrogen below -175 ° C, and thawed before resuscitation before reinfusion to subjects. .
  • tumor necrosis factor alpha TNF ⁇
  • IFN ⁇ interferon gamma
  • IL-10 IL-2
  • IL-6 cytokine release syndrome
  • tumor burden can be assessed by assessing the number of cells associated with cancer, such as in the patient's bone marrow or peripheral blood.
  • the tumor burden before treatment was assessed by assessing the bone marrow and the percentage of bone marrow blasts was determined. Subjects with at least 5% blasts in the bone marrow are considered to have a morphological disease (MD).
  • MD morphological disease
  • Cryopreserved anti-BCMA CAR-T cells were resuscitated by warming to about 38 ° C, and the patient was given by a single infusion. Continuous intravenous (IV) infusion was performed within about 2-30 minutes, and the median infusion duration was 5 minutes. .
  • the subject After administration of anti-BCMA CAR-T cells, the subject undergoes a physical examination and monitors for signs of any toxic or toxic results, such as fever, hypotension, hypoxia, neurological disorders or inflammatory cytokines or C-reactive protein (CRP) Elevated serum levels.
  • any toxic or toxic results such as fever, hypotension, hypoxia, neurological disorders or inflammatory cytokines or C-reactive protein (CRP) Elevated serum levels.
  • blood is obtained from the patient one or more times and the level of the serum factor indicating CRS is assessed by ELISA and / or MSD and / or CBA methods. The levels of serum factors were compared with the levels of serum factors obtained just prior to the first dose. If necessary, give anti-IL6 or other CRS treatments to reduce the symptoms of CRS.
  • Subjects were monitored regularly after administration of anti-BCMA CAR-T cells for several years. During follow-up, measure tumor burden, and / or detect anti-BCMA CAR-expressing cells by flow cytometry and quantitative polymerase chain reaction (qPCR) to measure the in vivo proliferation and persistence of the administered cells, and / or evaluate Development of anti-CAR immune response.
  • qPCR quantitative polymerase chain reaction
  • PFS Progression-free survival
  • DCR disease control rate
  • ORR objective response rate
  • OS overall survival
  • the subject is a multiple myeloma patient with positive BCMA expression, relapsed or refractory, and is divided into 4 groups to participate.
  • the subjects all overexpressed BCMA (the positive rate of BCMA expression was 53% -100%).
  • the median duration of multiple myeloma was between 0.3 and 10.7 years, and most were about 5.2 years (range: 0.4 to 10.7 years).
  • the median number of chemotherapy treatments was 6 (3-20), and some subjects had previously received stem cell transplants.
  • the average plasma cell proportion in the subject's bone marrow was about 25% (range: ⁇ 5% to 85%).
  • the subject's disease classification includes IgG ⁇ type, IgA ⁇ type, IgG ⁇ type, ⁇ light chain type, and IgA ⁇ type. So far, the median follow-up time of the subjects was about 135 days (range: 11 to 260 days).
  • the first group is the cyclophosphamide low-dose group.
  • the pretreatment dose of cyclophosphamide is not higher than 400 mg / m 2 / d (about 190-310 mg / m 2 / d), and about 20 mg / m 2 / d is given. Fludarabine.
  • BCMA CAR-T cells were administered to each subject at about 2 ⁇ 10 6 to 2.7 ⁇ 10 6 cells / kg.
  • each subject was infused with BCMA CAR-T cells at about 2 ⁇ 10 6 to 2.5 ⁇ 10 6 cells / kg.
  • the third group was a low-dose CAR-T group, which was given an anti-BCMA CAR-T cell infusion of less than 1.5 ⁇ 10 6 cells / kg, and the subject was infused with approximately 0.9 ⁇ 10 6 anti-BCMA CAR-T cells. Cells / kg.
  • the fourth group was a high-dose CAR-T group, which consisted of 3 patients.
  • the subjects in this group were infused with anti-BCMA CAR-T cells at about 2.7 ⁇ 10 6 to 3.3 ⁇ 10 6 cells / kg.
  • the day when the subject was administered CAR T cell therapy was designated as day 0.
  • Fludarabine and cyclophosphamide can be given on the same day or on different days.
  • groups 1 subjects were given fludarabine on days -6, -5, and -4, and cyclophosphamide on days -6, -5, -4, -3, and -2; or Subjects were given fludarabine and cyclophosphamide on -4, -3, and -2; or subjects were given fludarabine and cyclophosphamide on -3, -2, and -1 ; Or subjects were given fludarabine and cyclophosphamide on days -3 and -2.
  • subjects were given fludarabine on days -5, -4, -3, and -2, and cyclophosphamide on days -5, -4; or on days -5,- Subjects were given fludarabine on days 4 and -3 and cyclophosphamide on days -5 and -4; or subjects were given fludarabine on days -5, -4, and -3 , And cyclophosphamide on day -5; or fludarabine to subjects on days -4, -3, and -2, and cyclophosphamide on days -4 and -3; or Subjects were given fludarabine, cyclophosphamide on days -3 and -2.
  • group 3 subjects were given fludarabine on days -5, -4 and -3, and cyclophosphamide on days -5 and -4.
  • group 4 subjects were given fludarabine on days -6, -5, -4, and -3, and cyclophosphamide on days -6 and -5; or on days -3 and- Subjects were given fludarabine, cyclophosphamide on day 2; or subjects were given fludarabine, cyclophosphamide on days -3, -2, and -1
  • the dosage of each group is shown in Table 1.
  • the dosages of fludarabine, cyclophosphamide and CAR-T cells indicated in the table are the total dosages.
  • Probe783-P1 for the nucleotide sequence, see SEQ ID NO: 42
  • the upstream primer sequence was: Primer783P1 -F1 (see SEQ ID NO: 43 for the nucleotide sequence); Primer783P1-R3 (see SEQ ID NO: 44 for the nucleotide sequence) of the downstream primers, detect the copy number of anti-BCMA CAR DNA in peripheral blood until Any two consecutive tests were negative and recorded as continuous survival of anti-BCMA CAR-T cells.
  • BCMA CAR-T cells proliferated in all subjects. The cell copy number was detected on the third day, and the peak time was about the third. From 7 to 21 days, it is maintained until 2 to 3 months.
  • the number of copies of anti-BCMA CAR DNA in the peripheral blood of patients No. 14, 15, and 16 was tested, and the results showed that the number of CAR-T cells in patient No. 14 reached a peak on the 14th day after CAR-T administration , Can not be detected on day 56; the number of CAR-T cells in patient 15 reached a peak on day 14 after CAR-T administration, and was not detected on day 56; the number of CAR-T cells in patient 16 was on CAR-T It peaked on the 7th day after administration and was undetectable after 6 months.
  • the subject's disease status is assessed after administration of anti-BCMA CAR-T cells to assess response to treatment.
  • subjects are assessed and monitored for neurotoxicity (neurological complications including symptoms of confusion, aphasia, seizures, convulsions, lethargy and / or altered mental state), graded according to severity (use 1-5 Grade scales, for example, Guido Cavaletti and Paola Marmiroli Nature Reviews 6, 657-666 (December 2010), of which 3 (severe symptoms), 4 (life-threatening symptoms) or 5 (death) are considered severe Neurotoxicity.
  • Level 1 (Minor)-Not life threatening, only systemic treatments such as antipyretics and antiemetics (eg fever, nausea, fatigue, headache, myalgia, discomfort);
  • Oxygen requirement ⁇ 40%, or high-dose single vasopressor drugs eg noradrenaline ⁇ 20ug / kg / min, dopamine ⁇ 10ug / kg / min, phenylephrine ⁇ 200ug / kg / min, or adrenal Low blood pressure of ⁇ 10ug / kg / min
  • vasopressors for example, antidiuretic + one of the above agents, or vasopressors of norepinephrine equal to ⁇ 20ug / kg / min
  • Combination of drugs hypotension, or grade 3 organ toxicity, or grade 4 transaminitis (via CTCAE v4.0);
  • Level 4 life threatening-requires ventilator support, or level 4 organ toxicity (excluding transaminases);
  • Grade 1 (asymptomatic or mild)-mild or asymptomatic
  • Level 2 (Medium)-Symptoms that limit daily active activity (ADL), such as cooking, shopping for food or clothes, using the phone, managing money;
  • Level 3 severe-symptoms of restricted self-management ADL such as bathing, dressing or undressing, eating, using the toilet, taking medication;
  • the CAR shown in SEQ ID NO: 36 is selected as an example. However, it should be understood that other CARs targeting BCMA can also be applied to the technical solution described in this application. CAR as shown in SEQ ID NO: 30, 31, 32, 33, 34, 35, 39, 40, or 41.
  • the scFv of the CAR shown in SEQ ID NO: 30, 31, and 32 has the VH shown in SEQ ID NO: 15 and the VL shown in SEQ ID NO: 16 and the CDR regions are: SEQ ID NO: 1 HCDR1 shown in SEQ ID NO: 2 HCDR2 shown in SEQ ID NO: 2 HCDR3 shown in SEQ ID NO: 3 and LCDR1 shown in SEQ ID NO: 6 LCDR2 shown in SEQ ID NO: 7 LCDR2 shown in SEQ ID NO: 8 LCDR3 shown.
  • the scFv of the CAR shown in SEQ ID NO: 33, 34, and 35 has the VH shown in SEQ ID NO: 17 and the VL shown in SEQ ID NO: 18, and the CDR regions are: SEQ ID NO: 1 HCDR1 shown in SEQ ID No. 2: HCDR2 shown in SEQ ID NO: 2 HCDR3 shown in SEQ ID NO: 4 and LCDR1 shown in SEQ ID NO: 6 LCDR2 shown in SEQ ID NO: 7 LCDR2 shown in SEQ ID NO: 7 LCDR3 shown.
  • the scFv of the CAR shown in SEQ ID NO: 39, 40, 41 (the amino acid sequence is shown in SEQ ID NO: 29, the nucleotide sequence is shown in SEQ ID NO: 28) has SEQ ID ID: 23 VH, and VL shown in SEQ ID NO: 20, CDR regions are HCDR1 shown in SEQ ID NO: 13, HCDR1 shown in SEQ ID NO: 14, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 5, and SEQ IDR NO: 6 LCDR1, SEQ ID NO: 7 LCDR2, SEQ ID NO: 10 LCDR3.

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Abstract

提供一种给予个体特异性识别BCMA的表达嵌合抗原受体的免疫效应细胞，以及应用该免疫效应细胞的治疗方法。该方法能够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的方法 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
多发性骨髓瘤(MM)是一种恶性浆细胞肿瘤，占所有癌症死亡例的2％。主要病状是骨髓中的浆细胞无限度的扩增和富集，进而导致骨坏死。目前主要的治疗方案为化疗和干细胞移植，化疗药物主要是类固醇、沙利度胺、来那度胺、硼替佐米等。
BCMA(B-cell maturation antigen)是B细胞成熟抗原，由185个氨基酸残基组成的III型跨膜蛋白，属TNF受体超家族，其配体属于TNF超家族，如增殖诱导配体(APRIL)、B淋巴细胞刺激因子(BAFF)，BCMA与其配体结合后，可激活B细胞的增殖和存活。BCMA特异地高表达于浆细胞和多发性骨髓瘤细胞，而在造血干细胞和其他正常组织细胞中均不表达，因此BCMA可以作为MM的靶向性治疗的理想靶点。
发明内容
本发明的目的在于提供一种能够对BCMA阳性的肿瘤具备优异杀伤作用的技术手段。
在本发明的第一方面，提供了一种治疗BCMA阳性的肿瘤方法，所述方法包括给予受试者至少一个疗程的表达嵌合抗原受体(CAR)的免疫效应细胞，所述免疫效应细胞特异性识别BCMA。
在具体实施方式中，每个疗程的免疫效应细胞的剂量不超过约1x10 9细胞/千克受试者体重或总量不超过约1x10 10。优选地，每个疗程的免疫效应细胞的剂量不超过约1x10 8细胞/千克受试者体重或所述细胞总量不超过约1x10 9。优选的，每个疗程的免疫效应细胞的剂量不超过约1x10 7细胞/千克受试者体重或所述细胞总量不超过约5x10 8。
在具体实施方式中，所述每个疗程的免疫效应细胞的总剂量不低于1x10 5。优选的，所述每个疗程的免疫效应细胞的总剂量不低于1x10 6。优选的，所述每个疗程的免疫效应细胞的总剂量不低于1x10 7。
在具体实施方式中，给予所述受试者2-5个疗程的所述免疫效应细胞。
在具体实施方式中，当在先给予的免疫效应细胞在体内检测不到后，再给予在后疗程的免疫效应细胞。
在具体实施方式中，在所述在先疗程给予后约4周至24周的时间点处给予所述的在后疗程的免疫效应细胞。
在具体实施方式中，在后疗程给予的免疫效应细胞的剂量低于、等于、或高于在先疗程给予的免疫效应细胞。
在具体实施方式中，在后疗程给予的免疫效应细胞的剂量高于在先疗程给予的免疫效应细胞。优选的，所述在后疗程给予的免疫效应细胞的剂量是在先给予的免疫效应细胞的剂量的2倍、5倍、7倍或10倍。
在具体实施方式中，每个疗程的免疫效应细胞在15天内，分成N次给药，N为不小于1的自然数，在一优选方案中，N为1、2、3或4。
在具体实施方式中，给予在后疗程的免疫效应细胞时，所述受试者具有以下任一特征：
(i)指示细胞因子释放综合征(CRS)的因子在受试者中血清水平倍数比在给予在先疗程的免疫效应细胞之前即刻的受试者中的水平小约10倍、小约25倍、和/或小约50倍；
(ii)没有显示出3级或更高的神经毒性；
(iii)神经毒性或CRS水平与给予在先疗程的免疫效应细胞后的神经毒性或CRS水平的峰值水平相比，呈现降低；或者
(iv)所述受试者没有显示出针对由在先疗程的细胞表达的CAR的可检测的体液或细胞介导的免疫应答。
在具体实施方式中，上述给予在后疗程的免疫效应细胞时，所述受试者具有以下特征(iii)中，CRS水平与给予在先疗程的免疫效应细胞后的CRS的峰值水平相比，降低至少50％，优选的，降低至少20％，更优的，降低至少5％，或者CRS水平与给予在先疗程的免疫效应细胞之前的CRS水平相当。
在具体实施方式中，所述方法还包括在给予所述的免疫效应细胞的之前进行预处理，所述预处理包括给予所述受试者化疗剂或者辐射治疗，或其组合。
在具体实施方式中，所述预处理在给予免疫效应细胞前2-12天实施。在一优选方案中，在给予免疫效应细胞前2-7天实施。
在具体实施方式中，所述化疗剂选自以下任意一种或其组合：环磷酰胺、氟达拉滨。
在具体实施方式中，所述氟达拉滨的给予量约为10-50mg/m 2/天、或约15-40mg/m 2/天、或约15-35mg/m 2/天、或15-30mg/m 2/天、或约20-36mg/m 2/天、或约20-30mg/m 2/天。
在一优选方案中，所述氟达拉滨的给予量约为20-30mg/m 2/天。
在一优选方案中，所述氟达拉滨的给予量约为20-26mg/m 2/天。
在具体实施方式中，所述环磷酰胺的给予量约为100-700mg/m 2/天、或约150-600mg/m 2/天、或约190-600mg/m 2/天、或约190-560mg/m 2/天。
在具体实施方式中，所述环磷酰胺的给予量约为150-400mg/m 2/天，优选的，约为190-350mg/m 2/天。
在一优选方案中，所述环磷酰胺的给予量约为400-600mg/m 2/天，优选的，约为450-600mg/m 2/天，更优选的，约为450-560mg/m 2/天。
当同时给予氟达拉滨和环磷酰胺时，氟达拉滨和环磷酰胺各自的给予量也分别如上所述。
在具体实施方式中，所述化疗剂连续使用不超过6天。
在具体实施方式中，所述环磷酰胺连续使用1-5天。
在具体实施方式中，所述氟达拉滨连续使用2-4天。
在同时给予氟达拉滨和环磷酰胺时，氟达拉滨和环磷酰胺各自的连续使用时间如上所述。
在具体实施方式中，所述肿瘤为多发性骨髓瘤。
在具体实施方式中，所述嵌合抗原受体包括特异性结合BCMA的抗体、跨膜域及胞内域，所述抗体的重链可变区和轻链可变区具有：
SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:8所示的LCDR3；或者
SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:4所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:9所示的LCDR3；或者
SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者
SEQ ID NO:11所示的HCDR1、SEQ ID NO:12所示的HCDR2、SEQ ID NO:5所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者
SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:5所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3。
在具体实施方式中，所述抗体的重链可变区和轻链可变区具有SEQ ID NO:11所示的HCDR1、SEQ ID NO:12所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3。
在具体实施方式中，所述的所述嵌合抗原受体包括特异性结合BCMA的抗体、跨膜域及胞内域，所述抗体的轻链可变区具有SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3。
在具体实施方式中，所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列。
在具体实施方式中，所述抗体的重链可变区具有SEQ ID NO:5所示的HCDR3。
在具体实施方式中，所述的抗体的重链可变区具有SEQ ID NO：15所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：16所示的氨基酸序列；或者
所述的抗体的重链可变区具有SEQ ID NO：17所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：18所示的氨基酸序列；或者
所述的抗体的重链可变区具有SEQ ID NO：19所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列；或者
所述的抗体的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列；或者
所述的抗体的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列。
在具体实施例中，所述的抗体的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列。
在具体实施方式中，所述抗体具有SEQ ID NO：25、27、或29所示的scFv的序列。
在具体实施方式中，所述的嵌合抗原受体具有SEQ ID NO：30、31、32、33、34、35、36、37、38、39、40、或41所示氨基酸序列。
在具体实施例中，所述的嵌合抗原受体具有SEQ ID NO：36、37、38任一所示的氨基酸序列。
在一优选例中，所述的嵌合抗原受体具有SEQ ID NO：36所示的氨基酸序列。
在具体实施方式中，所述免疫效应细胞是T细胞、NK细胞或者NKT细胞；在一优选方案中，所述免疫效应细胞是T细胞。
在具体实施方式中，所述免疫效应细胞来自所述受试者自体。
在具体实施方式中，所述免疫效应细胞来自所述同种异体。
在具体实施方式中，每个疗程的免疫效应细胞的给药间隔是4周至24周。
在一优选例中，每个疗程的免疫细胞数量基本相同。
在一优选例中，在后疗程给予的免疫效应细胞的数量高于在先给的在后疗程的免疫细胞数。
在一优选例中，在后疗程给予的免疫效应细胞数量低于在先给的在后疗程的免疫细胞数。
在具体实施方式中，给予所述免疫效应细胞之前，所述受试者没有接受过靶向BCMA的表达嵌合抗原受体的免疫细胞的治疗。
在具体实施方式中，在给予所述免疫效应细胞治疗之前，所述受试者已经进行了手术治疗、化疗、或者不同于靶向BCMA的表达嵌合抗原受体的免疫细胞的免疫治疗。
在具体实施方式中，在给予每个疗程的免疫效应细胞之前，对所述受试者的指示CRS的因子、指示神经毒性的因子、指示肿瘤负荷的因子、和/或指示宿主抗-CAR免疫应答的因子的血清水平进行评价。
在具体实施方式中，所述的指示肿瘤负荷的因子为：所述受试者的肿瘤细胞总数、或者所述受试者的器官中肿瘤细胞总数、或者所述受试者的组织中的肿瘤细胞总数、或者肿瘤的质量或体积，或者肿瘤转移的程度、或者肿瘤数量。
在具体实施方式中，所述的指示肿瘤负荷的因子包括：
i)在给予在后疗程之前评价指示肿瘤负荷的因子；和
ii)基于所述评价的结果，确定在后疗程，并且
iii)如果评价确定所述受试者的肿瘤质量或体积稳定或下降，给予所述受试者包含少于或多于所述在先疗程中的CAR表达细胞的数量或与其大约相同的CAR表达细胞的数量的在后疗程。
在具体实施方式中，所述免疫效应细胞的给药剂量为约0.1x10 6细胞/kg受试者体重～5x10 7细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为约0.1x10 7细胞～1x10 10细胞。
优选的，为约0.5x10 6细胞/kg受试者体重～1x10 7细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为约0.1x10 8细胞～1x10 9细胞。
更优选的，为约0.9x10 6细胞/kg受试者体重～5x10 6细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为约0.1x10 8细胞～9x10 8细胞。
在具体实施方式中，所述受试者的BCMA的表达阳性率大于50％，优选的，大于70％，或者大于80％。更优选的，大于85％。更优选的，大于90％。
在具体实施方式中，所述受试者的疾病分型为IgGκ型、或者IgGλ型、或者IgAλ型、或者IgAκ型、或者λ轻链型。
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。
附图说明
图1显示了不同组别的患者的疗效结果。
图2显示了BCMA CAR-T细胞在受试者体内的增殖情况。
具体实施方式
发明人经过深入的临床研究，发现将靶向BCMA的表达嵌合抗原受体(CAR)的免疫效应细胞以特定量给予受试者，显著提高了利用免疫效应细胞进行肿瘤治疗的疗效。
除非另外定义，本文使用的所有专业术语、符号和其它技术和科学术语或专有词汇旨在具有本发明所属领域技术人员通常所理解的相同含义。在一些情况中，本文出于阐明和/或便于引用的目的对具有所常规理解的含义的术语加以进一步限定，本文中包括的此类进一步限定不应理解为表示与本领域常规理解的有实质上的差异。
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/m2，以及免疫效应细胞个数/m2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BCMA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。就本文而言，“剂量”是指对于在一个疗程内给药或者回输的过继细胞或者免疫效应细胞的总量。在一些实施方式中，在根据本文所述的方法包括多个疗程的情况下，每个疗程的剂量是相同的。在一些实施方式中，在根据本文所述的方法包括多个疗程的情况下，每个疗程的剂量不同的。
“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。在一些实施方式中，在一个疗程内的剂量被分为多次给予受试者的情况下，每次给药的分剂量是相同的。在一些实施方式中，在一个疗程内的剂量被分为多次给予受试者的情况下，每次给药的分剂量是不同的。就本文而言，若非特别指明，剂量均指在一个疗程内给药或者回输的过继细胞或者免疫效应细胞的总量。
在一些实施方式中，本文所述的方法包括在单疗程中回输所述过继细胞或者免疫效应细胞。单疗程是指在一定时间段内，回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，在所述疗程内一次回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，分两次或更多次回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，分三次或更多次回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，每次回输的过继细胞或者免疫效应细胞 是所要回输的过继细胞或者免疫效应细胞的等分量。在一些实施方式中，每次回输的过继细胞或者免疫效应细胞是所要回输的过继细胞或者免疫效应细胞的非等分量。在一些实施方案中，每次回输的过继细胞或者免疫效应细胞的量由医师根据受试者的具体情况确定。受试者的具体情况例如可以是受试者的整体健康情况、疾病的严重程度、对同疗程前次给药量的反应、对先前疗程的反应、受试者联合用药情况、毒性反应程度或可能性、并发症、以及医生所认为会影响到受试者适于回输的过继细胞或者免疫效应细胞的量的任何其他因素。在一些实施方式中，在所述多次回输一定总量的过继细胞或者免疫效应细胞中，每次回输的过继细胞或者免疫效应细胞的量呈递增趋势。在一些实施方式中，在所述多次回输一定总量的过继细胞或者免疫效应细胞中，每次回输的过继细胞或者免疫效应细胞的量呈递减趋势。在一些实施方式中，在所述多次回输一定总量的过继细胞或者免疫效应细胞中，每次回输的过继细胞或者免疫效应细胞的量呈先递增后递减的趋势。在一些实施方式中，在所述多次回输一定总量的过继细胞或者免疫效应细胞中，每次回输的过继细胞或者免疫效应细胞的量呈先递减后递增的趋势。
多疗程回输是指具有多个所述时间段，在每个时间段内回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，所述多个时间段的长度是一致的。在一些实施方式中，所述多个时间段的长度是不等的。在一些实施方式中，所述多疗程是指具有至少两个所述时间段。在一些实施方式中，所述多疗程是指具有至少三个或更多个所述时间段。
在一些实施方式中，在所述多个疗程中的一个疗程内一次回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，所述多个疗程中的一个疗程内分两次或更多次回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，所述多个疗程中的一个疗程内分三次或更多次回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，在所述多个疗程中的一个疗程内，每次回输的过继细胞或者免疫效应细胞是所要回输的过继细胞或者免疫效应细胞的等分量。在一些实施方式中，在所述多个疗程中的一个疗程内，每次回输的过继细胞或者免疫效应细胞是所要回输的过继细胞或者免疫效应细胞的非等分量。在一些实施方案中，在所述多个疗程中的一个疗程内，每次回输的过继细胞或者免疫效应细胞的量由医师根据受试者的具体情况确定。受试者的具体情况例如可以是受试者的整体健康情况、疾病的严重程度、对同疗程前次给药量的反应、对先前疗程的反应、受试者联合用药情况、毒性反应程度或可能性、并发症、癌症转移情况、以 及医师所认为会影响到受试者适于回输的过继细胞或者免疫效应细胞的量的任何其他因素。在一些实施方式中，在所述多个疗程中的每个疗程中以相同的次数回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，在所述多个疗程中的每个疗程中以不同的次数回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，在所述多个疗程中的每个疗程中以相同的次数回输一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，在所述多个疗程中的每个疗程中回输相同的一定总量的过继细胞或者免疫效应细胞。在一些实施方式中，在所述多个疗程中的每个疗程中回输不同的一定总量的过继细胞或者免疫效应细胞。
在一些实施方式中，在所述多个疗程中的每个疗程内所述过继细胞或者免疫效应细胞的总量呈递增趋势。在一些实施方式中，在所述多个疗程中的每个疗程内所述过继细胞或者免疫效应细胞的总量呈递减趋势。在一些实施方式中，在所述多个疗程中的每个疗程内所述过继细胞或者免疫效应细胞的总量呈先递增后递减的趋势。在所述多个疗程中的每个疗程内所述过继细胞或者免疫效应细胞的总量呈先递减后递增的趋势。
在一些实施方式中，本文所述的方法包括监控受试者对所述过继细胞或免疫效应细胞的暴露程度，并根据所述暴露程度确定后续分次给药或者后续疗程的给药剂量和间隔时间。在一些实施方式中，本文所述的方法包括监控受试者对所述过继细胞或免疫效应细胞的暴露程度，并根据所述暴露程度达到或者超过一定程度来维持或者降低后续分次给药或者后续疗程的给药剂量和/或维持或延长后续分次给药或者后续疗程之间的间隔时间。在一些实施方式中，本文所述的方法包括监控受试者对所述过继细胞或免疫效应细胞的暴露程度，并根据所述暴露程度低于一定程度来维持或者增加后续分次给药或者后续疗程的给药剂量和/或维持或缩短后续分次给药或者后续疗程之间的间隔时间。
在一些实施方式是中，本文所述的方法包括监控受试者对所述过继细胞或免疫效应细胞的毒性反应程度或者风险，并根据所述毒性程度或者风险确定后续分次给药或者后续疗程的给药剂量和间隔时间。在一些实施方式中，所述毒性反应程度或者风险包括但不限于例如CRS、神经毒性、巨噬细胞活化综合征和肿瘤裂解综合征等。
在一些实施方式中，在针对细胞的宿主适应性免疫应答未被检测到，尚未建立和/或尚未达到一定水平或程度或阶段时给予在后疗程的免疫效应细胞。
所谓肿瘤负荷，包括分型、分期，发病时骨髓中异常浆细胞的比例，细胞遗传学的改变，是否存在髓外浸润，以及对治疗后的反应，在一些实施方案中，肿瘤负荷通过血清、尿液蛋白电泳，骨髓涂片、骨髓活检、MRD、MRI和/或CT进行评估。
在一些实施方式中，每个疗程的免疫效应细胞的给药量包括足以降低受试者肿瘤负荷的量的细胞剂量。在给予在后疗程的细胞时，受试者中指示细胞因子-释放综合征(CRS)的因子的血清水平不超过该受试者在给予免疫效应细胞治疗之前的血清水平的10倍或25倍时，和/或在受试者中CRS相关结果峰值水平并且在给予免疫效应细胞之后开始下降，并且受试者没有由在先疗程的免疫效应细胞的细胞表达的嵌合抗原受体特异的可检测的适应性宿主免疫应答时。
在一些实施方式中，免疫效应细胞的给药剂量为不少于约0.5x10 5细胞/kg受试者体重或总剂量不低于1x10 5细胞；优选的，不少于约0.1x10 6细胞/kg体重或总剂量不低于1x10 6细胞；优选的，不少于约0.5x10 6细胞/kg受试者体重或总剂量不低于1x10 7细胞；优选的，不少于约0.9x10 6细胞/kg受试者体重或总剂量不低于1x10 7细胞。
在具体实施方式中，免疫效应细胞的给药剂量为不超过约1x10 9细胞/千克受试者体重或总量不超过约1x10 10细胞；优选的，不超过约1x10 8细胞或所述细胞总量不超过约1x10 9细胞；优选的，不超过约1x10 7细胞或所述细胞总量不超过约1x10 9细胞，优选的，不超过约5x10 6细胞或所述细胞总量不超过约9x10 8细胞。
在具体实施方式中，所述免疫效应细胞的给药剂量为约0.1x10 6细胞/kg受试者体重～5x10 7细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为约0.1x10 7细胞～1x10 10细胞。优选的，为约0.5x10 6细胞/kg受试者体重～1x10 7细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为约0.1x10 8细胞～1x10 9细胞。更优选的，为约0.9x10 6细胞/kg受试者体重～5x10 6细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为约0.1x10 8细胞～9x10 8细胞。
免疫效应细胞治疗
本发明提供的方法包括向表达BCMA的肿瘤受试者给予至少一个疗程的表达识别BCMA的嵌合抗原受体(如CAR、TCR、TFP、TAC)的免疫效应细胞。
本文中所用的“受试者”是哺乳动物，例如人或其它动物，通常是人。在一些实施方式中，给予在先疗程的免疫效应细胞和/或给予在后疗程的免疫效应细胞之 前，受试者已经采用化学治疗或放射治疗。在一些方面中，受试者对于其他治疗剂是难治性的或非响应性的。
在一些实施方式中，肿瘤具有持续性或复发性，例如，在用另一种治疗(包括化疗、放射)介入之后，肿瘤仍然进展或者控制后复发。。
在一些实施方式中，受试者对其他治疗剂有响应性，降低了肿瘤负荷。在一些方面中，受试者初始对治疗剂有响应性，但是随着时间显示出肿瘤的复发。在一些实施方式中，所述受试者经测定具有复发风险，例如处于复发高风险中，并且由此预防性地给予本发明CAR T细胞，以期减小复发可能性或预防复发。
在一些实施方式中，剂量的大小和回输时间由受试者的初始肿瘤负荷决定。例如，在一些情况中，一般给予受试者的在先疗程的免疫效应细胞的细胞数量较低，在较低肿瘤负荷的情况下，如实体肿瘤可以通过肿瘤标志物检测来评估肿瘤负荷大小和/或微小残留病灶，初始剂量可能较高。在其他情况中，在较高肿瘤负荷的受试者中，可采用每个疗程的细胞分针连续输注，如分为1、2、3、4、5、6、7、8、9或10次给药，优选的，为1-5次给药，更优的，为2-3次给药。每次给药之间间隔1、2、3、4、5、6更多天或不间隔。
本文中所用的术语“治疗”指完全或部分减轻或减少肿瘤或与其相关的症状，以及相关评估客观指标的好转。所需的治疗效果包括但不限于，预防肿瘤的发生或复发、减轻症状、减少肿瘤的任何直接或间接病理学结果、防止髓外病变、减缓肿瘤进展速率、改善或减轻肿瘤状态，和减轻或改善预后。本文中所用的“抑制”功能或活性意指，在与其它情况下的相同病症相比时或者相较于另一病症，功能或活性的减少。
在一些实施方式中，所述细胞治疗可以通过自体回输进行治疗。因此，在一些方面中，所述细胞源自需要治疗的受试者，并在分离和处理之后给予同一受试者。
在一些实施方式中，所述细胞治疗通过同种异体回输进行治疗，其中，所述细胞分离和/或在其它情况中从供者体内提取和制备，供者和回输细胞的受试者不同。在一些实施方式中，所述供者和受者是遗传学相同的。在一些实施方式中，所述供者和受者是遗传学相似的。在一些实施方式中，所述受者和供者相同的HLA类别或超类型。
所述细胞可通过任何合适的方式给予，例如，通过注射，如，静脉注射、眼内注射、眼底注射、视网膜下注射、玻璃体内注射、反间隔注射、巩膜下注射、 脉络膜内注射、前房注射、结膜下(subconjectval)注射、结膜下(subconjuntival)注射、巩膜上腔注射、球后注射、眼周注射或球周递送。在一些实施方式中，通过滴注给予细胞。在一些实施方式中，通过多次滴注给予细胞，例如，在不超过30天多次给药，或通过连续输注给予细胞。
在预防或治疗疾病，给药剂量可取决于疾病类型、嵌合抗原受体或细胞类型，疾病的严重程度和疾病病程、受试者的在先治疗史、和给予的细胞的响应，以及主治医师的判断。
在一些实施方式中，所述免疫效应细胞作为联合治疗的一部分，例如，与另一干预性治疗，例如抗体、经工程改造的免疫效应细胞、受体或试剂、细胞毒性药物或其他治疗方法联合，同时或依次地，以任何顺序给予。在一些实施方式中，免疫效应细胞与一种或多种其它治疗方法联用，或与另一干预性治疗方法联合，同时或以任何顺序依次进行。在一些情况中，所述免疫效应细胞与另一治疗在足够接近的时间共同给予，从而产生治疗作用大于上述免疫效应细胞群或者一种或多种其它治疗药物或者方法，反之亦然。在一些实施方式中，在一种或多种其它治疗剂之前给予所述免疫效应细胞。在一些实施方式中，在一种或多种其它治疗剂之后给予所述免疫效应细胞。在一些实施方式中，一种或多种其他治疗药物包括细胞因子，如IL-2、IL-12，以增强持久性。
在一些实施方式中，该方法包括在给予免疫效应细胞之前给予预处理，例如，化学治疗药物(化疗剂)、全身辐射、局部辐射治疗等或其组合。在一些实施方式中，所述方法包括采用一种或多种化疗剂对受试者进行预处理。在一些实施方式中，所述方法包括采用微管蛋白抑制剂与一种或多种其他化疗剂对受试者进行预处理。不受理论所限，预处理的作用被认为包括但不限于，淋巴细胞清除、降低肿瘤负荷等。所述化疗剂，是指化学治疗使用的药物，例如，环磷酰胺、氟达拉滨、蛋白酶抑制剂(如硼替佐米、卡非佐米等)、免疫调节剂(如沙利度胺、来那度胺、泊马度胺等)、马法兰、阿霉素、地塞米松、强的松等。
预处理可改善免疫效应细胞治疗的效果。将每个疗程给予免疫效应细胞(例如CAR T细胞)第一次输注之日定为第0日。在给予免疫效应细胞输注前的任何时候可给予预处理。在一些实施方式中，将氟达拉滨或环磷酰胺单独或其组合作为预处理，在CAR-T细胞输注前至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30天，优选地，至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、 17、18、19或20天，更优选地，至少2、3、4、5、6、7或8天给予受试者。在一些实施方式中，预处理包括在CAR-T细胞输注前2-12天给予氟达拉滨和环磷酰胺。在一些实施方式中，预处理包括在CAR-T细胞输注前7天给予氟达拉滨和环磷酰胺。
可以调整预处理的成分的给予时机是CAR T治疗疗效达到最大。通常，可每日给予氟达拉滨、环磷酰胺。在一些实施方式中，每日给予氟达拉滨、环磷酰胺给予约1日、约2日、约3日、约4日、约5日、约6日或约7日。在一些实施方式中，每日给予氟达拉滨，连续给予4日，和每日给予环磷酰胺，连续给予2日。在一些实施方式中，每日给予氟达拉滨，连续给予2日，和每日给予环磷酰胺，连续给予2日。在一些实施方式中，每日给予氟达拉滨，连续给予3日，和给予环磷酰胺一次，或连续给予环磷酰胺2日或3日或5日。
如上所述，将对患者每轮给予CAR T细胞疗法之日指定为第0日。在一些实施方式中，在第0日前的第6日(即第-6日)、-5日和-4日对患者给予氟达拉滨。在一些实施方式中，在第-5、-4和-3日对患者给予氟达拉滨。在一些实施方式中，在第-6、-5、-4和-3日对患者给予氟达拉滨。在一些实施方式中，在第-5、-4、-3和-2日对患者给予氟达拉滨。在一些实施方式中，在第-4、-3和-2日对患者给予氟达拉滨。在一些实施方式中，在第-3和-2日对患者给予氟达拉滨。在一些实施方式中，在第-3、-2和-1日对患者给予氟达拉滨。在一些实施方式中，在第-6、-5、-4、-3和-2日对患者给予环磷酰胺。在一些实施方式中，在第-6和-5日对患者给予环磷酰胺。在一些实施方式中，在第-4、-3和-2日对患者给予环磷酰胺。在一些实施方式中，在第-4和-3日对患者给予环磷酰胺。在一些实施方式中，在第-5和-4日对患者给予环磷酰胺。在一些实施方式中，在第-5日对患者给予环磷酰胺。在一些实施方式中，在第-3和-2日对患者给予环磷酰胺。在一些实施方式中，在第-3、-2和-1日对患者给予环磷酰胺。
可在同日或不同日开始给予氟达拉滨和环磷酰胺。
在某些实施方式中，可同时给予或依次给予氟达拉滨和环磷酰胺。
可利用任何途径(包括静脉滴注(I.V.))给予氟达拉滨、环磷酰胺。在一些实施方式中可以按照氟达拉滨和环磷酰胺的药品说明书给予氟达拉滨和环磷酰胺。
在一些实施方式中，过继细胞疗法或免疫效应细胞疗法选自肿瘤浸润淋巴球(TIL)免疫疗法、自体细胞疗法、工程化自体细胞疗法(eACT)、与同种异体 T细胞移植法。在一些实施方式中，免疫效应细胞疗法为给予靶向肿瘤抗原(如BCMA)的嵌合抗原受体修饰的T细胞。
在一些实施方式中，预处理包括给予氟达拉滨不高于约50、49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2、1mg/m 2/日；和/或给予环磷酰胺不高于约700、690、680、670、660、650、640、630、620、610、600、595、590、585、580、579、578、576、575、574、573、572、571、570、569、568、567、566、565、564、563、562、561、560、559、558、557、556、555、554、553、552、551、550、549、548、547、546、545、544、543、542、541、540、539、538、537、536、535、534、533、532、531、530、529、528、527、526、525、524、523、522、521、520、519、518、517、516、515、514、513、512、511、510、509、508、507、506、505、504、503、502、501、500、490、480、470、460、450、440、430、420、410、400、390、380、370、360、350、340、330、320、310、300、290、280、270、260、250、240、230、220、210、200、190、180、170、160、150、140、130、120、110、或100mg/m 2/日。
在一些实施方案中，氟达拉滨的给予量约为10-50mg/m 2/天、或约15-40mg/m 2/天、或约15-35mg/m 2/天、或15-30mg/m 2/天、或约20-30mg/m 2/天。
在一些实施方案中，环磷酰胺的给予量约为100-700mg/m 2/天、或约150-600mg/m 2/天、、或约190-600mg/m 2/天、或约190-560mg/m 2/天。
在一优选方案中，环磷酰胺的给予量约为150-400mg/m 2/天，优选的，约为190-350mg/m 2/天。
在一优选方案中，环磷酰胺的给予量约为400-600mg/m 2/天，优选的，约为450-600mg/m 2/天，更优选的，约为450-560mg/m 2/天。
在此处所述的方法中可包括各种其他干预。例如，环磷酰胺与氟达拉滨在给予后可能引起患者的不良反应。本发明的范围包括对患者给予组成物以使这些不良事件中的某些事件减少。在某些实施方式中，该方法包含对患者给予生理盐水。可在给予环磷酰胺和/或氟达拉滨前或后，或在给予环磷酰胺和/或氟达拉滨前与后对患者给予生理盐水。在一些实施方式中，在每个输注日给予环磷酰胺和/或氟达拉滨前，及在给予环磷酰胺和/或氟达拉滨后对患者给予生理盐水。另外，还可 对患者给予佐剂与赋形剂。例如，美司钠(2-巯基乙烷磺酸钠)。此外，还可对患者给予外源性细胞激素。
在一些实施方式中，在输注在先疗程的免疫效应细胞或在后疗程的免疫效应细胞之前给予预处理改善了治疗的结果。例如，在一些方面，预处理改善了用在先疗程的免疫效应细胞或在后疗程的免疫效应细胞治疗的功效，或增加了受试者中免疫效应细胞(例如CAR-T细胞)的持久性。在一些实施方式中，预处理治疗增加疾病稳定期。
一旦向受试者给予免疫效应细胞，在一些方面中，通过多种已知方法中的任一种来测量免疫效应细胞群在体内的持续存活期及其生物活性。如，CAR-T细胞在体内持续存活期为CAR-T细胞“植入”体内后，在体内的持续存活期，CAR-T的持续存活时间的检测可以从初次植入结束后，在每个访视点采用Q-PCR的方法检测外周血中含有CAR DNA的拷贝数，直到任何2次连续的检测为阴性，记录为CAR-T细胞持续存活期。
免疫效应细胞的生物活性可以通过工程改造的或天然T细胞或其它免疫细胞与抗原的特异性结合来评估，如可以通过ELISA或流式细胞术等评估。
在某些实施方式中，工程改造的免疫效应细胞的细胞杀伤能力可采用本领域已知的任何合适的方法来检测，如可通过测定某些细胞因子，例如CD107a，IFNγ，IL-2和TNF的表达和/或分泌，来测量免疫效应细胞的生物活性。在一些方面中，生物活性通过临床结果来评估，例如肿瘤负荷或负载的减少。。在一些方面，评估细胞的毒性结果、持久性和/或增殖，和/或存在或不存在宿主免疫应答。
在免疫效应细胞治疗的情况下，给予给定的“剂量”包括以单一组合物和/或单次不间断给药，例如以单次注射或连续输注给定量或数量的免疫效应细胞，并且还包括在不超过30、29、28、27、26、25、24、23、22、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3或2天特定时间段内，以分剂量，在多个单独组合物或输注物中提供，给予给定量或数量的免疫效应细胞。因此，每个疗程的免疫效应细胞是在单个时间点给予或开始的指定数量的免疫效应细胞的单次或连续给予。然而，在一些情况下，每个疗程的免疫效应细胞在不超过30、29、28、27、26、25、24、23、22、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3或2天的时间段内进行多次注射或输注，例 如每天一次持续三天或两天，或通过在一天时间内的多次输注。在一些方面，在先疗程的免疫效应细胞的免疫效应细胞以单一药物组合物给予。
相对于第一疗程的给药，术语“在后疗程”是指在第一疗程之后期间没有向受试者给予任何间插剂量的情况下给予受试者的剂量。尽管如此，该术语不包括在单次分剂量内包含的一系列输注或注射中的第二、第三和/或其他注射或输注。因此，除非另有说明，否则在一天、两天或三天内的第二输注不被认为是本文所用的“在后疗程“。类似地，在分剂量内的多个剂量系列中的第二，第三和其他的剂量也不被认为是“在后疗程”剂量意义内容中的“间插”剂量。因此，除非另有说明，即使在受试者在第一疗程开始之后接受第二或后续疗程的输注，在第一疗程或在先疗程开始之后，在超过30天的一定时间段给予的剂量被认为是“在后疗程”的剂量。除非另有说明，否则在多达30天的时间段内将相同免疫效应细胞的多次给予视为单一剂量，并且在初始给药后30天(每个疗程的第一次输注(即初始给药)当天为第1天)内给予免疫效应细胞不被认为是在后疗程给药，并且不被认为是以确定第二剂量是否与在先疗程的免疫效应细胞“连续”为目的的间插剂量。
如本文所用，“第一疗程”用于描述根据本文所述的方法进行治疗所进行的首个疗程中所给予的总剂量。该剂量等于单疗程给药中该疗程中给药的总剂量，或者多疗程给药中首个疗程中给药的总剂量。该术语并不一定意味着受试者之前从未接受过免疫效应细胞治疗，或者受试者之前没有接受靶向相同抗原的免疫效应细胞的治疗。
通常设计在先疗程的免疫效应细胞和/或一个或多个在后疗程的免疫效应细胞剂量的大小以提供改善的功效和/或降低的毒性风险。在一些实施方式中，每个疗程的免疫细胞剂量为高于、低于或等于约0.5x10 6细胞/kg受试者体重～1x10 7细胞/kg受试者体重，如高于、低于或者等于约0.6×10 6、0.7×10 6、0.8×10 6、0.9×10 6、1.0×10 6、1.1×10 6、1.2×10 6、1.3×10 6、1.4×10 6、1.5×10 6、1.6×10 6、1.7×10 6、1.8×10 6、1.9×10 6、2.0×10 6、2.1×10 6、2.2×10 6、2.3×10 6、2.4×10 6、2.5×10 6、2.6×10 6、2.7×10 6、2.8×10 6、2.9×10 6、3.0×10 6、3.1×10 6、3.2×10 6、3.3×10 6、3.4×10 6、3.5×10 6、3.6×10 6、3.7×10 6、3.8×10 6、3.9×10 6、4.0×10 6、4.1×10 6、4.2×10 6、4.3×10 6、4.4×10 6、4.5×10 6、4.6×10 6、4.7×10 6、4.8×10 6、4.9×10 6、5.0×10 6、5.1×10 6、5.2×10 6、5.3×10 6、5.4×10 6、5.5×10 6、5.6×10 6、5.7×10 6、5.8×10 6、5.9×10 6、6.0×10 6、6.1×10 6、6.2×10 6、6.3×10 6、6.4×10 6、6.5×10 6、 6.6×10 6、6.7×10 6、6.8×10 6、6.9×10 6、7.0×10 6、7.1×10 6、7.2×10 6、7.3×10 6、7.4×10 6、7.5×10 6、7.6×10 6、7.7×10 6、7.8×10 6、7.9×10 6、8.0×10 6、8.1×10 6、8.2×10 6、8.3×10 6、8.4×10 6、8.5×10 6、8.6×10 6、8.7×10 6、8.8×10 6、8.9×10 6、9.0×10 6、9.1×10 6、9.2×10 6、9.3×10 6、9.4×10 6、9.5×10 6、9.6×10 6、9.7×10 6、9.8×10 6、9.9×10 6、1.0×10 7细胞/kg受试者体重。在一些实施方式中，每个疗程的免疫细胞给药总剂量为高于、低于或等于约0.1x10 8细胞～1x10 10细胞。优选的，为高于、低于或等于约1x10 6细胞/kg受试者体重～1x10 7细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为高于、低于或等于约0.5x10 8细胞～1x10 9细胞。更有选的，为高于、低于或等于约1.5x10 6细胞/kg受试者体重～3x10 6细胞/kg受试者体重，或者所述免疫效应细胞的给药总剂量为约0.5x10 8细胞～5x10 8细胞。
在具体实施方式中，免疫效应细胞的数量是指表达嵌合抗原受体的免疫效应细胞的数量，如CAR-T细胞的数量。在其它实施方式中，免疫效应细胞的数量还可以指给予的T细胞或PBMC或总细胞的数量。
在一些实施方式中，在后疗程的剂量足以减少肿瘤负荷或其指标，和/或疾病或病症的一种或多种症状。
在一些实施方式中，与给予第一疗程的免疫效应细胞之前时刻相比，给予免疫效应细胞后，肿瘤负荷，如骨髓中浆细胞比例、血/尿M蛋白、髓外软组织浆细胞瘤减少约25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或100％。
M蛋白是浆细胞或B淋巴细胞单克隆恶性增殖所产生的一种大量的异常免疫球蛋白。
基于本发明的教导，本领域技术人员应该理解，本发明具体公开的剂量是本发明人经过研究得到是安全有效的剂量，本领域技术人员，例如临床医师可以根据各种实际情况，例如患者的肿瘤负荷、患者本身的身体状况等因素决定在先疗程的免疫效应细胞的剂量；如果需要进一步给予在后疗程的免疫效应细胞，本领域技术人员可以根据，例如给予免疫效应细胞之后的肿瘤负荷变化来决定在后疗程的免疫效应细胞的剂量。
在一些实施方式中，每个疗程的免疫效应细胞的剂量包括不引起毒性或者降低毒性的量，所述的毒性可以是细胞因子释放综合征(CRS)、严重CRS(sCRS)、巨噬细胞活化综合征、肿瘤溶解综合征、持续三天或更多天的至少38摄氏度或约38摄氏度的发热，CRP血浆水平至少等于约20mg/dL，和/或神经毒性。在一些方 面，基于在给予细胞后受试者将显示毒性或毒性结果的可能性来确定在后疗程的免疫效应细胞中给予的细胞的数量。例如，在一些实施方式中，基于肿瘤负荷预测受试者中发展毒性结果的可能性。在一些实施方式中，该方法包括在给予免疫效应细胞之前检测或评估毒性结果和/或肿瘤负荷。
给药时间
在一些实施方式中，在后疗程的给药时间是从在先疗程完成(在先疗程的总剂量输注完成日定为0日)开始起算。
在一些实施方式中，在受试者中指示CRS的因子的血清水平不超过给予在先疗程的免疫效应细胞后受试者中该指示物的血清水平的约10倍、25倍、50倍或100倍时，给予在后疗程的免疫效应细胞。
在一些实施方式中，在受试者中与CRS相关的结果(例如与CRS相关或指示CRS的血清因子)或其临床迹象或症状如发热、缺氧、低血压或神经障碍已经达到峰值水平，并且在给予免疫效应细胞治疗后开始下降时，给予在后疗程的免疫效应细胞。在一些实施方式中，在观察到给予后与这类结果的最高水平相比下降时，或在给予后达到结果的最大值或水平之后水平下降时给予在后疗程的免疫效应细胞。
在一些实施方式中，当毒性结果的指示物(例如CRS的血清指示物)的水平下降到在先疗程的免疫效应细胞给予之前时刻的指示物水平的约25倍以下时，给予在后疗程的免疫效应细胞。在一些方面，在后疗程的免疫效应细胞在受试者不显示CRS或不显示严重CRS时给予。
在一些方面，在后疗程的免疫效应细胞在与给予在先疗程的免疫效应细胞之前的肿瘤负荷相比患者中的肿瘤负荷下降的时间点给予。在一些实施方式中，在后疗程的免疫效应细胞在给予在先疗程的免疫效应细胞后，当肿瘤负荷如骨髓中浆细胞比例、血/尿M蛋白、髓外软组织浆细胞瘤已经减少了约25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或更多时给予。
在一些实施方式中，在受试者的疾病或病症在对在先疗程的免疫效应细胞或在先剂量的应答降低后没有复发时给予在后疗程的免疫效应细胞。在一些实施方式中，减轻肿瘤负荷由一个或多个指标的降低来指示，如分型、分期，发病时骨髓中异常浆细胞的比例，细胞遗传学的改变，是否存在髓外浸润，以及对治疗后 的反应。在一些实施方式中，在宿主适应性免疫应答未被检测到、尚未建立、或尚未达到一定水平、程度或阶段时给予在后疗程的免疫效应细胞。在一些方面，在受试者的记忆免疫应答发展之前给予在后疗程的免疫效应细胞。
在一些方面，给予在先疗程的免疫效应细胞与给予在后疗程的免疫效应细胞之间的时间为约4周至约24周。
在一些实施方式中，在给予在后疗程的免疫效应细胞后，给予额外或进一步的在后疗程的免疫效应细胞，例如，第二在后疗程的免疫效应细胞(第三疗程剂量)、第三在后疗程的免疫效应细胞(第四疗程剂量)，以此类推。
在一些实施方式中，与给予在先疗程的免疫效应细胞或在后疗程的免疫效应细胞之前时刻相比，给予在后疗程的免疫效应细胞后，肿瘤负荷减少至少等于约25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或更多。在一些实施方案中，肿瘤负荷可以通过血清、尿液蛋白电泳，骨髓涂片、骨髓活检、MRD、MRI和/或CT进行评估。
针对回输的细胞的宿主免疫应答
在一些实施方式中，一个或多个剂量，例如在后疗程的免疫效应细胞在受试者的免疫应答(例如对转基因受体或细胞的适应性或特异性免疫应答)不存在，不可检测，或在一定水平以上无法检测时给予。对转基因的特异性免疫应答的存在或程度通常与受体的免疫原性(例如由细胞表达的CAR或转基因TCR)和/或受试者暴露于细胞的时间有关。在一些实施方式中，在受试者中已经发展针对嵌合抗原受体或细胞的免疫应答，适应性或特异性免疫应答，可检测的免疫应答和/或记忆应答之前给予在后疗程的免疫效应细胞。在这方面，与其他与在先或在先疗程的免疫效应细胞相比，在较晚时间点给予在后疗程的免疫效应细胞的其它方法相比，在后疗程的免疫效应细胞的细胞增殖和/或持续存在于受试者中的能力得到改善。在一些实施方式中，何时和/或是否给予在后疗程的免疫效应细胞的决定取决于受试者是否表现出这样的免疫应答或其可检测的读数，例如对细胞或嵌合抗原受体特异的可检测的特异性或适应性宿主免疫应答，例如由在先疗程的免疫效应细胞的细胞表达的CAR，和/或是否在某一水平上检测到这样的反应。在一些实施方式中，在检测到这样的反应的情况下，不向受试者给予在后疗程的免疫效应细胞。在受试者不表现出针对受体(例如由在先疗程的免疫效应细胞的细胞表达的CAR)的特异性或适应性(例如体液或细胞介导的)免疫应答时，或者不表现出在可 检测水平或高于可接受水平的这种反应或指示物时给予在后疗程的免疫效应细胞。在一些方面，在给予在后疗程的免疫效应细胞时，与初始剂量较大时相比，受试者表现出减少的针对在先疗程的免疫效应细胞的细胞表达的CAR的体液或细胞介导的免疫应答。
细胞持久性
在一些实施方案中，所提供的方法增加受试者对所施用的免疫效应细胞的持久性，例如，随时间增加的细胞数量或持续时间，和/或改善免疫细胞疗法中的功效和治疗结果。在一些方面，该方法的优点在于，与其他方法相比，表达嵌合抗原受体的细胞(例如CAR-T细胞)能更大和/或更长程度地改善治疗结果。这些结果可包括患者的生存和缓解，甚至在具有严重肿瘤负荷的受试者中。
在一些实施方式中，检测在在先疗程的免疫效应细胞和/或在后疗程的免疫效应细胞之后在受试者中表达嵌合抗原受体的细胞(例如CAR-表达细胞)的存在和/或量。在一些方面，使用定量PCR(qPCR)来评估在受试者的血液或血清或器官或组织(例如疾病部位)中表达嵌合抗原受体的细胞(例如CAR-T)的量。在一些方面，持续性被定量为每微克DNA中编码受体，例如CAR的DNA或质粒的拷贝，或作为每微升样品例如，血液或血清的受体表达，例如CAR表达细胞的数量，或每微升样品的外周血单核细胞(PBMC)或白细胞或T细胞的总数。
在一些实施方式中，在给予在先疗程的免疫效应细胞之后或至少在第1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30天，在受试者中检测到细胞。在一些方面，在或者至少在给予首次或在后疗程的免疫效应细胞后2、4或6周，或者3、6或12、18或24、或30或36个月，或者1、2、3、4、5或更多年检测到细胞。
在一些实施方式中，该方法导致在受试者的血液或血清或其他体液或器官或组织中至少10、20、30、40、50、60、70、80、90、100、500、1000、1500、2000、5000、10000或15000个拷贝的或编码受体的核酸的最大浓度，例如每微克DNA的CAR，或至少0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8或0.9的受体表达，例如CAR表达的细胞/外周血单个核细胞(PBMC)总数、单核细胞总数、T细胞总数或总微升数。在一些实施方式中，表达受体的细胞被检测为受试者血液中总PBMC的至少10％、20％、30％、40％、50％或60％，和/或在该水平在首次 给药或后续给药后持续至少1、2、3、4、5、6、7、8、9、10、11、12、24、36、48、或52周，或者这种给药后持续1、2、3、4或5年或更多年。
在一些方面，该方法导致例如，在受试者的血清中编码嵌合抗原受体，例如CAR的核酸的拷贝每微克DNA增加至少2倍、至少4倍、至少10倍或至少20倍。
在一些实施方式中，表达受体的细胞可在受试者的血液或血清中检测到，例如通过指定的方法，例如qPCR或基于流式细胞术的检测方法，给予在先疗程的免疫效应细胞后或在给予在后疗程的免疫效应细胞后至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59或60或更多天，或者在给予在先疗程的免疫效应细胞或在后疗程的免疫效应细胞之后持续至少或约2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24或更多周。
在一些方面，通过免疫组织化学、PCR和/或流式细胞术测量，每100个细胞中编码嵌合抗原受体的核酸的拷贝数，例如载体拷贝数，例如在外周血或骨髓或其他隔室中，为至少0.01、至少0.1、至少1、或至少10，在给予细胞，例如，在在先疗程的免疫效应细胞或在后疗程的免疫效应细胞后约1周、约2周、约3周、约4周、约5周、或至少约6周，或至少约2、3、4、5、6、7、8、9、10、11或12个月，或至少2或3年时。
表达嵌合抗原受体的细胞
所述的免疫效应细胞表达有识别BCMA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。
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:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:8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；或者SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:4 所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:9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；或者SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10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；或者SEQ ID NO:11所示的HCDR1、SEQ ID NO:12所示的HCDR2、SEQ ID NO:5所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10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；或者SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:5所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10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。在一具体实施方式中，所述抗体的重链可变区和轻链可变区具有SEQ ID NO:11所示的HCDR1、SEQ ID NO:12所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10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。
在一些实施方式中，所述抗体的轻链可变区具有SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10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，所述抗体的轻链可变区具有SEQ ID NO：20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。在一优选例中，所述抗体的重链可变区具有SEQ ID NO:5所示的HCDR3，轻链可变区具有SEQ ID NO：20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，所述的抗体的重链可变区具有SEQ ID NO：15所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：16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；或者所述的抗体的重链可变区具有SEQ ID NO：17所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：18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；或者所述的抗体的重链可变区具有SEQ ID NO：19所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20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；或者所述的抗体的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20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；或者所述的抗体的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20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。在具体实施例中，所述的抗体的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20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：25、27、或29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：30、31、32、33、34、35、36、37、38、39、40、或41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。在具体实施例中，所述的嵌合抗原受体 具有SEQ ID NO：36、37、38任一所示的氨基酸序列，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。在一优选例中，所述的嵌合抗原受体具有SEQ ID NO：36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，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。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。
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。在一个实施方式中，使用天然关联受体(例如CAR)中的结构域之一的跨膜结构域。在一些情况中，所述跨膜结构域通过氨基酸取代来选择或修饰，以避免所述结构域结合至相同或不同表面膜蛋白的跨膜结构域，以使与受体复合物的其它成员的相互作用最小化。
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。在一些方面中，苯丙氨酸、色氨酸和缬氨酸的三聚体将出现在合成的跨膜结构域的各末端。在一些实施方式中，所述连接通过接头、间隔物和/或跨膜结构域发生。
所述胞内信号转导结构域包括模拟或近似通过天然抗原受体的信号、通过此类受体联合共刺激受体的信号，和/或通过单独共刺激受体的信号的那些。在一些 实施方式中，存在短的寡肽或多肽接头，例如，长度为2-10个氨基酸的接头，例如包含甘氨酸和丝氨酸，例如，甘氨酸-丝氨酸双联体的接头，并在CAR的胞质信号转导结构域和跨膜结构域之间形成连接。
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，例如包含示于SEQ ID NO:35的氨基酸序列，或与SEQ ID NO:35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的实例包括首胞质信号转导序列，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CAR。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CD3-ζ、CD28和CD137的胞内部分。
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，是或含有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，是包含或含有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BCMA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BCMA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BCMA-CAR-T细胞的给予次数以及剂量。
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。
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。
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。在一些实例中，两种部分均保留用于进一步应用。在一些方面中，当没有可用于在异质群中特异性地鉴定细胞类型的抗体时，负选择可能是特别有用的，从而分离最好基于通过与所需群不同的细胞表达的标志物进行。
所述分离不需导致具体细胞群或表达具体标志物的细胞的100％的富集或移除。例如，正选择或富集具体类型的细胞，例如表达标志物的那些，指的是，增加所述细胞的数量或百分数，但不需要导致不表达所述标志物的细胞完全不存在。同样地，负选择、移除或消耗具体类型的细胞，例如表达标志物的那些，指的是，减少所述细胞的数量或百分数，但不需要导致所有此类细胞的完全移除。
在一些实例中，进行多轮分离步骤，其中，对来自一个步骤的正或负选择的部分进行另一分离步骤，例如后续正或负选择。在一些实例中，单一分离步骤同时消耗表达多重标志物的细胞，例如通过孵育使细胞与各自对负选择靶向的标志物具有特异性的多种抗体或结合伴侣孵育。同样地，可通过将细胞与各种细胞类型上表达的多种抗体或结合伴侣孵育来对多重细胞类型进行同时正选择。
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，分离通过如下方式进行：通过正选择富集具体细胞群或通过负选择消耗具体细胞群。在一些实施方式中，正或负选择通过将细胞与特异性地结合至一种或多种表面标志物的一种或多种抗体或其它结合试剂孵育来完成，所述一种或多种表面标志物分别在正选择或负选择的细胞上表达(标志物+)或以相对较高水平表达(标志物高)。
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。
在一些实施方式中，CD8+细胞针对原初、中心记忆、效应记忆和/或中心记忆干细胞进行进一步富集或消耗，例如通过基于与对应亚群相关联的表面抗原进行正或负选择。在一些实施方式中，进行针对中心记忆T(TCM)细胞的富集，以增加功效，例如以改善长期存活、扩增和/或给予后的植入，在一些方面中，其在 此类亚群中特别强健。参见Terakura等，(2012)Blood.1:72–82；Wang等，(2012)J Immunother.35(9):689-701。在一些实施方式中，将TCM-富集的CD8+T细胞与CD4+T细胞合并以进一步增强功效。
在实施方式中，记忆T细胞在CD8+外周血淋巴细胞的CD62L+和CD62L-亚组中存在。PBMC可以针对CD62L-CD8+和/或CD62L+CD8+部分进行富集或消耗，例如采用抗CD8和抗CD62L抗体。
在一些实施方式中，针对中心记忆T(TCM)细胞的富集基于CD45RO、CD62L、CCR7、CD28、CD3、和/或CD127阳性或高表面表达；在一些方面中，其基于CD45RA和/或粒酶B表达或高度表达的细胞进行负选择。在一些方面中，针对TCM细胞富集的CD8+群的分离通过如下方式进行：消耗表达CD4、CD14、CD45RA的细胞，和正选择或针对表达CD62L的细胞进行富集。一方面，针对中心记忆T(TCM)细胞的富集通过如下方式进行：由基于CD4表达选择的细胞的阴性部分起始，其基于CD14和CD45RA的表达进行负选择，和基于CD62L进行正选择。在一些方面中，此类选择同时进行，且在其它方面中，此类选择以某一顺序依次进行。在一些方面中，相同的基于CD4表达的选择步骤用于制备CD8+细胞群或亚群，也用以产生CD4+细胞群或亚群，从而保留来自基于CD4分离的阳性和阴性部分，并任选地在一种或多种进一步正或负选择步骤之后，用于所述方法的后续步骤。
在具体实例中，对PBMC样品或其它血液白细胞样品进行CD4+细胞选择，其中保留阴性和阳性部分。然后，基于CD14和CD45RA或CD19的表达对阴性部分进行负选择，并基于中心记忆T细胞特征性标志物，例如CD62L或CCR7、进行正选择，其中，所述正和负选择以某一顺序进行。
通过鉴定具有细胞表面抗原的细胞群，将辅助性CD4+T细胞分选成原初、中心记忆和效应细胞。CD4+淋巴细胞可通过标准方法获得。在一些实施方式中，原初CD4+T淋巴细胞是CD45RO-、CD45RA+、CD62L+、CD4+T细胞。在一些实施方式中，中心记忆CD4+细胞是CD62L+和CD45RO+。在一些实施方式中，效应CD4+细胞是CD62L-和CD45RO-。
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。
在一些实施方式中，提供的方法包括培育、孵育、培养，和/或遗传工程改造步骤。例如，在一些实施方式中，提供了用于对消耗的细胞群和培养起始组合物进行孵育和/或工程改造的方法。
在一些实施方式中，所述细胞群在培养起始组合物中孵育。可在培养器皿，如单元、腔室、孔、柱、管、管组、阀、小瓶、培养皿、袋或用于培养或培育细胞的其它容器中进行孵育和/或工程改造。
在一些实施方式中，在遗传工程改造之前或与遗传工程改造一起孵育和/或培养细胞。孵育步骤可包括培养、培育、刺激、活化，和/或增殖。在一些实施方式中，在刺激条件或刺激性试剂存在下孵育细胞或组合物。这类条件包括设计成在群中诱导细胞增殖、繁殖、活化，和/或存活以模拟抗原接触，和/或引发细胞用于遗传工程改造，如用于导入重组抗原受体的那些。
所述条件可包括如下一种或多种：具体培养基、温度、含氧量、二氧化碳含量、时间、试剂，例如，营养物、氨基酸、抗生素、离子，和/或刺激因子，例如细胞因子、趋化因子、抗原、结合伴侣、融合蛋白、重组可溶性受体，和经设计可以维持活化细胞状态的任何其他物质。
在一些实施方式中，刺激条件或试剂包括一种或多种物质，例如，配体，其能够活化TCR复合物的胞内信号转导结构域。在一些方面中，所述物质开启或启动T细胞中的TCR/CD3胞内信号转导级联反应。此类物质可包括抗体，例如对TCR组分和/或共刺激受体具有特异性的那些，例如，抗CD3、抗CD28，例如，其结合至固体支持物，例如珠，和/或一种或多种细胞因子。任选地，扩增方法还可包括如下步骤：向培养基(例如，以至少约0.5ng/ml的浓度)添加抗CD3和/或 抗CD28抗体。在一些实施方式中，刺激剂包括1L-2和/或IL-15和/或IL-7和/或IL-21，例如，至少约10单位/mL浓度的IL-2。
在一些方面中，孵育按照一些技术，例如授予Riddell等的美国专利号6,040,177，Klebanoff等，(2012)J Immunother.35(9):651-660,Terakura等，(2012)Blood.1:72-82和/或Wang等，(2012)J Immunother.35(9):689-701中所述的那些进行。
在一些实施方式中，T细胞群通过如下方式扩增：添加至培养起始组合物饲养层细胞，例如非分裂型外周血单核细胞(PBMC)，(例如，从而针对待扩增的初始群中的各T淋巴细胞，所得细胞群包含至少约5、10、20或40或更多PBMC饲养层细胞)；并孵育所述培养物(例如足以扩增所述数量的T细胞的时间)。在一些方面中，所述非分裂型饲养层细胞可包括γ射线辐照的PBMC饲养层细胞。在一些实施方式中，所述PBMC用约3000-3600拉德范围内的γ射线辐照以防止细胞分裂。在一些方面中，所述饲养层细胞在添加T细胞的群之前添加至培养基。
在一些实施方式中，所述刺激条件包括适于人T淋巴细胞生长的温度，例如，至少约25摄氏度，一般至少约30度，且一般是或约是37摄氏度。任选地，孵育还可包括添加非分裂型EBV-转化的类淋巴母细胞(LCL)作为饲养层细胞。LCL可以用约6000-10000拉德范围内的γ射线辐照。在一些方面中，LCL饲养层细胞以任何合适的量提供，例如LCL饲养层细胞与初始T淋巴细胞的比率是至少约10:1。
在某实施方式中，通过用抗原刺激天然或抗原特异性T淋巴细胞来获得抗原特异性T细胞，如抗原特异性CD4+和/或CD8+T细胞。例如，抗原特异性T细胞系或克隆可针对巨细胞病毒抗原产生，通过从感染的受试者分离T细胞并采用相同抗原体外刺激细胞来进行。
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。示例性方法具体描述于，例如，《雷明顿：药物科学与实践》(Remington:The Science and Practice of Pharmacy)，LWW公司(Lippincott Williams&Wilkins)；第21版(2005年5月1日)。
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。在一些方面中，可查阅标准教科书来制备合适制备物。
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。
该容器可由各种材料如玻璃或塑料制成。在一些实施方式中，容器具有一个或多个端口，例如，无菌浸入端口，例如，用于通过管或导管连接至一个或多个管，例如，用于静脉内或其他输注和/或用于出于从其他容器和向其他容器转移的目的连接，如细胞培养和/或储存袋或其他容器。示例性的容器包括冷冻袋、静脉内溶液袋、小瓶，包括具有可通过注射用针头穿破的塞盖的那些。
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，说明指定了通过进行首次给药和连续给药向受试者给予多个单位剂量。在一些实施方式中，首次给药包括向受试者递送所述单位剂量之一并且后续给药包括向受试者给予所述单位剂量中的一个或多个。
在一些实施方式中，标签或包装插页或包装包含标识符，以指示从其衍生细胞的受试者和/或将待给予的受试者的身份。在自体移植的情况下，细胞衍生自待给予细胞的受试者。因此，识别信息可以指定将细胞给予特定患者，这样的信息可以以条形码或其他编码标识符的形式存在于包装材料和/或标签中，或者可以指示受试者的姓名和/或其他识别特征。
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”从患有癌症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癌症表达的抗原，其是肿瘤相关或肿瘤特异性抗原。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在先疗程的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。
在给予在先疗程的免疫效应细胞后，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，在给予在先疗程的免疫效应细胞后，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。
在给予在先疗程的免疫效应细胞后，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。
通过在先疗程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。
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。
表达嵌合抗原受体的免疫效应细胞的制备方法，参考例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、国际专利申请公开号WO2018006882A1、WO2015172339A8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:27所示，核苷酸序列如SEQ ID NO:26所示，scFv具有SEQ ID NO：21所示的重链可变区以及SEQ ID NO：20所示的轻链可变区，嵌合抗原受体具有SEQ ID NO：36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：37或38所示的序列。
SEQ ID NO:27所示的scFv具有SEQ ID NO：11所示的HCDR1、SEQ ID NO：12所示的HCDR2、SEQ ID NO：5所示的HCDR3、及SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2、SEQ ID NO：10所示的LCDR3。
实施例1多发性骨髓瘤患者的治疗
给予BCMA阳性的多发性骨髓瘤患者表达抗-BCMA嵌合抗原受体(CAR)的自体T细胞。在给予细胞之前，患者接受“去单个核细胞分离术”的单采分离技术，并进行预处理治疗。为了获得自体CAR-T细胞，通过分离受试者的外周血PBMC获得T细胞，通过编码靶向BCMA的CAR的病毒载体转导，并进行大量扩增后由冷冻介质制剂获得靶向BCMA的CAR-T细胞，随后分装于冷冻袋中并于低于-175℃液氮条件下保存，在输注前解冻复苏后自体回输受试者。。
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)的一种或多种因子的水平，例如肿瘤坏死因子α(TNFα)、干扰素γ(IFNγ)、IL-10、IL-2和IL-6。在治疗开始之前，可以通过评估与癌症相关的例如患者骨髓或外周血中的细胞数目来评估肿瘤负荷。通过评估骨髓对治疗前的肿瘤负荷进行评估，并确定骨髓胚细胞的百分比。在骨髓中具有至少5％的胚细胞的对象被认为具有形态学疾病(MD)。
冷冻保存的抗BCMA CAR-T细胞通过升温至38℃左右进行复苏，并且患者通过单次输注给予，约2-30min分钟内连续静脉(I.V.)滴注，中位输注持续时间为5分钟。
在给予抗BCMA CAR-T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，在给予抗BCMA CAR-T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首剂量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。
从给予抗BCMA CAR-T细胞后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 抗BCMA 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。
抗BCMA CAR-T细胞的对复发/难治骨髓瘤的反应(参考IMWG2016版，治疗评价为CR、sCR、ICR、MCR或VGPR):
a.参考《多发性骨髓瘤的诊断标准》(IMWG2016版)反应标准进行疗效评价；
b.疗效评估统计学指标：
无进展生存期(PFS)、疾病控制率(DCR)和客观缓解率(ORR)、总生存期(OS)。
本发明中，受试者为BCMA表达阳性、复发或难治的多发性骨髓瘤患者，分成4组参与。受试者均高表达BCMA(BCMA表达阳性率为53％-100％)，多发性骨髓瘤中位病程在0.3-10.7年之间，大多为约5.2年(范围：0.4～10.7年)，之前接受化疗次数的中位数为6次(3-20次)，部分受试者之前还接受过干细胞移植。受试者骨髓中平均浆细胞比例约25％(范围：<5％～85％)。受试者疾病分型包括IgGκ型、IgAλ型、IgGλ型、λ轻链型、IgAκ型。到目前为止，受试者中位随访时间约135天(范围：11～260天)。
第一组为环磷酰胺低剂量组，预处理给予环磷酰胺的剂量不高于400mg/m 2/d(约为190-310mg/m 2/d)，并给予约20mg/m 2/d的氟达拉滨。给予预处理剂后，给予每个受试者BCMA CAR-T细胞约2×10 6至2.7×10 6个细胞/kg。
第二组为环磷酰胺高剂量组，预处理给与环磷酰胺的剂量高于400mg/m 2/d(约为450-560mg/m 2/d)，并给予约20-30mg/m 2/d的氟达拉滨。给予预处理剂后，给予每个受试者输注BCMA CAR-T细胞约2×10 6至2.5×10 6个细胞/kg。
第三组为CAR-T低剂量组，给予其输注抗BCMA CAR-T细胞小于1.5×10 6个细胞/kg，给予该受试者输注抗BCMA CAR-T细胞约0.9×10 6个细胞/kg。
第四组为CAR-T高剂量组，包括3个患者，给予该组受试者输注抗BCMA CAR-T细胞约为2.7×10 6至3.3×10 6个细胞/kg。
将对受试者给予CAR T细胞疗法之日指定为第0日。可在同日或不同日给予氟达拉滨、环磷酰胺。第1组中，在第-6、-5和-4日对受试者给予氟达拉滨，第-6、-5、-4、-3和-2日给予环磷酰胺；或在第-4、-3、和-2日对受试者给予氟达拉滨、环磷酰胺；或在第-3、-2、和-1日对受试者给予氟达拉滨、环磷酰胺；或在第-3和-2日对受试者给予氟达拉滨、环磷酰胺。第2组中，在第-5、-4、-3和-2 日对受试者给予氟达拉滨，和在第-5、-4日给予环磷酰胺；或在第-5、-4和-3日对受试者给予氟达拉滨，和在第-5和-4日给予环磷酰胺；或在第-5、-4和-3日对受试者给予氟达拉滨，和在第-5日给予环磷酰胺；或在第-4、-3和-2日对受试者给予氟达拉滨，和在第-4和-3日给予环磷酰胺；或在第-3和-2日对受试者给予氟达拉滨，环磷酰胺。第3组中，在第-5、-4和-3日对受试者给予氟达拉滨，和在第-5和-4日给予环磷酰胺。第4组中，在第-6、-5、-4和-3日对受试者给予氟达拉滨，和在第-6和-5日给予环磷酰胺；或在第-3和-2日对受试者给予氟达拉滨，环磷酰胺；或在第-3、-2和-1日对受试者给予氟达拉滨，环磷酰胺
每组的给药情况见表1所示，表中注明的氟达拉滨、环磷酰胺、CAR-T细胞给药量为总给药剂量。
表1
Figure PCTCN2019097453-appb-000001
Figure PCTCN2019097453-appb-000002
每组患者的疗效见图1，ORR为100％；CR 8例(约50％)。其中，第2组的CR率为75％。为了进一步评估给予抗BCMA CAR-T细胞的功效，通过检测抗BCMA 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Probe783-P1(核苷酸序列见SEQ ID NO:42)；上游引物序列为：Primer783P1-F1(核苷酸序列见SEQ ID NO:43)；下游引物序列为：Primer783P1-R3(核苷酸序列见SEQ ID NO:44)，检测外周血中含有抗BCMA CAR DNA的拷贝数，直到任何2次连续的检测为阴性，记录为抗BCMA CAR-T细胞持续存活期。
对1号～13号患者进行了检测，结果如表2及图2所示，BCMA CAR-T细胞在所有受试者中增殖，约第3天检测到细胞拷贝数，达峰时间约在第7～21天，维持至第2～3个月。
对14号、15号、16号患者的外周血中含有抗BCMA CAR DNA的拷贝数进行了检测，结果显示，14号患者的CAR-T细胞数量在CAR-T给药后第14天达 到峰值，第56天检测不到；15号患者的CAR-T细胞数量在CAR-T给药后第14天达到峰值，第56天检测不到；16号患者的CAR-T细胞数量在CAR-T给药后第7天达到峰值，6个月后检测不到。
表2BCMA CART拷贝数
Figure PCTCN2019097453-appb-000003
在给予抗BCMA 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)-需要并响应中等介入：
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级转氨酶炎(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(排除转氨酶炎)；
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。
受试者均没有严重神经毒性，但均发生了至少1次不良事件；最常见的与本发明抗BCMA 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。如，第2组中有1例受试者发生2级CRS、1例受试者发生3级CRS，均经解热、IL-6R单抗(托珠单抗)、抗生素抗感染治疗后已恢复；1例受试者发生4级血小板降低SAE，经对症支持治疗已恢复。
在上述实施例中，示例性的，选择了SEQ ID NO：36所示CAR，然而，应理解，其他靶向BCMA的CAR，也可以应用于本申请所述的技术方案。如SEQ ID NO：30、31、32、33、34、35、39、40、或41所示的CAR。
其中，SEQ ID NO：30、31、32所示的CAR的scFv具有SEQ ID NO：15所示的VH，和SEQ ID NO：16所示的VL，CDR区分别为：SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3、及SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2、SEQ ID NO：8所示的LCDR3。
其中，SEQ ID NO：33、34、35所示的CAR的scFv具有SEQ ID NO：17所示的VH，和SEQ ID NO：18所示的VL，CDR区分别为：SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：4所示的HCDR3、及SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2、SEQ ID NO：9所示的LCDR3。
其中，SEQ ID NO：39、40、41所示的CAR的scFv(氨基酸序列如SEQ ID NO：29所示，核苷酸序列如SEQ ID NO：28所示)具有SEQ ID NO：23所示的VH，和SEQ ID NO：20所示的VL，CDR区分别为SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：5所示的HCDR3、及SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2、SEQ ID NO：10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。
以下是本专利文本涉及的序列：
Figure PCTCN2019097453-appb-000004
Figure PCTCN2019097453-appb-000005
Figure PCTCN2019097453-appb-000006
Figure PCTCN2019097453-appb-000007
Figure PCTCN2019097453-appb-000008

Claims (27)

  1. 一种治疗BCMA阳性的肿瘤方法，其特征在于，所述方法包括给予受试者至少一个疗程的表达嵌合抗原受体(CAR)的免疫效应细胞，所述免疫效应细胞特异性识别BCMA。
  2. 根据权利要求1所述的方法，其特征在于，每个疗程的免疫效应细胞的剂量不超过约1x10 9细胞/千克受试者体重或总量不超过约1x10 10细胞；
    优选地，每个疗程的免疫效应细胞的剂量不超过约1x10 8细胞/千克受试者体重或所述细胞总量不超过约1x10 9，
    优选的，每个疗程的免疫效应细胞的剂量不超过约1x10 7细胞/千克受试者体重或所述细胞总量不超过约5x10 8。
  3. 如权利要求1所述的方法，其特征在于，所述每个疗程的免疫效应细胞的总剂量不低于1x10 5；
    优选的，所述每个疗程的免疫效应细胞的总剂量不低于1x10 6；
    优选的，所述每个疗程的免疫效应细胞的总剂量不低于1x10 7。
  4. 如权利要求1所述的方法，其特征在于，给予所述受试者2-5个疗程的所述免疫效应细胞，
    进一步优选，每个疗程的免疫效应细胞在15天内，分成N次给药，N为不小于1的自然数，在一优选方案中，N为1、2、3或4。
  5. 如权利要求4所述的方法，其特征在于，当在先给予的免疫效应细胞在体内检测不到后，再给予在后疗程的免疫效应细胞；或者
    在所述在先疗程给予后约4周至24周的时间点处给予所述的在后疗程的免疫效应细胞。
  6. 如权利要求4所述的方法，其特征在于，在后疗程给予的免疫效应细胞的剂量低于、等于、或高于在先疗程给予的免疫效应细胞，
    优选地，在后疗程给予的免疫效应细胞的剂量高于在先疗程给予的免疫效应细胞，
    更优选地，所述在后疗程给予的免疫效应细胞的剂量是在先给予的免疫效应细胞的剂量的2倍、5倍、7倍或10倍。
  7. 如权利要求4所述的方法，其特征在于，给予在后疗程的免疫效应细胞时，所述受试者具有以下任一特征：
    (i)指示细胞因子释放综合征(CRS)的因子在受试者中血清水平倍数比在给予在先疗程的免疫效应细胞之前即刻的受试者中的水平小约10倍、小约25倍、和/或小约50倍；
    (ii)没有显示出3级或更高的神经毒性；
    (iii)神经毒性或CRS水平与给予在先疗程的免疫效应细胞后的神经毒性或CRS水平的峰值水平相比，呈现降低；或者
    (iv)所述受试者没有显示出针对由在先疗程的细胞表达的CAR的可检测的体液或细胞介导的免疫应答。
  8. 如权利要求7所述的方法，其特征在于，所述(iii)中，CRS水平与给予在先疗程的免疫效应细胞后的CRS的峰值水平相比，降低至少50％，优选的，降低至少20％，更优的，降低至少5％，或者CRS水平与给予在先疗程的免疫效应细胞之前的CRS水平相当。
  9. 如权利要求1所述的方法，其特征在于，所述方法还包括在给予所述的免疫效应细胞的之前进行预处理，所述预处理包括给予所述受试者化疗剂或者辐射治疗，或其组合，优选所述预处理在给予免疫效应细胞前2-12天实施，
    进一步优选，在给予免疫效应细胞前2-7天实施预处理。
  10. 如权利要求9所述的方法，其特征在于，所述化疗剂包含以下任意一种或其组合：环磷酰胺、氟达拉滨。
  11. 如权利要求10所述的方法，其特征在于，当使用氟达拉滨时，所述氟达拉滨的给予量约为10-50mg/m 2/天、或约15-40mg/m 2/天、或约15-35mg/m 2/天、或 15-30mg/m 2/天、或约20-30mg/m 2/天；
    优选的，所述氟达拉滨的给予量约为20-30mg/m 2/天；
    优选的，所述氟达拉滨的给予量约为20-26mg/m 2/天；
    当使用环磷酰胺时，所述环磷酰胺的给予量约为100-700mg/m 2/天、或约150-600mg/m 2/天、或约190-600mg/m 2/天、或约190-560mg/m 2/天；
    优选的，所述环磷酰胺的给予量约为150-400mg/m 2/天，优选的，约为190-350mg/m 2/天；
    优选的，所述环磷酰胺的给予量约为400-600mg/m 2/天，优选的，约为450-600mg/m 2/天，更优选的，约为450-560mg/m 2/天。
  12. 如权利要求10所述的方法，其特征在于，所述化疗剂连续使用不超过6天；当使用氟达拉滨时，优选所述环磷酰胺连续使用1-5天，当使用氟达拉滨时，优选所述氟达拉滨连续使用2-4天。
  13. 如权利要求1所述的方法，其特征在于，所述肿瘤为多发性骨髓瘤。
  14. 如权利要求1所述的方法，其特征在于，所述嵌合抗原受体包括特异性结合BCMA的抗体、跨膜域及胞内域，所述抗体的重链可变区和轻链可变区具有：
    SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:8所示的LCDR3；或者
    SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:4所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:9所示的LCDR3；或者
    SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:5所示的HCDR3以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者
    SEQ ID NO:11所示的HCDR1、SEQ ID NO:12所示的HCDR2、SEQ ID NO:5所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3；或者
    SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:5所示的HCDR3，以及SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3。
  15. 如权利要求1所述的方法，其特征在于，所述的所述嵌合抗原受体包括特异性结合BCMA的抗体、跨膜域及胞内域，所述抗体的轻链可变区具有SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、SEQ ID NO:10所示的LCDR3。
  16. 如权利要求15所述的方法，其特征在于，所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列；或者
    所述抗体的重链可变区具有SEQ ID NO:5所示的HCDR3。
  17. 如权利要求14所述的方法，其特征在于：
    所述的抗体的重链可变区具有SEQ ID NO：15所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：16所示的氨基酸序列；或者
    所述的抗体的重链可变区具有SEQ ID NO：17所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：18所示的氨基酸序列；或者
    所述的抗体的重链可变区具有SEQ ID NO：19所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列；或者
    所述的抗体的重链可变区具有SEQ ID NO：21所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列；或者
    所述的抗体的重链可变区具有SEQ ID NO：23所示的氨基酸序列并且所述抗体的轻链可变区具有SEQ ID NO：20所示的氨基酸序列。
  18. 如权利要求17所述的方法，其特征在于，所述抗体具有SEQ ID NO：25、27、或29所示的scFv的序列。
  19. 如权利要求14所述的方法，其特征在于，所述的嵌合抗原受体具有SEQ ID NO：30、31、32、33、34、35、36、37、38、39、40、或41所示氨基酸序列；
    优选地，所述的嵌合抗原受体具有SEQ ID NO：36、37、38任一所示的氨基 酸序列；
    更优选地，所述的嵌合抗原受体具有SEQ ID NO：36所示的氨基酸序列。
  20. 如权利要求1-19中任一项所述的方法，其特征在于，所述免疫效应细胞是T细胞、NK细胞或者NKT细胞；
    优选地，所述免疫效应细胞是T细胞，进一步优选，所述免疫效应细胞来自所述受试者自体。
  21. 如权利要求1所述的方法，其特征在于，每个疗程的免疫效应细胞的给药间隔是约4周至24周；
    优选的，每个疗程的免疫细胞数量基本相同；
    优选的，在后疗程给予的免疫效应细胞的数量高于在先给的在后疗程的免疫细胞数；
    优选的，在后疗程给予的免疫效应细胞数量低于在先给的在后疗程的免疫细胞数。
  22. 如权利要求1所述的方法，其特征在于，给予所述免疫效应细胞之前，所述受试者没有接受过靶向BCMA的表达嵌合抗原受体的免疫细胞的治疗；或者
    在给予所述免疫效应细胞治疗之前，所述受试者已经进行了手术治疗、化疗、或者不同于权利要求1所述的免疫治疗。
  23. 如权利要求1所述的方法，其特征在于，在给予每个疗程的免疫效应细胞之前，对所述受试者的指示CRS的因子、指示神经毒性的因子、指示肿瘤负荷的因子、和/或指示宿主抗-CAR免疫应答的因子的血清水平进行评价；
    其中，所述的指示肿瘤负荷的因子为：所述受试者的肿瘤细胞总数、或者所述受试者的器官中肿瘤细胞总数、或者所述受试者的组织中的肿瘤细胞总数、或者肿瘤的质量或体积，或者肿瘤转移的程度、或者肿瘤数量。
  24. 如权利要求23所述的方法，其特征在于，包括：
    i)在给予在后疗程之前评价指示肿瘤负荷的因子；和
    ii)基于所述评价的结果，确定在后疗程，并且
    iii)如果评价确定所述受试者的肿瘤质量或体积稳定或下降，给予所述受试者包含少于或多于所述在先疗程中的CAR表达细胞的数量或与其大约相同的CAR表达细胞的数量的在后疗程。
  25. 如权利要求1所述的方法，其特征在于，所述免疫效应细胞的给药剂量为约0.1x10 6细胞/kg受试者体重～5x10 7细胞/kg，或者所述免疫效应细胞的给药总剂量为约0.1x10 7细胞～1x10 10细胞；
    优选的，为约0.5x10 6细胞/kg～1x10 7细胞/kg，或者所述免疫效应细胞的给药总剂量为约0.1x10 8细胞～1x10 9细胞；
    更优选的，为约0.9x10 6细胞/kg～5x10 6细胞/kg，或者所述免疫效应细胞的给药总剂量为约0.1x10 8细胞～9x10 8细胞。
  26. 如权利要求1所述的方法，其特征在于，所述受试者的BCMA的表达阳性率大于50％，优选的，大于70％，或者大于80％；
    更优选的，大于85％；
    更优选的，大于90％。
  27. 如权利要求1所述的方法，其特征在于，所述受试者的疾病分型为IgGκ型、或者IgGλ型、或者IgAλ型、或者IgAκ型、或者λ轻链型。
PCT/CN2019/097453 2018-07-24 2019-07-24 免疫效应细胞治疗肿瘤的方法 WO2020020210A1 (zh)

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