WO2019206326A1 - 改进的t细胞治疗方法 - Google Patents

改进的t细胞治疗方法 Download PDF

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WO2019206326A1
WO2019206326A1 PCT/CN2019/084773 CN2019084773W WO2019206326A1 WO 2019206326 A1 WO2019206326 A1 WO 2019206326A1 CN 2019084773 W CN2019084773 W CN 2019084773W WO 2019206326 A1 WO2019206326 A1 WO 2019206326A1
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Prior art keywords
cancer
cells
cell
associated antigen
car
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PCT/CN2019/084773
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English (en)
French (fr)
Inventor
王歈
李贤秀
郑南哲
王佑春
孙磊
刘强
张永华
王萌
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IMMUNOTECH BEIJING Ltd
National Institutes for Food and Drug Control
Immunotech Biopharm Co Ltd
Pharos Vaccine Inc
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IMMUNOTECH BEIJING Ltd
National Institutes for Food and Drug Control
Immunotech Biopharm Co Ltd
Pharos Vaccine Inc
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Priority to CA3098635A priority Critical patent/CA3098635A1/en
Priority to EP19791829.5A priority patent/EP3805366A4/en
Priority to US17/051,410 priority patent/US12427181B2/en
Priority to SG11202010676TA priority patent/SG11202010676TA/en
Priority to JP2021508054A priority patent/JP7467414B2/ja
Priority to KR1020207034343A priority patent/KR20210033941A/ko
Publication of WO2019206326A1 publication Critical patent/WO2019206326A1/zh
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Definitions

  • the invention belongs to the field of biomedicine.
  • the invention relates to improved methods of T cell therapy. More specifically, the invention relates to enhancing the efficacy of cancer treatment of therapeutic T cells, such as CAR-T or TCR-T cells, by stimulation with living cells expressing a cancer associated antigen.
  • B-ALL B-cell acute lymphoblastic leukemia
  • multi-drug chemotherapy and/or targeted therapy have achieved a high rate of complete remission, but still have a poor prognosis in adults, and relapse / refractory Sexual B-ALL is a big challenge for all patients.
  • Lymphocyte depletion chemotherapy followed by CD19-targeted chimeric antigen receptor-modified T (CAR-T) cell therapy is a novel therapeutic approach and demonstrates relapsed/refractory B-ALL, chronic in adults and children Patients with lymphocytic leukemia and lymphoma have a strong response. New data from these studies indicate that significantly small doses of CAR-T cells delivered can eliminate large disease burdens.
  • CAR generally comprises an extracellular antigen binding domain (single chain variable fragment of a monoclonal antibody (scFV)) and an intracellular signaling domain of activated T cells.
  • scFV single chain variable fragment of a monoclonal antibody
  • MHC major histocompatibility complex
  • One of the possible methods is to modify the structure of the CAR, for example by applying a second or third generation or even a fourth generation CAR, which combines an activation signal domain and a costimulatory signal domain, and can improve T cell expansion and persistence.
  • CD19-targeting CAR-T cells prepared from purified primary T cells or central memory T cells were more effective in eliminating CD19+ tumors than CD19-targeting CAR-T cells prepared according to effector memory T cells.
  • synergistic enhancement of efficiency can be achieved by delivering a defined proportion of CAR-T cells derived from CD8/CD4 T cells.
  • the CAR-T cells currently used retain the endogenous T cell receptor (TCR), and the biology of TCR and CAR crosstalk is very complex.
  • CAR and TCR greatly attenuates the in vivo efficacy of CD8+ CAR-T cells; in contrast, CD4+ CAR-T cells retain their ability to survive in vivo in the presence of TCR and CAR antigens.
  • the invention provides a method of treating cancer in a subject, comprising the steps of:
  • the invention provides a method of preventing cancer progression or recurrence in a subject, wherein the subject has been subjected to cancer treatment using therapeutic T cells that specifically target a cancer associated antigen, the method comprising The subject administers a cell that expresses a cancer associated antigen.
  • the invention provides a combination for treating cancer in a subject comprising a therapeutic T cell that specifically targets a cancer associated antigen and a cell expressing the cancer associated antigen.
  • the invention provides the use of a combination of the third aspect of the invention in the manufacture of a medicament for the treatment of cancer in a subject.
  • a cell expressing a cancer associated antigen for the preparation of a medicament for treating cancer in a subject, wherein the medicament further comprises a therapeutic T cell that specifically targets the cancer associated antigen.
  • the invention provides the use of a cell expressing a cancer associated antigen for the preparation of a medicament for preventing cancer progression or recurrence in a subject, wherein the subject has been subjected to treatment using a specific targeting of the cancer associated antigen Cancer treatment for sex T cells.
  • the invention provides the use of a therapeutic T cell that specifically targets a cancer associated antigen for the preparation of a medicament for treating cancer in a subject, wherein the medicament further comprises a cell expressing the cancer associated antigen.
  • the invention provides a cell expressing a cancer associated antigen for use in treating cancer in a subject in combination with a therapeutic T cell that specifically targets the cancer associated antigen.
  • the invention provides a cell expressing a cancer-associated antigen for preventing cancer progression or recurrence in a subject, wherein the subject has been subjected to cancer using a therapeutic T cell that specifically targets the cancer-associated antigen treatment.
  • the invention provides a therapeutic T cell that specifically targets a cancer associated antigen for use in treating cancer in a subject in combination with a cell expressing the cancer associated antigen.
  • the invention provides a kit comprising a therapeutic T cell that specifically targets a cancer associated antigen and/or a cell expressing the cancer associated antigen, the kit being used by the method of the invention Treating cancer or preventing cancer progression or recurrence in a subject.
  • FIG. 1 Production and characterization of B-NDG mice.
  • A Schematic representation of the Il2rg gene deletion targeting strategy by CRISPR/Cas9 genome editing technology.
  • B Detection of different lymphocyte populations in peripheral blood and spleen of NOD-scid and B-NDG mice by flow cytometry. Numbers indicate the percentage of positive cells for a given phenotype.
  • C 6-week-old C57BL/6, nude mice, Rag2-/-, NOD-scid and B-NSG mice were implanted with 5 ⁇ 10 5 Raji cells by tail vein injection, respectively. The relative levels of bioluminescence are shown in pseudo-color, with red and blue representing the strongest and weakest flux.
  • F B-NDG mice died on day 17 or 18, but other mice survived on day 30.
  • G CD19 is highly expressed on the surface of Raji-Fluc cells, and the luminescence intensity of Fluc is positively correlated with the number of Raji-Fluc cells.
  • FIG. 1 Efficiency of in vitro CD19 CAR-T cells.
  • A Structure of anti-CD19 CAR.
  • B Summary of in vitro culture of CAR-T cells from PBMC donors, including percentage of viable cells, total cell count of CAR-T cells, and percentage of CAR-T cells.
  • C Cytotoxic effects of CAR-T cells on Raji cells in vitro at CAR-T/Raji ratios of 1:1, 3:1 and 6:1.
  • FIG. 3 Re-stimulation of low dose live tumor cells promotes the production of immune memory in CAR-T cells.
  • A Separate measurement of flux in organs and tissues in different groups.
  • B Reimplantation of low dose live tumor cells promotes tumor clearance and prolongs overall survival.
  • C Reimplantation of low-dose live tumor cells can delay recurrence.
  • D Average total flux per group.
  • LT low dose reimplantation
  • MT medium dose reimplantation
  • HT high dose reimplantation.
  • FIG. 4 Reimplantation of tumor cells that died after irradiation did not work.
  • A Separate measurement of flux in organs and tissues in different groups. Reimplantation of tumor cells that died after (B) and (C) radiation did not prolong overall survival and recurrence-free survival. The effect of dual treatment of CAR-T cells is the same as for a single treatment.
  • D The number of CAR-T cells in peripheral blood of mice was measured by qPCR.
  • FIG. 1 Characterization of CAR-T-19 and aT19 cells.
  • A Schematic diagram showing the structure of the CAR-19 gene construct and the CAR-T-19 transduction efficiency of primary activated T cells.
  • B Structure of human CD19 gene construct and CD19 transduction efficiency of primary activated T cells.
  • C Cytotoxic effects of CAR-T-19 cells on CD19-K562 or K562 cells at a ratio of CAR-T-19:CD19-K562 or K562 of 1:1, 3:1 and 6:1; and CAR- The cytotoxic effect of T-19 cells on Raji-Fluc or aT19 cells at a CAR-T-19:Raji-Fluc or aT19 ratio of 0.3:1, 1:1 and 3:1.
  • FIG. 6 Re-stimulation of CAR-T cells by live Raji tumor cells increases survival and delays tumor recurrence.
  • Raji-Fluc cells (2 x 10 5 cells per mouse; A1, A2, A3, NT and P groups) were injected intravenously (iv) at D0.
  • mice received iv injection of CAR-T-19 cells (2 x 10 7 cells per mouse; groups A1, 2, 3, NT).
  • mice were injected iv with different doses of Raji-Fluc cells (A1 and PA1 groups: 5 x 10 4 ; A2 and PA2 groups: 1.5 x 10 5 ; and A3 and PA3 groups: 5 x 10 5 ).
  • the PA group was used as a control for the group A.
  • A Schematic showing a schedule of animal experiments.
  • B Bioluminescence images showing total flux in organs and tissues of different groups of mice. Kaplan-Meier survival curves were constructed from different doses of reimplanted Raji-Fluc cells to monitor eradication of systemic disease (C) and to estimate recurrence-free survival (D).
  • FIG. 7 Reactivation of CAR-T cells by tumor cells that died after irradiation was ineffective.
  • Raji-Fluc cells (2 x 10 5 cells per mouse; A1, A2, A3, NT and P groups) were injected intravenously (iv) at D0.
  • mice received iv injection of CAR-T-19 cells (2 x 10 7 cells per mouse; A1, A2, A3, NT groups).
  • mice were iv injected with different doses of irradiated Raji-Fluc cells (A1 and PA1 groups: 5 x 10 4 ; A2 and PA2 groups: 1.5 x 10 5 ; and A3 and PA3 groups: 5 x 10 5 ).
  • the PA group was used as a control for the group A.
  • A Schematic showing a schedule of animal experiments.
  • B Bioluminescence images showing total flux in organs and tissues of different groups of mice. Kaplan-Meier survival curves were constructed from different doses of reimplanted irradiated Raji-Fluc cells to monitor eradication of systemic disease (C) and to estimate recurrence-free survival (D).
  • E Quantitative polymerase chain reaction was used to detect CAR+ cells in the blood of group A and group NT after implantation of Raji-Fluc cells. The graph shows the number of copies of the CAR gene in the blood samples from each group.
  • FIG. 8 Continuous treatment with CAR-T-19 and aT19 cells promotes the production of memory CAR-T cells.
  • Raji-Fluc cells (2 x 10 5 cells per mouse; A1, NT, aT19 and P groups) were injected intravenously (iv) at D0.
  • mice received iv injection of CAR-T-19 cells (2 x 10 7 cells per mouse; A1, NT, aT19 groups).
  • mice in group A1 received 5 ⁇ 10 4 cells in iv
  • mice in group aT19 received 5 ⁇ 10 5 cells in iv.
  • the other groups did not receive injection.
  • the CAR-T treatment for CD19 can achieve a remission rate of 67% to 90% in patients with relapsed/refractory B-ALL; however, the recurrence-free period varies from 2 months to 3 years.
  • There are two recurrence patterns after CD19 CAR-T treatment one is due to the recurrence of CD19-positive tumors due to insufficient persistence of CAR-T cells; the other is due to the presence of CAR-T in approximately 20% of patients.
  • Recurrence caused by loss of CD19- in cells such loss may be caused by clonal selection or tumor evolution.
  • the inventors successfully constructed a Raji-B-NDG mouse model to study the efficiency of CD19 CAR-T therapy.
  • the alleviated mice showed different degrees of recurrence on day 20 (D20) and then died within D28-36, indicating that after initial contact with antigen and elimination of tumor cells, injected CAR- The T cells gradually decrease, resulting in a final recurrence that is not effectively controlled.
  • the cause of the recurrence may be due to the following two points.
  • the problem of therapeutic cell life and antigen stimulation should be addressed in clinical applications.
  • the effect of re-inoculation of tumor cells (Raji-Fluc) or cells expressing tumor antigens such as CD19 (such as T cells) is tested when D10 tumor cells have been substantially eliminated by CAR-T.
  • the low-dose re-implantation group (re-stimulation) maintained a longer period of tumor-free recurrence than the single-dose group (tumor-forming), with more than half of the mice surviving for more than 36 days, indicating Immunological memory occurs under repeated stimulation of the same antigen, and rapid response to repeated injection of the same antigen results in sustained inhibition of tumor cells.
  • the inventors investigated whether antigens from Raji cells that died after irradiation can stimulate CAR-T cells.
  • the results indicate that re-implantation of dead tumor cells does not awaken the memory of CAR-T cells, resulting in a short survival time in mice. This suggests that CAR-T cells can only be activated by living cells carrying antigen.
  • the inventors have also surprisingly found that repeated administration of CAR-T therapy does not delay tumor recurrence, which provides additional evidence for the ineffectiveness of multiple CAR-T infusions. Without wishing to be bound by any theory, it is believed that stimulation of live cells of low doses of cancer-associated antigens promotes the production of CAR-T cellular immune memory, resulting in delayed relapse and prolonged survival.
  • the invention provides a method of treating cancer in a subject comprising the steps of:
  • the therapeutic T cell is administered one or more times, preferably once. In some embodiments, the cell expressing the cancer associated antigen is administered one or more times, preferably once.
  • the cell expressing a cancer associated antigen is administered after administration of the therapeutic T cell.
  • administration of the therapeutic T cells for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days
  • Expression of the cancer-associated antigen is about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days later. Cell.
  • the cells expressing the cancer associated antigen are administered after administration of the therapeutic T cells such that the cancer cell load is reduced.
  • the cancer cell load is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80.
  • cells expressing the cancer associated antigen are administered.
  • the cell expressing the cancer associated antigen is administered after the cancer is completely relieved by administration of the therapeutic T cell.
  • Changes in cancer cell load in a subject can be determined by various methods known in the art. For example, for hematological cancer, cancer cell load can be determined by flow cytometry of circulating cells.
  • tumor burden can be determined by clinically commonly used imaging methods (such as nuclear magnetic resonance, CT, PET-CT, etc.).
  • tumor burden can be determined by detecting tumor markers.
  • the change in tumor burden can be determined by detecting the amount of the therapeutic T cell prior to administration of the cell expressing the cancer associated antigen, eg, the content of the therapeutic T cell is inversely related to tumor burden .
  • a cell expressing the cancer associated antigen is administered when the amount of the therapeutic T cell in the subject is decreased.
  • the amount of the therapeutic T cell in the subject can be detected by, for example, flow cytometry or quantitative PCR.
  • the therapeutic T cell is a CAR-T cell
  • the copy number of the CAR-encoding gene in the sample from the subject can be detected by PCR.
  • cells expressing the cancer-associated antigen are administered.
  • the method further comprises the step of monitoring the tumor burden and/or the amount of the therapeutic T cells in the subject after administration of the therapeutic T cells.
  • one or more of the cells expressing the cancer associated antigen are administered after administration of one or more of the therapeutic T cells. In some embodiments, the one or more cells expressing the cancer associated antigen are administered after administration of the therapeutic T cell once. In some embodiments, one or more of the therapeutic T cells are used and one or more of the cells expressing the cancer associated antigen are administered after each administration of the therapeutic T cells. In some embodiments, the cancer-expressing antigen-expressing cells are administered once after administration of the therapeutic T cells. In some embodiments, the one or more cells expressing the cancer associated antigen are administered after administration of the therapeutic T cells. In some embodiments, the cancer-expressing antigen-expressing cells are administered once after administration of the therapeutic T cells.
  • the therapeutic T cell is administered in a therapeutically effective amount.
  • a therapeutically effective amount of a therapeutic T cell refers to an amount of therapeutic T cell capable of reducing the burden of a cancer cell after use, for example, reducing the cancer cell load by at least about 5%, at least about 10%, at least about 20%, At least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or an amount that completely reduces cancer.
  • the therapeutic T cell is effective in an amount of from about 10 4 to about 10 9 cells, such as about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 or about 10 9 cells.
  • the amount of therapeutic T cell administered is determined based on the subject's body weight, for example, from about 10 4 cells/kg body weight to about 10 9 cells/kg body weight, such as about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 or about 10 9 cells/kg body weight.
  • the cell expressing the cancer associated antigen is administered in a stimulating effective amount.
  • a stimulatory effective amount of a cell expressing a cancer associated antigen refers to the ability to prolong the in vivo persistence (enhance immune memory) of a previously administered therapeutic T cell after administration and/or to prolong the overall survival of the subject and/or prolong the subject. The amount of recurrence-free survival.
  • the stimulatory effective amount of the cancer-associated antigen-expressing cell is from about 10 3 to about 10 6 cells, such as about 10 3 , about 10 4 , about 10 5 , or about 10 6 Cells.
  • a low dose of said cell expressing a cancer associated antigen for example about 10 4 cells, is administered.
  • the amount of cells expressing the cancer associated antigen is determined based on the subject's body weight, from about 10 3 to about 10 6 cells/kg body weight, such as about 10 3 , about 10 4 , about 10 5 , or about 10 6 Cell/kg body weight.
  • the amount of cells expressing a cancer associated antigen is determined based on the amount of therapeutic T cells administered.
  • the ratio of the amount of therapeutic T cells administered to the amount of cells expressing the cancer associated antigen is any ratio between about 1:1 to about 1000:1 or higher, such as about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 50:1, about 100:1 or about 1000:1 or higher.
  • the in vivo persistence of therapeutic T cells can be increased by stimulating existing therapeutic T cells (such as CAR-T cells) in vivo by administering living cells expressing a cancer associated antigen. At least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 100%, at least about 150%, at least about 200%, at least about 300% or more.
  • existing therapeutic T cells such as CAR-T cells
  • stimulation of existing therapeutic T cells in vivo by administration of living cells expressing a cancer associated antigen can extend the overall survival of the subject by at least about 5%. At least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% At least about 150%, at least about 200%, at least about 300% or longer.
  • stimulation of existing therapeutic T cells in vivo by administration of a living cell expressing a cancer associated antigen may result in prolonged relapse-free survival after remission in the subject At least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 100%, at least about 150%, at least about 200%, at least about 300% or longer.
  • existing therapeutic T cells eg, CAR-T cells
  • a living cell expressing a cancer associated antigen may result in prolonged relapse-free survival after remission in the subject At least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 100%, at least about 150%, at least about 200%, at least about 300% or longer.
  • the therapeutic T cell is a T cell (TCR-T cell) comprising an exogenous T cell receptor (TCR).
  • TCR T cell receptor
  • the exogenous TCR typically comprising alpha and beta chains specifically binds to a cancer associated antigen.
  • the therapeutic T cell is a T cell (CAR-T cell) comprising a chimeric antigen receptor (CAR).
  • CAR-T cell T cell
  • CAR-T cells a T cell comprising a chimeric antigen receptor (CAR).
  • CAR-T cells have been developed in the art, such as first to fourth generation CAR-T cells, CAR-T cells modified to remove inhibitory signals, and the like, which can be applied to the present invention.
  • the CAR comprises an extracellular antigen binding domain directed against a cancer associated antigen.
  • the extracellular antigen binding domain can be, for example, a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, an antibody single chain variable region (scFV), and an antigen binding fragment thereof.
  • the extracellular antigen binding domain is a scFV.
  • the extracellular antigen binding domain can be derived from one or more known antibodies including any commercially available antibodies, such as FMC63, rituximab, alemtuzumab, Epratuzumab, trastuzumab, bivatuzumab, cetuximab, labetuzumab, palivizumab ( Palivizumab), sevirumab, tuvirumab, basiliximab, daclizumab, infliximab, omalizumab ( Omalizumab), efalizumab, Keliximab, siplizumab, natalizumab, clenoliximab, and pemabizumab ( Pemtumomab), Edrecolomab, Cantuzumab, and the like.
  • FMC63 FMC63
  • rituximab alemtuzumab
  • Epratuzumab trastuzumab
  • the CAR further comprises a transmembrane domain and an intracellular signal transduction domain.
  • the intracellular signal transduction domain of a CAR according to the invention is responsible for intracellular signal transduction after binding of the extracellular ligand binding domain to the target, resulting in activation of immune cells and immune responses.
  • the intracellular signal transduction domain has the ability to activate at least one normal effector function of the immune cells expressing the CAR.
  • the effector function of a T cell can be cytolytic or helper activity, including secretion of cytokines.
  • An intracellular signal transduction domain for a CAR can be a cytoplasmic sequence, such as, but not limited to, a cytoplasmic sequence of a T cell receptor and a co-receptor that act in concert to initiate signal transduction after antigen receptor engagement, And derivatives or variants of any of these sequences as well as any synthetic sequences having the same functional ability.
  • the intracellular signal transduction domain includes two different types of cytoplasmic signal transduction sequences: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or costimulatory signals.
  • the primary cytoplasmic signal transduction sequence can include a signal transduction motif called the immunoreceptor tyrosine activation motif of ITAM.
  • Non-limiting examples of ITAM used in the present invention may include those derived from TCR(R), FcR[gamma], FcR[beta], FcR[epsilon], CD3[gamma], CD3[delta], CD3[epsilon], CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signal transduction domain of a CAR can comprise a CD3 ⁇ signal transduction domain.
  • the intracellular signal transduction domain of a CAR of the invention further comprises a costimulatory domain.
  • the costimulatory domain is selected from the group consisting of a 41BB costimulatory domain or a CD28 costimulatory domain.
  • a CAR is expressed on the surface of the cell.
  • a CAR can include a transmembrane domain.
  • Suitable transmembrane domains of the CAR of the invention have the ability to: (a) express on the cell surface, preferably immune cells such as, but not limited to, lymphocytes or natural killer (NK) cells, and (b) ligand binding domains Interacting with an intracellular signal transduction domain for directing cellular responses to immune cells to a predetermined target cell.
  • Transmembrane domains can be derived from natural or synthetic sources.
  • the transmembrane domain can be derived from any membrane-bound protein or transmembrane protein.
  • the transmembrane domain may be derived from a subunit of a T cell receptor such as an alpha subunit, a beta subunit, a gamma or delta subunit, a polypeptide comprising a CD3 complex, and a p55 of an IL-2 receptor ( ⁇ chain), p75 ( ⁇ chain) or ⁇ , a subunit chain of an Fc receptor, particularly an Fc ⁇ receptor III or CD protein.
  • the transmembrane domain can be synthetic and can include primarily hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from a human CD8 alpha chain.
  • the transmembrane domain can further comprise a hinge region located between the extracellular ligand binding domain and the transmembrane domain.
  • the hinge region is for example from the extracellular region of CD8, CD4 or CD28.
  • the hinge region is part of a human CD8 alpha chain.
  • a CAR for use in the invention may comprise an extracellular antigen binding domain that specifically binds to a cancer associated antigen, a CD8 alpha hinge and a transmembrane domain, a CD3 ⁇ signal transduction domain, and 4-1BB co-stimulatory domain.
  • the CAR of the invention (for CD19) comprises the amino acid sequence of SEQ ID NO: 1 as follows:
  • the CAR of the invention (for CD19) is encoded by the nucleotide sequence of SEQ ID NO: 2:
  • the cell expressing the cancer associated antigen is a living cell. In some embodiments, the cancer associated antigen is expressed on the surface of the living cell. In some embodiments, the cell expressing the cancer associated antigen is not a cancer cell. In some embodiments, the cell expressing the cancer associated antigen is a cell that is genetically engineered to express the antigen.
  • the type of the cell is not particularly limited, and it may be derived from an isolated primary cell or derived from a cell line. In some embodiments, the cell expressing the cancer associated antigen is derived from a peripheral blood cell, such as a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the cell expressing the cancer associated antigen is derived from an immune cell, such as a macrophage, dendritic cell, plasma cell, granulocyte, mast cell, lymphocyte (inflammatory T lymphocyte, cytotoxic T Lymphocytes, regulatory T lymphocytes, or helper T lymphocytes, B lymphocytes, and the like.
  • the cells can be derived from CD34+ cells.
  • the cells can be derived from T lymphocytes such as CD4+ T lymphocytes or CD8+ T lymphocytes.
  • the cells can be derived from B lymphocytes.
  • the therapeutic T cell and/or the cell expressing a cancer associated antigen are derived from an autologous cell of the subject.
  • autologous refers to a cell, cell line or population of cells used to treat a subject from which the subject is derived.
  • the therapeutic T cell and/or the cell expressing a cancer associated antigen is derived from an allogeneic cell, eg, from a donor compatible with the human leukocyte antigen (HLA) of the subject. Cells from the donor can be converted to non-allogene-reactive cells using standard protocols and replicated as needed to produce cells that can be administered to one or more patients.
  • HLA human leukocyte antigen
  • the therapeutic T cells of the invention and/or cells expressing a cancer associated antigen can be prepared by a variety of means known in the art.
  • T cells can be transduced with an expression construct comprising a CAR or TCR coding sequence to obtain CAR-T cells or TCR-T cells.
  • an expression construct comprising a coding sequence for a cancer associated antigen can be transduced into a cell to obtain a cell expressing the cancer associated antigen.
  • One of skill in the art can readily construct expression constructs suitable for protein expression.
  • the cells of the invention can be obtained from a variety of non-limiting sources by a variety of non-limiting methods, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors.
  • cell lines that are available and known to those of skill in the art can be used.
  • the cells can be derived from a healthy donor or from a patient diagnosed with cancer.
  • the cells can be part of a mixed population of cells that exhibit different phenotypic characteristics.
  • T cells of the invention can be activated and expanded before or after genetic modification.
  • T cells can be expanded in vitro or in vivo.
  • T cells of the invention can be expanded, for example, by contact with an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of a T cell to produce a T cell activation signal.
  • an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of a T cell to produce a T cell activation signal.
  • a chemical such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or a mitotic lectin such as phytohemagglutinin (PHA) can be used to generate T cell activation signals. .
  • a population of T cells can be activated by contact with, for example, an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by a protein kinase C activator (eg, moss)
  • the enzyme is activated by contact with a calcium ionophore.
  • a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation.
  • Conditions suitable for T cell culture include suitable media (eg, Minimal Essential Media or RPMI Media 1640, or X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, wherein the necessary factors include serum (eg, Fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp and TNF, Or additives for cell growth known to those skilled in the art.
  • Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder, and reducing agents such as N-acetyl-cysteine and 2-mercaptoacetic acid.
  • the medium may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer, amino acids, sodium pyruvate and vitamins, serum-free or appropriate supplemented serum. (or plasma) or a defined group of hormones, and / or a certain amount of cytokines sufficient for T cell growth and expansion.
  • the target cells can be maintained under conditions necessary to support growth, such as a suitable temperature (eg, 37 ° C) and an environment (eg, air plus 5% CO 2 ). T cells exposed to different stimulation times may exhibit different characteristics.
  • Subject refers to an organism that has or is susceptible to a disease or condition that can be treated by the methods, combinations or pharmaceutical compositions of the invention.
  • Non-limiting examples include humans, cows, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals.
  • the subject is a human.
  • the present invention provides a method of preventing cancer progression or recurrence in a subject, wherein the subject has been subjected to treatment (cancer treatment) for administering therapeutic T cells that specifically target a cancer-associated antigen,
  • the method comprises administering to the subject a cell that expresses a cancer associated antigen.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the cell expressing the cancer associated antigen is administered one or more times, preferably once.
  • the therapeutic T cells are administered about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, Administration of the expression for about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days later A cell that describes a cancer-associated antigen.
  • the cell expressing the cancer associated antigen is administered after the therapeutic T cell causes a decrease in cancer cell load.
  • the cancer cell load is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80.
  • cells expressing the cancer associated antigen are administered.
  • the cells expressing the cancer associated antigen are administered after the cancer is completely relieved by administration of the therapeutic T cells.
  • a cell expressing the cancer associated antigen is administered when the amount of the therapeutic T cell in the subject is decreased.
  • the amount of the therapeutic T cell in the subject can be detected by, for example, flow cytometry or quantitative PCR.
  • the therapeutic T cell is a CAR-T cell
  • the copy number of the CAR-encoding gene in the sample from the subject can be detected by PCR.
  • cells expressing the cancer-associated antigen are administered.
  • the method further comprises the step of monitoring the tumor burden and/or the amount of the therapeutic T cells in the subject after administration of the therapeutic T cells.
  • the cell expressing a cancer associated antigen is administered in a stimulating effective amount.
  • a stimulatory effective amount of a cell expressing a cancer associated antigen refers to the ability to prolong the in vivo persistence (enhance immune memory) of a previously administered therapeutic T cell after administration and/or to prolong the overall survival of the subject and/or prolong the subject. The amount of recurrence-free survival.
  • the stimulation of cell expressing a cancer-associated antigen effective amount is from about 103 to about 106 cells, for example, about 103, about 104, about 105, or about 106 cells.
  • a low dose of said cell expressing a cancer associated antigen for example about 10 4 cells, is administered.
  • the amount of cells expressing the cancer associated antigen is determined based on the subject's body weight, from about 10 3 to about 10 6 cells/kg body weight, such as about 10 3 , about 10 4 , about 10 5 , or about 10 6 Cell/kg body weight. In some embodiments, the amount of cells expressing a cancer associated antigen is determined based on the amount of therapeutic T cells previously received by the patient.
  • the ratio of the amount of therapeutic T cells administered to the amount of cells expressing the cancer associated antigen is any ratio between about 1:1 to about 1000:1 or higher, such as about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 50:1, about 100:1 or about 1000:1 or higher.
  • the invention provides a combination for treating cancer in a subject comprising a therapeutic T cell that specifically targets a cancer associated antigen and a cell expressing the cancer associated antigen.
  • the combination is for treating cancer in a subject by the method of the first aspect of the invention.
  • the combination comprises a therapeutically effective amount of the therapeutic T cell and a stimulating effective amount of the cell expressing a cancer associated antigen.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the invention provides the use of a combination of the third aspect of the invention in the manufacture of a medicament for the treatment of cancer in a subject.
  • the medicament is for treating cancer in a subject by the method of the first aspect of the invention.
  • the invention provides the use of a cell expressing a cancer associated antigen for the manufacture of a medicament for the treatment of cancer in a subject.
  • the medicament is for use in treating cancer in a subject in combination with a therapeutic T cell that specifically targets the cancer associated antigen.
  • the medicament further comprises a therapeutic T cell that specifically targets the cancer associated antigen.
  • a cell expressing a cancer associated antigen is independently present in the drug with a therapeutic T cell that specifically targets the cancer associated antigen, eg, independently placed in a different container.
  • the medicament is for treating cancer in a subject by the method of the first aspect of the invention.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the invention provides the use of a cell expressing a cancer associated antigen for the preparation of a medicament for preventing cancer progression or recurrence in a subject, wherein the subject has undergone administration of a treatment that specifically targets the cancer associated antigen Treatment of sexual T cells (cancer treatment).
  • the medicament comprises a stimulating effective amount of the cell expressing a cancer associated antigen.
  • the medicament is for preventing cancer progression or relapse in a subject by the method of the second aspect of the invention.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the invention provides the use of a therapeutic T cell that specifically targets a cancer associated antigen for the preparation of a medicament for treating cancer in a subject, wherein the medicament further comprises a cell expressing the cancer associated antigen.
  • therapeutic T cells that specifically target a cancer associated antigen are independently present in the drug with cells expressing a cancer associated antigen, eg, independently placed in separate containers.
  • the medicament is for treating cancer in a subject by the method of the first aspect of the invention.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the invention provides a cell expressing a cancer associated antigen for use in treating cancer in a subject in combination with a therapeutic T cell that specifically targets the cancer associated antigen.
  • the cell expressing a cancer associated antigen is for use in treating cancer in a subject by the method of the first aspect of the invention.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the invention provides a cell expressing a cancer associated antigen for use in preventing cancer progression or recurrence in a subject, wherein the subject has been subjected to treatment with a therapeutic T cell that specifically targets the cancer associated antigen (cancer treatment).
  • the cell expressing a cancer associated antigen is used to prevent cancer progression or relapse in a subject by the method of the second aspect of the invention.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the invention provides a therapeutic T cell that specifically targets a cancer associated antigen for use in treating cancer in a subject in combination with a cell expressing the cancer associated antigen.
  • the therapeutic T cell that specifically targets a cancer associated antigen is for use in treating cancer in a subject by the method of the first aspect of the invention.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the invention provides a kit comprising a therapeutic T cell that specifically targets a cancer associated antigen and/or a cell expressing the cancer associated antigen, the kit being used by the method of the invention Treating cancer or preventing cancer progression or recurrence in a subject.
  • a cell expressing a cancer associated antigen is independently present in the kit independently of a therapeutic T cell that specifically targets the cancer associated antigen, eg, independently placed in a different container.
  • the therapeutic T cell or cell expressing a cancer associated antigen is as defined herein.
  • the cell or the medicament of the present invention may further comprise a "pharmaceutically acceptable excipient" or a “pharmaceutically acceptable carrier”, which means a substance which facilitates administration of the active substance to a subject and absorption by the subject, which may be included in the present invention.
  • a pharmaceutical composition does not cause significant toxic side effects of the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, Aromatizers, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and colorants.
  • This preparation may be sterilized, if desired, with auxiliary preparations such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts which affect osmotic pressure, buffers, colorants and/or fragrances, etc.
  • auxiliary preparations such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts which affect osmotic pressure, buffers, colorants and/or fragrances, etc.
  • these auxiliary preparations do not adversely react with the cells of the present invention.
  • auxiliary preparations such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts which affect osmotic pressure, buffers, colorants and/or fragrances, etc.
  • auxiliary preparations do not adversely react with the cells of the present invention.
  • Those skilled in the art understand that other pharmaceutical excipients can also be used in the present invention.
  • the cancer-associated antigen of the present invention is preferably a cancer-specific antigen including, but not limited to, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), carcinoma Embryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialyl ganglioside GD2, catheter Epithelial mucin, gp36, TAG-72, glycosphingolipid, glioma-associated antigen, ⁇ -human chorionic gonadotropin, alpha fetoglobulin (AFP), lectin-reactive AFP, thyroglobulin , RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal
  • Non-limiting examples of the cancer of the present invention include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, and colloid. Tumor, stomach, nasopharyngeal, laryngeal, cervical, uterine, and osteosarcoma.
  • cancers examples include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer , endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer , penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), solid tumors of children, lymphocytic lymphoma, bladder cancer, kidney or Ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal
  • the method of treatment or prevention may be combined with one or more of the following treatments for cancer: antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy , laser treatment and radiation therapy.
  • the cells or combinations or medicaments according to the invention can be carried out in any convenient manner, including by injection, infusion, implantation or transplantation.
  • the cells or combinations or drug administrations described herein can be administered intravenously, intralymphically, intradermally, intratumorally, intramedullaryly, intramuscularly or intraperitoneally.
  • the cells or combinations or medicaments of the invention are preferably administered by intravenous injection.
  • the CRISPR/Cas9-mediated B-NDG mouse was generated by Beijing Biocytogen Co., Ltd. Briefly, the sgRNA is involved in targeting the target region flanking the first and last exons of the Il2rg locus. For each target site, four candidate sgRNAs were identified by the CRISPR design tool (http://crispr.mit.edu/) and the targeting activity was screened using the UCA kit (Beijing Biocytogen). Two sgRNAs were selected for injection into the mouse fertilized eggs. In vitro transcribed Cas9 mRNA and sgRNA were mixed at different concentrations and co-injected into fertilized eggs of NOD-scid mice.
  • mice heterozygous and homozygous mice with Il2rg deficiency were further produced. All mice were maintained in a specific pathogen free facility.
  • 293-T cells (ATCC, CRL-3216) were cultured in DMEM (Hyclone, Logan, Utah, USA) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin.
  • Raji (ATCC, CCL-86), Raji-Fluc (School of Military Medical Sciences, Beijing; derived from Raji cells by transfection of lentiviral vectors encoding fluorescein and puromycin resistance), CD19-K562 and K562 cells (Genomeditech , Shanghai) Cultured in RPMI1640 supplemented with 10% FBS and 100 U/ml penicillin/streptomycin.
  • Irradiated Raji-Fluc cells were obtained by exposing the cells to 40 Gy cobalt radiation. All media and antibiotics were purchased from Gibco Life Technologies (Carlsbad, CA, USA).
  • Anti-CD19 CAR includes the scFv of the CD19-specific FMC63 mAb, the hinge and transmembrane regions of CD8, the 4-1BB costimulatory domain and the intracellular CD3 ⁇ chain of the TCR complex.
  • the anti-CD19 CAR gene was cloned into the lentiviral vector pPVLV2 (Pharosvaccine Inc., Gyeonggi, Republic of Korea). This vector was used to generate CAR-T-19 cells.
  • the aT19 lentiviral expression construct was generated by substituting the anti-CD19 CAR coding sequence with the CD19 coding sequence. This vector was used to generate aT19 cells.
  • the lentivirus expressing the anti-CD19 CAR or CD19 was generated as follows: 293T cells grown in DMEM containing 10% FBS were subjected to PEI using a lentiviral vector plasmid and pMDLg/pRRE, pRSV-Rev and pMD.G packaging plasmids (Polysciences Inc) ., Warrington, PA, USA) Transfection reagents were co-transfected. After 24 hours of transfection, the medium was replaced with DMEM supplemented with 2% FBS, 1 mM sodium pyruvate and 2 mM L-glutamine.
  • the virus supernatant was collected, concentrated by ultrafiltration and titrated with 293T cells.
  • 293T cells were transduced with serially diluted concentrated lentiviral preparations. After 72 hours, cells were harvested and analyzed by flow cytometry.
  • PBMC peripheral blood mononuclear cells
  • the multiplicity of infection (MOI) was 0.5-2 and transferred to a 12-well plate.
  • the plates were centrifuged at 1200 g for 2 hours at ambient temperature using a horizontal rotor centrifuge and then transferred to a 37 ° C, 5% CO 2 incubator.
  • the transduced cells were collected and washed with IMSF100 medium, and then the cells were incubated in a medium supplemented with 500 IU/mL IL-2 (BMI, Korea).
  • the medium and IL-2 300 IU/mL were then changed every 2 or 3 days and the culture was maintained at 37 ° C / 5% CO 2 until injected into the mice.
  • CAR-T-19 cells are T cells expressing anti-CD19 CAR.
  • the aT19 cells are T cells expressing CD19.
  • BD Becton Dickinson
  • FACS Fluorescence Activated Cell Sorting
  • Immunophenotypic analysis was performed by flow cytometry. The efficacy of CAR gene transduction was monitored by staining cells with the following specific antibodies: CD45 (PerCP-Vio770), CD3 (FITC), CD4 (Vio Green), CD16 (APC), CD56 (APC), CD19 (APC-Vio770) ), CD14 (Vio Blue; Miltenyi Biotec, Bergisch Gladbach, Germany).
  • Transduced T cells were labeled with biotin-SP (long spacer) AffiniPure F (ab') fragment goat anti-mouse IgG, F(ab')2 fragment specificity (Jackson ImmunoResearch Lab), and then with streptavidin Protein-PE (BD Biosciences) or CARTEST-19 (CytoCares Inc., Shanghai, China) was labeled and then detected by CytoFLEX flow cytometry. Data was analyzed using CytExpert (2.0) software.
  • Leukocytes and spleen cells harvested from mice were stained with FITC-CD45, PE-CAR, FITC-CD19 and APC-CD45RO (BD, NJ) and examined in a FACSCanto II flow cytometer (BD). Splenocytes were collected and filtered through a BD cell filter. Red blood cells in blood and spleen cell preparations were depleted by incubation in a lysis solution (BD) for 5 minutes.
  • BD lysis solution
  • mice in this study were housed and operated according to guidelines established by the Association of Laboratory Animal Care Assessment and Identification. The study was approved by the NIFDC Institutional Animal Care and Use Committee.
  • Four-week-old female BALB/c, C57BL/6, NOD-scid, and B-NDG mice were obtained from the NIFDC Laboratory Animal Resources Institute (Beijing, China).
  • D5 transplanted mice were injected with 5 ⁇ 10 5 th Raji-Fluc cells through the tail vein, then at day 5 (D5) or day 6 (D6) implant 2 ⁇ 10 7 th CAR-T-19 cells.
  • bioluminescence imaging of mice was obtained using an IVIS-Lumina II imaging system (PerkinElmer, Baltimore, MD). Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg body weight). Then, after 10 minutes, it was intraperitoneally injected with D-luciferin (75 mg/kg body weight; PE). Images were analyzed using Living Image software (Caliper Life Sciences) and the data was expressed as total flux (photons/s). If the maximum bioluminescence value produced exceeds 1 x 10 6 flux, and the bioluminescence area is no longer less than 1 x 10 6 flux at a subsequent time point, then the time point is recorded as the time of tumor recurrence.
  • CAR-T-19 cells isolated from peripheral blood and spleen of mice was detected by qPCR. Briefly, DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, GER) and quantified in an IMPLEN N50 ultra-violet spectrophotometer (IMPLEN, N80TOUCH). The extracted DNA was stored at -20 ° C until needed.
  • DNA 100 ng was PCR amplified using 4-1BB primer (Invitrogen) and probe (Thermo Fisher Scientific) to detect expression of CAR gene (CD19 4-1BB F primer, 5'-TGCCGATTTCCAGAAGAAGAAGAAG-3'; CD19 4 -1BB R primer, 5'-GCGCTCCTGCTGAACTTC-3'; CD19 4-1BB MGB probe, 5'-ACTCTCAGTTCACATCCTC-3')).
  • 4-1BB primer Invitrogen
  • probe Thermo Fisher Scientific
  • Non-obese diabetic severe combined immunodeficiency mice with ineffective Il2rg lack mature T cells, B cells, and natural killer (NK) cells, and lack cytokine signaling, resulting in better implantation of multiple cancer types .
  • the Il2rg gene was deleted from NOD-scid mice using the CRISPR/Cas9 genome editing technique to prepare B-NDG mice (Fig. 1A).
  • B-NDG mice were observed to be viable, reproducible and did not show any significant physical abnormalities.
  • FACS analysis showed that the spleen and peripheral blood of B-NDG mice had lower numbers of CD19+ B cells, CD4+ and CD8+ T cells compared to NOD-scid mice (Fig. IB).
  • NOD-scid mice had a high percentage of CD49b+ NK cells (20.90% of spleen, 24.10% of peripheral blood), while B-NDG mice had no functional NK cells (1.45% spleen, 5.95% peripheral blood). Therefore, B-NDG mice produced by genome editing are phenotypically similar to NSG mice.
  • Raji-Fluc cells could not be detected in CB57/L, rag2-/-, nude mice, or even NOD-scid mice. All four mice survived at D30PI, while 7 B-NDG mice died at D18PI and 3 died at D19 ( Figures 1C, D and F). These data demonstrate that Raji cells are implanted much better in B-NDG mice and can be used as a model for testing therapeutic agents such as CAR-T cells.
  • PBMC isolated from volunteers were transduced with a second generation CAR (Fig. 2A).
  • the results showed that the proportion of CD19 CAR-T cells increased with the prolongation of culture time, reaching 20% at D12, and the total number of cells was greater than 1 ⁇ 10 8 after infection (Fig. 2B).
  • CD19 CAR-T cells cultured for 12 days showed more than 50% killing of Raji cells at an effector/target cell ratio of approximately 3:1. .
  • B-NDG mice were injected with 5 ⁇ 10 5 Raji-Fluc cells through the tail vein, followed by implantation of 2 ⁇ 10 7 CD19 CAR-T cells 5 days later.
  • D9 Four days after CD19 CAR-T treatment (D9), the bioluminescence signals of these treated mice were close to those of non-tumored mice (Fig. 3A), indicating that CD19 CAR-T cells successfully mediate tumor clearance in vivo.
  • CAR-T cells are very effective, B-NDG mice eventually die from tumors.
  • bioluminescence of all major organs of the mouse was tested. The results indicate that Raji cells are significantly cleared by the CD19 CAR-T cells in the spleen, heart and skin.
  • CAR-T cells were reduced in bioluminescence by 1000-fold after treatment (Fig. 3A).
  • CD19 CAR-T is not as effective in other organs, especially in the liver and brain (Fig. 3A). Residual Raji cells hidden in these organs that are not effectively cleared by CAR-T may be responsible for tumor recurrence.
  • Example 4 stimulation of low-dose live tumor cells contributes to the production of immune memory in CAR T cells
  • the signal in the low-dose re-implantation group approached the signal from the blank control group (Fig. 3D), while the signals in the other groups increased to varying degrees, indicating that the recurrence was caused by poor persistence of CAR and insufficient ability, and low-dose tumors.
  • Antigen stimulation of cells promotes the production of immune memory in CAR-T cells.
  • mice vaccinated with the same dose of rRaji-Fluc were used as the irradiated cell control group (PA1/PA2/PA3).
  • Mice without rRaji-FLuc implantation were treated as single treatment groups (SNT).
  • Dual treated mice groups (the DNT) injection of 2 ⁇ 10 7 th CAR-T cells in D10. Bioluminescence signals were observed in mice until all mice in the SNT group died.
  • Reimplantation of rRaji-Fluc cells did not prolong overall survival and recurrence-free survival in mice compared to the SNT group (Fig. 4B and C). There was no significant difference between the SNT group and the DNT group, indicating that the double treatment was ineffective.
  • the CAR gene construct contains the scFv derived from the CD19-specific FMC63 mAb, the hinge and transmembrane regions of CD8, and the 4-1BB cytoplasmic domain, as well as the intracellular CD3 ⁇ chain of the TCR complex (Fig. 5A).
  • pEF1 ⁇ is a CAR gene promoter.
  • the aT19 construct was generated by replacing the CAR transgene with human CD19 (Fig. 5B). T cells isolated from PBMCs obtained from healthy volunteers were transduced with CAR-19 or CD19 construct (aT19). Table 1 shows the phenotype and number of CAR-T-19, live Raji-Fluc, irradiated Raji-Fluc and aT19 cells used in the experiments.
  • CAR-T-19 cells killed more than 50% of Raji-Fluc cells or aT19 at an effector/target ratio of >3:1, while T cells and controls Little cytotoxicity was observed when K562 cells were co-cultured (Fig. 5C).
  • Figure 6A shows the timeline of use of tumor cells, CAR-T cells, and restimulatory cells.
  • mice For the A1 group of mice, the fluorescent signal gradually decreased from D11 to D24, and no Raji-Fluc cells were detected in D34 (Fig. 6B); three mice in this group had no tumor at D200. In the A2 group, three mice died of D96, while the other two mice survived to D100 without tumor (Fig. 6B). In the A3 group, one mouse died of unexplained cause at D40, three died of D56, D80, and D86 due to tumor recurrence, and the other died of D96. Taken together, these results indicate that mice re-stimulated with tumor cells survived longer than mice that were not stimulated with tumor cells (Fig. 6C, P ⁇ 0.05).
  • the CAR copy number (representing the number of CAR+ cells) of blood obtained from mouse sputum was measured on D0/D5/D10/D15/D20.
  • Raji-Fluc cells are tumor cells with high proliferative capacity. Thus, if a patient receives a stimulating dose of Raji cells, there is a risk that these cells will form a tumor. This is obviously unacceptable. Therefore, T cells (aT19 cells) carrying the human CD19 gene were used to replace the Raji-Fluc cells to re-stimulate CAR-T-19 cells.
  • the injection time and the number of cells used are listed in Table 4.
  • Mice in the positive/NC group P and N groups
  • Mice in the NT group received a single dose of CAR-T-19 cells with no subsequent dose of stimulator cells.
  • blood was drawn from the inside on D10, D20, and D30 (Fig. 8D). The results showed that the survival time of the mice in the aT19 group was significantly longer than that in the NT group; the three mice in the aT19 group survived to D160 (Fig. 8C).
  • qPCR was used to measure CAR copy number and use flow cytometry The percentage of CAR+ and CD45RO+ cells in peripheral blood and spleen of D10, D20 and D30 was measured.
  • QPCR showed that the number of CAR copies in the blood of NT group mice decreased with time (from 1175 copies to 295 copies), while the number of CAR copies in the blood of aT19 group mice increased with time (from 1224 copies to 6824 copies). Especially after D10 (Fig. 8D).
  • the percentage of CAR+ cells in the blood of the NT group decreased (from 3.28% to 0.29%), while the percentage of CAR+ cells in the blood of the aT19 group increased (from 9.6% to 13.0%) (data not shown) .
  • the percentage of CD45RO+ (memory) cells was also measured. As shown in Fig. 8F, the percentage of CAR+CD45RO+ cells in the blood of the NT group decreased slightly from 0.44% (D20) to 0.37% (D30), while the percentage of blood in the aT19 group increased from 0.34% (D20) to 0.86% (D30). ), it was confirmed that memory CAR-T-19 cells were produced after re-stimulation of aT19 cells.
  • CAR-T cell therapy has progressed; however, many obstacles have yet to be overcome. Among them, prolonging the efficacy of CAR-T cells in vivo and reducing the side effects of CAR-T treatment are the most important.
  • the efficacy of CAR-T cells can be extended by optimizing extracellular recognition 19,20 and CAR-mediated intracellular signal transduction 21 (by adjusting the ratio and type of CAR+ cells) 22 and using strategies such as targeting drug 23 at the same time.
  • the dose of CAR-T cells and using immunosuppressive drugs can be reduced 24 .
  • a similar approach has been taken to vaccines that are continually optimized to provide effective protection over a longer period of time while reducing adverse effects25 .
  • the most common vaccination regimens involve multiple immunizations or even the use of different immunogens to enhance the immune response.
  • primary and booster immunization contain the virus antigen and nucleic acid, respectively, increase the duration of antibody protection and increased immune activation 26.
  • multiple vaccinations increase cost and toxicity but reduce efficacy.
  • Loss or shorter effectiveness of tumor antigens means that tumors tend to recur after treatment with CAR-T cells. Loss of tumor antigens is a common immune escape strategy, which is common in ALL. Potential mechanisms involved in phenotypic spectrum and selective splicing conversion 27. In addition, the number of CAR-T cells must be maintained at the minimum level required to control the tumor for long periods of time. There are many complex reasons that make it difficult to maintain the number of CAR-T cells; these include immune responses against CAR, loss of proliferative capacity induced by cellular senescence, and activation-induced cell death. By optimizing the structure and conditions used to generate the CAR, the immune response to CAR can be reduced.
  • mice are injected with CAR-T cells at D5 and then exposed to large doses of tumor (antigen positive) cells.
  • CAR-T cells rapidly proliferate and kill tumor cells until D9, when few tumor cells are detectable in the blood.
  • the proliferative capacity of T cells is reduced. Therefore, it was found that the number of CAR-T cells decreased with the passage of time, and eventually the proliferation of residual cancer cells could not be inhibited.
  • the percentage of CAR+ cells in the blood of SNT group gradually decreased from D20 to D30.
  • aT19 cells a second "antigen"
  • aT19 cells a second "antigen”
  • the results of the present application indicate that not only is the number of CAR+ cells significantly higher than D20 at D30 but also the percentage of CD45RO+ memory cells is significantly higher than D20, which further consolidates CAR-T mediated immunity. Therefore, aT19 cells act similarly to boosting immunity.
  • the recurrence-free survival and overall survival of mice receiving continuous treatment were superior to those receiving monotherapy (CAR-T cells alone), suggesting that continuous immunization may be a safer and more effective extension of CAR-T Strategies for the effective time of cells in a clinical setting.
  • CAR-T treatment may lead to severe cases of cytokine release syndrome (CRS; commonly referred to as "cytokine storm”), which can be life threatening 3, 28, 29, 30 . This is one of the most serious side effects of CAR-T therapy in addition to off-target effects.
  • CAR-T cells can be activated by tumor cells, and the immune system can in turn be activated by cytokines released by CAR-T cells. If the release of a large number of inflammatory factors, which may cause fever, hypotension, respiratory distress, multiple organ dysfunction, neurological disorders and other symptoms of regulation 28.
  • CRS usually occurs within the first 2 weeks after CAR-T injection, although the underlying mechanism remains unclear.
  • CRS31 Early intervention with anti-cytokine monoclonal antibodies or glucocorticoids reduces the risk of CRS31 .
  • severity of CRS is positively correlated with the number of vaccinated CAR-T cells and tumor burden 4, 22, 32, 33, 34 .
  • a sustained immune response is produced in mice by continuous "immunization" with CAR-T cells followed by aT19 cells. This can reduce the number of CAR-T cells required for each vaccination and the number of vaccinations required over time, thereby reducing the likelihood and/or severity of CRS.
  • aT19 cells have clinical utility. This is because these cells are from the same source as the CAR-T-19 cells.
  • autologous PBMC can be used to produce these cells, which can effectively prevent grafts from fighting host diseases.
  • CAR-T cells recognize aT19 cells by CD19 molecules rather than other immune signals, thereby ensuring specificity.
  • the aT19 transduction fragment was constructed by substituting the CD19 gene for the Fab fragment of the CAR antibody; this would eliminate any off-target effects in normal tissues in vivo. For example, CAR-T-Her2 can attack the lung mucosal epithelium and cause pulmonary edema, which can be fatal in severe cases35 .
  • mice were injected with aT19 cells at D10 because no tumor was detected at D9. At this point, loss of target cells may cause CAR-T cells to attack host tissues. However, the proliferation rate of CAR-T-19 cells may be reduced due to loss of antigen-specific stimulation, and T cells that are not specifically attacking the host may continuously proliferate. Therefore, aT19 cells were injected at D10 to maintain CAR-T-19 cells.
  • the time of administration of aT19 can be adjusted according to the number of CAR-T cells.
  • the number of CAR-T cells was estimated by measuring the number of CAR copies in blood by qPCR. The results are consistent with the results provided by flow cytometry.
  • the fluorescence value total flux
  • the circulatory system in humans is longer and more complex than mice, and the dose of CAR-T administered in the clinic is much larger than the dose administered to mice. Therefore, within the first week after CAR-T cell therapy, cancer cells in the human body may not be completely eliminated, and the number of CAR copies in the blood will continue to increase.
  • aT19 cells should be considered for immunization. Continuous monitoring of CAR+ copy number and subsequent inoculation of aT19 cells may be a viable method for clinical application of aT19 cells in this setting.
  • mice treated with CAR-T cells were injected with Raji-Fluc cells, irradiated Raji-Fluc cells or aT19 cells.
  • the results showed that re-stimulation of CAR-T cells with live Raji-Fluc cells and aT19 cells prolonged recurrence-free survival and overall survival, whereas re-stimulation with irradiated (dead) Raji-Fluc cells did not prolong recurrence-free survival and Overall survival rate.
  • Continuous treatment of aT19 increased the percentage of CAR+ and CD45RO+ cells in the blood, confirming the production of memory CAR-T cells.

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Abstract

本发明属于生物医药领域。具体而言，本发明公开了改进的T细胞治疗方法。更具体而言，本发明公开了通过表达癌症相关抗原的活细胞刺激来增强治疗性T细胞（如CAR-T或TCR-T细胞）的癌症治疗效力。

Description

改进的T细胞治疗方法 技术领域
本发明属于生物医药领域。具体而言，本发明涉及改进的T细胞治疗方法。更具体而言，本发明涉及通过用表达癌症相关抗原的活细胞刺激来增强治疗性T细胞(如CAR-T或TCR-T细胞)的癌症治疗效力。
发明背景
目前，对于B细胞急性淋巴细胞白血病(B-ALL)的儿童患者，多药化疗方案和/或靶向治疗已实现高的完全缓解率，但在成人中仍具有不良预后，并且复发/难治性B-ALL对所有患者来说都是一个很大的挑战。淋巴细胞耗竭化疗随后进行CD19靶向性嵌合抗原受体修饰的T(CAR-T)细胞疗法是一种新型治疗方法，并且证明在成人和儿童患有复发/难治性B-ALL、慢性淋巴细胞性白血病和淋巴瘤的患者中具有很强的响应。来自这些研究的新数据表明，所递送的显著小剂量的CAR-T细胞可以消除很大的疾病负荷。然而，长期随访表明，大部分患者的缓解不能持久。由于CAR-T细胞持续性差，CD19阳性肿瘤会复发。因此，目前认为延长功能性CAR-T细胞在体内的存在时间对维持持续缓解可能很重要。
CAR一般包含细胞外抗原结合结构域(单克隆抗体的单链可变片段(scFV))和活化T细胞的胞内信号传导结构域。基因工程化改造的CAR-T细胞以主要组织相容性复合物(MHC)非依赖性方式高效对抗抗原阳性肿瘤细胞。目前，研究人员致力于寻求改善CAR-T细胞效能的方法。其中一种可行的方法是修改CAR的结构，例如应用第二代或第三代甚至第四代CAR，其组合了激活信号域和共刺激信号域，并且能够改善T细胞扩增和持续性。在体外扩增和过继输注后，源自不同T细胞亚群的CAR-T细胞在体内增殖和持续的能力是不同的。与根据效应记忆T细胞制备的CD19靶向性CAR-T细胞相比，由纯化的初始T细胞或中央记忆T细胞制备的CD19靶向性CAR-T细胞在消除CD19+肿瘤中更有效。并且效率的协同增强可以通过递送具有确定比例的衍生自CD8/CD4T细胞的CAR-T细胞来实现。目前使用 的CAR-T细胞保留内源性T细胞受体(TCR)，而TCR和CAR串扰的生物学十分复杂。CAR和TCR的伴随激活极大地削弱了CD8+CAR-T细胞的体内功效；相反，CD4+CAR-T细胞在存在TCR和CAR抗原的情况下保持其体内持续存活的能力。
尽管开发新的CAR结构并且精确确定CAR-T细胞的来源可能有助于改进CAR-T的应用，这些策略却不能从根本上促进CAR-T细胞的免疫记忆的产生，免疫记忆的产生对于适应性免疫应答是必不可少的。而且CAR-T细胞的重复施用倾向于诱导针对那些经工程化的T细胞的免疫清除。因此，本领域仍然需要新的CAR-T细胞治疗策略，特别是能够诱导CAR-T细胞免疫记忆的策略。
发明简述
在第一方面，本发明提供了一种在对象中治疗癌症的方法，包括以下步骤：
(a)给所述对象施用特异性靶向癌症相关抗原的治疗性T细胞；和
(b)给所述对象施用表达所述癌症相关抗原的细胞。
在第二方面，本发明提供了一种在对象中预防癌症进展或复发的方法，其中所述对象已经经历使用特异性靶向癌症相关抗原的治疗性T细胞的癌症治疗，所述方法包括给所述对象施用表达癌症相关抗原的细胞。
在第三方面，本发明提供一种用于在对象中治疗癌症的组合，其包含特异性靶向癌症相关抗原的治疗性T细胞和表达所述癌症相关抗原的细胞。
在第四方面，本发明提供本发明第三方面的组合在制备用于在对象中治疗癌症的药物中的用途。
在第五方面，表达癌症相关抗原的细胞在制备用于在对象中治疗癌症的药物中的用途，其中所述药物还包含特异性靶向所述癌症相关抗原的治疗性T细胞。
在第六方面，本发明提供表达癌症相关抗原的细胞在制备用于在对象中预防癌症进展或复发的药物中的用途，其中所述对象已经经历使用特异性靶向所述癌症相关抗原的治疗性T细胞的癌症治疗。
在第七方面，本发明提供特异性靶向癌症相关抗原的治疗性T细胞在制备用于在对象中治疗癌症的药物中的用途，其中所述药物还包含表达所 述癌症相关抗原的细胞。
在第八方面，本发明提供表达癌症相关抗原的细胞，其用于与特异性靶向所述癌症相关抗原的治疗性T细胞组合在对象中治疗癌症。
在第九方面，本发明提供表达癌症相关抗原的细胞，其用于在对象中预防癌症进展或复发，其中所述对象已经经历使用特异性靶向所述癌症相关抗原的治疗性T细胞的癌症治疗。
在第十方面，本发明提供特异性靶向癌症相关抗原的治疗性T细胞，其用于与表达所述癌症相关抗原的细胞组合在对象中治疗癌症。
在第十一方面，本发明提供药盒，其包含特异性靶向癌症相关抗原的治疗性T细胞和/或表达所述癌症相关抗原的细胞，所述药盒用于通过本发明的方法在对象中治疗癌症或预防癌症进展或复发。
附图简述
图1.B-NDG小鼠的产生和表征。(A)通过CRISPR/Cas9基因组编辑技术的Il2rg基因缺失靶向策略的示意图。(B)通过流式细胞术检测NOD-scid和B-NDG小鼠的外周血和脾脏中的不同淋巴细胞群体。数字表示给定表型的阳性细胞的百分比。(C)6周龄C57BL/6、裸鼠、Rag2-/-，NOD-scid和B-NSG小鼠分别通过尾静脉注射植入5×10 5个Raji细胞。生物发光的相对水平以伪彩显示，红色和蓝色代表最强和最弱的光通量。(D)显示每个时间点的总通量并且每个数据点表示平均值(n＝10)。(E)CD19-FITC识别的Raji细胞计数显示为通过细胞计数记录的图。通过拟合对数曲线计算Raji细胞计数与总通量之间的相关性，R 2＝0.90。(F)B-NDG小鼠在第17或18天死亡，但其他小鼠在第30天存活。(G)CD19在Raji-Fluc细胞表面高度表达，Fluc的发光强度与Raji-Fluc细胞的数量正相关。
图2.体外CD19 CAR-T细胞的效率。(A)抗CD19 CAR的结构。(B)来自PBMC供体的CAR-T细胞的体外培养的总结，包括活细胞百分比、CAR-T细胞的总细胞计数和CAR-T细胞的百分比。(C)在CAR-T/Raji比率为1:1、3:1和6:1时CAR-T细胞对体外Raji细胞的细胞毒效应。
图3.低剂量活肿瘤细胞的再刺激促进了CAR-T细胞的免疫记忆的产生。(A)分开测量不同组中器官和组织的通量。(B)低剂量活肿瘤细胞的再植入可促进肿瘤清除并延长总体存活。(C)低剂量活肿瘤细胞的再植入可延 缓复发。(D)每组的平均总通量。LT：低剂量再植入；MT：中剂量再植入；HT：高剂量再植入。
图4.辐射后死亡的肿瘤细胞的再植入不起作用。(A)分开测量不同组中器官和组织的通量。(B)和(C)辐射后死亡的肿瘤细胞的再植入不能延长总体存活和无复发存活。CAR-T细胞双重处理的效果与单一处理相同。(D)通过qPCR测量小鼠外周血中CAR-T细胞的数量。
图5.CAR-T-19和aT19细胞的表征。(A)显示CAR-19基因构建体的结构和原代活化的T细胞的CAR-T-19转导效率的示意图。(B)人CD19基因构建体的结构和原代活化的T细胞的CD19转导效率。(C)CAR-T-19细胞在CAR-T-19:CD19-K562或K562比例为1:1、3:1和6:1时对CD19-K562或K562细胞的细胞毒性作用；以及CAR-T-19细胞在CAR-T-19:Raji-Fluc或aT19比率为0.3:1、1:1和3:1时对Raji-Fluc或aT19细胞的细胞毒性作用。
图6.活Raji肿瘤细胞对CAR-T细胞的再刺激增加存活率并延迟肿瘤复发。在D0静脉内(i.v.)注射Raji-Fluc细胞(每只小鼠2×10 5个细胞；A1、A2、A3、NT和P组)。在注射Raji-Fluc细胞后的D5，小鼠接受i.v.注射CAR-T-19细胞(每只小鼠2×10 7个细胞；A1、2、3、NT组)。在D10，给小鼠i.v.注射不同剂量的Raji-Fluc细胞(A1和PA1组：5×10 4；A2和PA2组：1.5×10 5；和A3和PA3组：5×10 5)。PA组用作A组的对照。(A)显示动物实验的时间表的示意图。(B)生物发光图像，显示不同组小鼠的器官和组织中的总通量。根据不同剂量的再植入的Raji-Fluc细胞构建Kaplan-Meier存活曲线以监测全身性疾病的根除(C)以及估计无复发的存活(D)。
图7.用辐射后死亡的肿瘤细胞再刺激CAR-T细胞是无效的。在D0静脉内(i.v.)注射Raji-Fluc细胞(每只小鼠2×10 5个细胞；A1、A2、A3、NT和P组)。在注射Raji-Fluc细胞后的D5，小鼠接受i.v.注射CAR-T-19细胞(每只小鼠2×10 7个细胞；A1、A2、A3、NT组)。在D10，给小鼠i.v.注射不同剂量的辐射的Raji-Fluc细胞(A1和PA1组：5×10 4；A2和PA2组：1.5×10 5；和A3和PA3组：5×10 5)。PA组用作A组的对照。(A)显示动物实验的时间表的示意图。(B)生物发光图像，显示不同组小鼠的器官和组织中的总通量。根据不同剂量的再植入的经辐射的Raji-Fluc细胞的构建 Kaplan-Meier存活曲线以监测全身性疾病的根除(C)并估计无复发的存活(D)。(E)定量聚合酶链式反应用于在植入Raji-Fluc细胞后检测A组和NT组血液中的CAR+细胞。图表显示来自每组的血液样品中的CAR基因拷贝数。
图8.用CAR-T-19和aT19细胞的连续处理促进记忆CAR-T细胞的产生。在D0静脉内(i.v.)注射Raji-Fluc细胞(每只小鼠2×10 5个细胞；A1、NT、aT19和P组)。在注射Raji-Fluc细胞后的D7，小鼠接受i.v.注射CAR-T-19细胞(每只小鼠2×10 7个细胞；A1、NT、aT19组)。在D10，A1组小鼠接受了i.v.注射5×10 4个细胞，aT19组小鼠接受i.v.注射5×10 5个细胞，其他组没有接受注射。(A)显示动物实验的时间表的示意图。(B)生物发光图像，显示不同组小鼠的器官和组织中的总通量。(C)构建Kaplan-Meier生存曲线以监测全身性疾病的根除。定量聚合酶链式反应用于检测在D10、D20和D30的血液(D)和脾脏(E)中的CAR+细胞，图表显示来自每组的样品中的CAR基因拷贝数。(F)外周血中CAR+和CD45RO+细胞的百分比。
发明详述
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook et al.,“Molecular Cloning:A Laboratory Manual”；Lewin,“Genes VIII”；及Roitt et al.,“Immunology”(第8版)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
先前的临床试验已经确定了CAR-T免疫疗法在癌症治疗特别是B细胞恶性肿瘤中的有效性。通过CAR-T细胞治疗癌症的下一步是延长缓解的持续时间。
针对CD19的CAR-T治疗对复发/难治性B-ALL患者的缓解率可达67％～90％；然而，无复发期只有2个月到3年不等。CD19 CAR-T治疗后有两种复发模式，一种是由于CAR-T细胞体内持续性不够造成的CD19阳性肿瘤复发；另一种是在大约20％患者中出现的由于在体内存在CAR-T细胞时CD19-丢失而造成的复发，这样的丢失可能是由克隆选择或肿瘤演变 造成。当前研究人员倾向于在患者外周血中的CAR-T细胞减少或消失时向患者额外再注射CAR-T细胞，但此种方案临床效果差异很大，表明CAR-T剂量增加可能不是CAR-T能力体内持续存在的关键。
在本发明的具体实施例中，发明人成功构建了Raji-B-NDG小鼠模型以研究CD19 CAR-T疗法的效率。在用CD19 CAR-T细胞处理后，缓解的小鼠在第20天(D20)出现不同程度的复发，然后在D28-36内死亡，表明在初始接触抗原并消除肿瘤细胞后，注入的CAR-T细胞逐渐减少，导致最终复发而没能有效地进行控制。复发的原因可能在于以下两点。首先，小鼠的荧光检测显示仅在具有丰富血流的器官(如脾脏和心脏)中实现了理想的肿瘤抑制效果，而在具有较少的血管分支的器官(如肝和脑)中没有有效杀死肿瘤细胞，这表明复发可能来自这些组织。其次，肿瘤复发的时间很重要，大多数PBMC的寿命不超过4周。CAR-T细胞在注射前的长达12天的体外增殖时间在加上注射后到复发前的20天大致与PBMC的27天寿命一致。因此，起始剂量的CAR-T消除了大部分Raji细胞，导致缺乏抗原刺激，因而增殖受阻且寿命被消耗掉，CAR-T数量减少直到肿瘤复发。
为了最大化CAR-T的特异性杀伤效应，应在临床应用中解决治疗性细胞寿命和抗原刺激的问题。在本发明的具体实施例中，当在D10肿瘤细胞基本上已经被CAR-T消除时，测试了肿瘤细胞(Raji-Fluc)或表达肿瘤抗原如CD19的细胞(如T细胞)再接种的效果。出乎意料的是，与单次植入组(成瘤)相比较，低剂量再植入组(再刺激)维持更长时间的无肿瘤复发状态，一半以上的小鼠存活36天以上，表明在同一抗原重复刺激下发生免疫记忆，对重复注入的同一抗原的快速反应导致肿瘤细胞的持续抑制。随后，发明人研究了来自辐射后死亡的Raji细胞的抗原是否可以刺激CAR-T细胞。再次出乎意料的是，结果表明再植入死亡的肿瘤细胞不能唤醒CAR-T细胞的记忆，导致小鼠的短存活时间。这表明CAR-T细胞只能被携带抗原的活细胞激活。本发明人还令人惊奇地发现重复性施用CAR-T的疗法不能延迟肿瘤的复发，这为多次CAR-T输注的无效提供了更多证据。不期望受任何理论限制，认为低剂量的表达癌症相关抗原的活细胞的刺激可促进CAR-T细胞免疫记忆的产生，从而导致延迟复发和延长存活。
因此在第一方面，本发明提供了一种在对象中治疗癌症的方法，包括以下步骤：
(a)给所述对象施用特异性靶向癌症相关抗原的治疗性T细胞；和
(b)给所述对象施用表达所述癌症相关抗原的细胞。
在一些实施方案中，所述治疗性T细胞施用一或多次，优选一次。在一些实施方案中，表达所述癌症相关抗原的细胞施用一或多次，优选一次。
在一些实施方案中，在施用所述治疗性T细胞之后施用所述表达癌症相关抗原的细胞。例如，在施用所述治疗性T细胞约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天或约21天后施用表达所述癌症相关抗原的细胞。
在一些实施方案中，在施用所述治疗性T细胞使得癌症细胞负荷降低后，施用表达所述癌症相关抗原的细胞。例如，在癌症细胞负荷降低至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％后，施用表达所述癌症相关抗原的细胞。在一些实施方案中，在癌症通过施用所述治疗性T细胞而完全缓解之后，施用表达所述癌症相关抗原的细胞。对象中癌症细胞负荷的变化可以通过本领域已知的各种方法确定。例如，对于血液癌症，可以通过对循环细胞进行流式细胞术确定癌症细胞负荷。又例如，对于实体瘤，可以通过临床上常用的成像方法(如核磁共振、CT、PET-CT等)确定肿瘤负荷。或者，可以通过检测肿瘤标志物来确定肿瘤负荷。在一些实施方案中，在施用表达所述癌症相关抗原的细胞前，可以通过检测所述治疗性T细胞的含量来确定肿瘤负荷的变化，例如所述治疗性T细胞的含量与肿瘤负荷负相关。
在一些实施方案中，在施用所述治疗性T细胞后，当所述治疗性T细胞在所述对象体内的量下降时，施用表达所述癌症相关抗原的细胞。所述治疗性T细胞在所述对象体内的量可以通过例如流式细胞术或定量PCR的方法检测。例如，如果所述治疗性T细胞是CAR-T细胞，可以通过PCR检测来自对象的样品中CAR编码基因的拷贝数。当CAR编码基因的拷贝数出现下降时，施用表达所述癌症相关抗原的细胞。
因此，在一些实施方案中，所述方法还包括在施用所述治疗性T细胞后，监测所述对象体内的肿瘤负荷和/或所述治疗性T细胞的量的步骤。
在一些实施方案中，在施用一或多次所述治疗性T细胞之后，施用一 或多次所述表达癌症相关抗原的细胞。在一些实施方案中，在施用一次所述治疗性T细胞之后，施用一或多次所述表达癌症相关抗原的细胞。在一些实施方案中，使用一或多次所述治疗性T细胞且在每次施用所述治疗性T细胞之后，施用一或多次所述表达癌症相关抗原的细胞。在一些实施方案中，在施用一次所述治疗性T细胞之后，施用一次所述表达癌症相关抗原的细胞。在一些实施方案中，在施用所述治疗性T细胞之后，施用一或多次所述表达癌症相关抗原的细胞。在一些实施方案中，在施用所述治疗性T细胞之后，施用一次所述表达癌症相关抗原的细胞。
在一些实施方案中，以治疗有效量施用所述治疗性T细胞。如本文所用，治疗性T细胞的治疗有效量是指使用后能够降低癌症细胞负荷的治疗性T细胞的量，例如使癌症细胞负荷降低至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％或者使癌症完全缓解的量。在本发明各方面的一些实施方案中，所述治疗性T细胞的有效量为约10 4至约10 9个细胞，例如约10 4、约10 5、约10 6、约10 7、约10 8或约10 9个细胞。在一些实施方案中，根据对象体重确定治疗性T细胞的施用量，例如约10 4细胞/kg体重至约10 9个细胞/kg体重，例如约10 4、约10 5、约10 6、约10 7、约10 8或约10 9个细胞/kg体重。
在一些实施方案中，以刺激有效量施用表达癌症相关抗原的细胞。如本文所用，表达癌症相关抗原的细胞的刺激有效量是指在施用后能够延长之前施用的治疗性T细胞的体内持续性(增强免疫记忆)和/或延长对象整体存活期和/或延长对象无复发存活期的量。在本发明各方面的一些实施方案中，所述表达癌症相关抗原的细胞的刺激有效量为约10 3至约10 6个细胞，例如约10 3、约10 4、约10 5或约10 6个细胞。优选施用低剂量的所述表达癌症相关抗原的细胞，例如约10 4个细胞。在一些实施方案中，根据对象体重确定表达癌症相关抗原的细胞的施用量，约10 3至约10 6个细胞/kg体重，例如约10 3、约10 4、约10 5或约10 6个细胞/kg体重。在一些实施方案中，根据所施用的治疗性T细胞的量确定表达癌症相关抗原的细胞的施用量。例如，所施用的治疗性T细胞的量与表达癌症相关抗原的细胞的量的比例为约1:1至约1000:1之间的任意比例或更高的比例，例如约1:1、约2:1、约3:1、约4:1、约5:1、约6:1、约7:1、约8:1、约9:1、约10:1、约50:1、 约100:1或约1000:1或更高。
在本发明各个方面的一些实施方案中，通过施用表达癌症相关抗原的活细胞对体内已有的治疗性T细胞(如CAR-T细胞)进行刺激，可以使治疗性T细胞的体内持续性增加至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％、至少大约100％、至少大约150％、至少大约200％、至少大约300％或更高。在本发明各个方面的一些实施方案中，通过施用表达癌症相关抗原的活细胞对体内已有的治疗性T细胞(如CAR-T细胞)进行刺激，可以使对象整体存活期延长至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％、至少大约100％、至少大约150％、至少大约200％、至少大约300％或更长。在本发明各个方面的一些实施方案中，通过施用表达癌症相关抗原的活细胞对体内已有的治疗性T细胞(如CAR-T细胞)进行刺激，可以使对象缓解后的无复发存活期延长至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％、至少大约100％、至少大约150％、至少大约200％、至少大约300％或更长。
在本发明各方面的一些实施方案中，治疗性T细胞是包含外源T细胞受体(TCR)的T细胞(TCR-T细胞)。在一些实施方案中，所述外源TCR(通常包含α和β链)特异性结合癌症相关抗原。
在本发明各方面的一些实施方案中，治疗性T细胞是包含嵌合抗原受体(CAR)的T细胞(CAR-T细胞)。本领域已经开发了多种的CAR-T细胞，例如第一至第四代CAR-T细胞、被修饰去除抑制性信号的CAR-T细胞等，这些都可以应用于本发明。
在本发明各方面的一些实施方案中，CAR包含针对癌症相关抗原的细胞外抗原结合结构域。所述细胞外抗原结合结构域例如可以是单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区(scFV)，以及其抗原结合片段。在一些优选实施方式中，所述细胞外抗原结合结构域是scFV。例如，所述细胞外抗原结合结构域可以衍生自一或多种已知抗体包括任何商业可获得的抗体，如FMC63、利妥昔单抗(rituximab)、阿仑珠 单抗(alemtuzumab)、依帕珠单抗(epratuzumab)、曲妥珠单抗(trastuzumab)、比伐珠单抗(bivatuzumab)、西妥昔单抗(cetuximab)、拉贝珠单抗(labetuzumab)、帕利珠单抗(palivizumab)、司韦单抗(sevirumab)、妥韦单抗(tuvirumab)、巴利昔单抗(basiliximab)、达克珠单抗(daclizumab)、英利昔单抗(infliximab)、奥马珠单抗(omalizumab)、依法珠单抗(efalizumab)、凯利昔单抗(Keliximab)、西利珠单抗(siplizumab)、那他珠单抗(natalizumab)、克立昔单抗(clenoliximab)、培马单抗(pemtumomab)、依屈洛单抗(Edrecolomab)、坎妥珠单抗(Cantuzumab)等。
在本发明各方面的一些实施方案中，所述CAR还包括跨膜结构域和细胞内信号转导结构域。根据本发明的CAR的细胞内信号转导结构域负责细胞外配体结合结构域与靶结合之后的细胞内信号转导，导致激活免疫细胞和免疫应答。细胞内信号转导结构域具有激活表达CAR的免疫细胞的至少一种正常效应子功能的能力。例如，T细胞的效应子功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。
用于CAR的细胞内信号转导结构域可以是细胞质序列，例如但不限于T细胞受体和共同受体(它们一致地起作用从而在抗原受体接合之后启动信号转导)的细胞质序列，以及任何这些序列的衍生物或变体以及具有相同功能能力的任何合成序列。细胞内信号转导结构域包括两种不同类型的细胞质信号转导序列：那些启动抗原依赖性初级激活的序列，以及那些以抗原非依赖性方式作用以提供次级或共刺激信号的序列。初级细胞质信号转导序列可以包括称为ITAM的免疫受体酪氨酸激活基序的信号转导基序。本发明中使用的ITAM的非限制性实例可以包括衍生自TCRζ、FcRγ、FcRβ、FcRε、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。在一些实施方案中，CAR的细胞内信号转导结构域可以包括CD3ζ信号转导结构域。在一些实施方案中，本发明的CAR的细胞内信号转导结构域还包括共刺激结构域。在一些实施方案中，所述共刺激结构域选自41BB共刺激结构域或CD28共刺激结构域。
CAR在细胞的表面上表达。因此，CAR可以包括跨膜结构域。本发明的CAR的合适跨膜结构域具有以下能力：(a)在细胞表面表达，优选免疫细胞，例如但不限于淋巴细胞或自然杀伤(NK)细胞，和(b)与配体结合结构域和细胞内信号转导结构域相互作用，用于指导免疫细胞对预定靶细胞的细 胞应答。跨膜结构域可以衍生自天然来源或合成来源。跨膜结构域可以衍生自任何膜结合蛋白或跨膜蛋白。作为非限制性实例，跨膜结构域可以衍生自T细胞受体的亚基如α亚基、β亚基、γ或δ亚基，组成CD3复合物的多肽，IL-2受体的p55(α链)、p75(β链)或γ，Fc受体的亚基链，特别是Fcγ受体III或CD蛋白。或者，跨膜结构域可以是合成的，并且可以主要包括疏水性残基，如亮氨酸和缬氨酸。在一些实施方案中，所述跨膜结构域衍生自人CD8α链。跨膜结构域可以进一步包括位于细胞外配体结合结构域和所述跨膜结构域之间的铰链区域。所述铰链区域例如来自CD8、CD4或CD28的胞外区。在一些实施方案中，所述铰链区域是人CD8α链的一部分。
在本发明各方面的一些具体实施方案中，本发明中使用的CAR可以包括特异性结合癌症相关抗原的细胞外抗原结合结构域、CD8α铰链和跨膜结构域、CD3ζ信号转导结构域、和4-1BB共刺激结构域。
在一具体实施方式中，本发明的CAR(针对CD19)包含如下SEQ ID NO:1的氨基酸序列：
Figure PCTCN2019084773-appb-000001
在一具体实施方式中，本发明的CAR(针对CD19)由如下SEQ ID NO:2的核苷酸序列编码：
Figure PCTCN2019084773-appb-000002
Figure PCTCN2019084773-appb-000003
在本发明各方面的一些实施方案中，表达所述癌症相关抗原的细胞是活细胞。在一些实施方案中，所述癌症相关抗原表达于所述活细胞的表面。在一些实施方案中，表达所述癌症相关抗原的细胞不是癌症细胞。在一些实施方案中，表达所述癌症相关抗原的细胞是被遗传工程化改造为表达所述抗原的细胞。所述细胞的类型不特别限制，其可以衍生自分离的原代细胞，或衍生自细胞系。在一些实施方案中，表达所述癌症相关抗原的细胞衍生自外周血细胞例如外周血单个核细胞(PBMC)。在一些实施方案中，表达所述癌症相关抗原的细胞衍生自免疫细胞，例如巨噬细胞、树突状细胞、浆细胞、粒细胞、肥大细胞、淋巴细胞(炎性T淋巴细胞、细胞毒性T淋巴 细胞、调节性T淋巴细胞、或辅助性T淋巴细胞、B淋巴细胞)等。在一些实施方案中，细胞可以衍生自CD34+细胞。在一些实施方案中，细胞可以衍生自T淋巴细胞例如CD4+T淋巴细胞或CD8+T淋巴细胞。在一些实施方案中，细胞可以衍生自B淋巴细胞。
在本发明各方面的一些实施方案中，所述治疗性T细胞和/或所述表达癌症相关抗原的细胞衍生自对象的自体细胞。如本文所用，“自体”是指用于治疗对象的细胞、细胞系或细胞群源自所述对象。在一些实施方案中，所述治疗性T细胞和/或所述表达癌症相关抗原的细胞衍生自异体细胞，例如源自与所述对象人类白细胞抗原(HLA)相容的供体。可以使用标准方案将来自供体的细胞转化为非同种异体反应性细胞，并根据需要进行复制，从而产生可以施用至一个或多个患者的细胞。
本发明的治疗性T细胞和/或表达癌症相关抗原的细胞可以通过本领域已知的多种手段制备。例如，可以用包含CAR或TCR编码序列的表达构建体转导T细胞来获得CAR-T细胞或TCR-T细胞。例如，可以将包含癌症相关抗原的编码序列的表达构建体转导细胞来获得表达所述癌症相关抗原的细胞。本领域技术人员能够容易地构建适合于蛋白表达的表达构建体。
本发明的细胞可以通过各种非限制性方法从许多非限制性来源获得，包括外周血单个核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、腹水、胸腔积液、脾组织和肿瘤。在一些实施方案中，可以使用本领域技术人员可用和已知的细胞系。在一些实施方案中，细胞可以衍生自健康供体或来自诊断为癌症的患者。在一些实施方案中，细胞可以是呈现不同表型特征的细胞的混合群体的一部分。
本发明的细胞如T细胞可以在遗传修饰之前或之后被活化和扩增。T细胞可以在体外或体内扩增。通常，本发明的T细胞可以例如通过与刺激CD3 TCR复合物和T细胞表面上的共刺激分子以产生T细胞活化信号的试剂接触来扩增。例如，可以使用诸如钙离子载体A23187、佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)、或有丝分裂凝集素如植物血凝素(PHA)的化学品来产生T细胞的活化信号。在一些实施方案中，T细胞群体可以通过在体外与例如抗CD3抗体或其抗原结合片段、或固定在表面上的抗CD2抗体接触被活化，或通过与蛋白激酶C激活剂(例如，苔藓抑素)连同钙离子载体的接触来活化。例如，在适合于刺激T细胞增殖的条件下，T细胞群可与抗 CD3抗体和抗CD28抗体接触。适用于T细胞培养的条件包括可能含有增殖和活力所必需的因子的合适培养基(例如Minimal Essential Media或RPMI Media 1640、或X-vivo 5、(Lonza))，其中必需的因子包括血清(例如胎牛或人类血清)、白介素-2(IL-2)、胰岛素、IFN-γ、IL-4、IL-7、GM-CSF、IL-10、IL-2、IL-15、TGFp和TNF，或本领域技术人员已知的用于细胞生长的添加剂。其它用于细胞生长的添加剂包括但不限于表面活性剂、人血浆蛋白粉、和还原剂如N-乙酰-半胱氨酸和2-巯基乙酸。培养基可以包括RPMI 1640、A1M-V、DMEM、MEM、a-MEM、F-12、X-Vivo 1和X-Vivo 20、Optimizer、氨基酸、丙酮酸钠和维生素、无血清或适量补充的血清(或血浆)或一组明确的激素、和/或一定量的足以使T细胞生长和扩增的细胞因子。靶细胞可以保持在支持生长所必需的条件下，例如适当的温度(例如37℃)和环境(例如，空气加5％CO 2)。暴露于不同刺激时间的T细胞可能表现出不同的特征。
如本文所用“对象”是指患有或者易于患有可以通过本发明的方法、组合或药物组合物治疗的疾病或病症的生物体。非限制性例子包括人、牛、大鼠、小鼠、狗、猴、山羊、绵羊、母牛、鹿，及其它非哺乳动物。在优选实施方案中，对象是人。
在第二方面，本发明提供了一种在对象中预防癌症进展或复发的方法，其中所述对象已经经历施用特异性靶向癌症相关抗原的治疗性T细胞的治疗(癌症治疗)，所述方法包括给所述对象施用表达癌症相关抗原的细胞。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在一些实施方案中，表达所述癌症相关抗原的细胞施用一或多次，优选一次。
在一些实施方案中，在施用所述治疗性T细胞约1天、约2天、约3天、约4天、约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天或约21天后施用表达所述癌症相关抗原的细胞。
在一些实施方案中，在所述治疗性T细胞使得癌症细胞负荷降低后，施用表达所述癌症相关抗原的细胞。例如，在癌症细胞负荷降低至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％后，施用表达所述癌症相关抗原的细胞。在一些实施方案中，在癌症通过 施用所述治疗性T细胞而完全缓解之后，施用表达所述癌症相关抗原的细胞。
在一些实施方案中，在施用所述治疗性T细胞后，当所述治疗性T细胞在所述对象体内的量下降时，施用表达所述癌症相关抗原的细胞。所述治疗性T细胞在所述对象体内的量可以通过例如流式细胞术或定量PCR的方法检测。例如，如果所述治疗性T细胞是CAR-T细胞，可以通过PCR检测来自对象的样品中CAR编码基因的拷贝数。当CAR编码基因的拷贝数出现下降时，施用表达所述癌症相关抗原的细胞。
因此，在一些实施方案中，所述方法还包括在施用所述治疗性T细胞后，监测所述对象体内的肿瘤负荷和/或所述治疗性T细胞的量的步骤。
在一些实施方案中，以刺激有效量施用所述表达癌症相关抗原的细胞。如本文所用，表达癌症相关抗原的细胞的刺激有效量是指在施用后能够延长之前施用的治疗性T细胞的体内持续性(增强免疫记忆)和/或延长对象整体存活期和/或延长对象无复发存活期的量。在一些实施方案中，所述表达癌症相关抗原的细胞的刺激有效量为约10 3至约10 6个细胞，例如约10 3、约10 4、约10 5或约10 6个细胞。优选施用低剂量的所述表达癌症相关抗原的细胞，例如约10 4个细胞。在一些实施方案中，根据对象体重确定表达癌症相关抗原的细胞的施用量，约10 3至约10 6个细胞/kg体重，例如约10 3、约10 4、约10 5或约10 6个细胞/kg体重。在一些实施方案中，根据之前患者所接受的治疗性T细胞的量确定表达癌症相关抗原的细胞的施用量。例如，所施用的治疗性T细胞的量与表达癌症相关抗原的细胞的量的比例为约1:1至约1000:1之间的任意比例或更高的比例，例如约1:1、约2:1、约3:1、约4:1、约5:1、约6:1、约7:1、约8:1、约9:1、约10:1、约50:1、约100:1或约1000:1或更高。
在第三方面，本发明提供一种用于在对象中治疗癌症的组合，其包含特异性靶向癌症相关抗原的治疗性T细胞和表达所述癌症相关抗原的细胞。在一些实施方案中，所述组合用于通过本发明第一方面的方法在对象中治疗癌症。在一些实施方案中，所述组合包含治疗有效量的所述治疗性T细胞和刺激有效量的所述表达癌症相关抗原的细胞。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在第四方面，本发明提供本发明第三方面的组合在制备用于在对象中 治疗癌症的药物中的用途。在一些实施方案中，所述药物用于通过本发明第一方面的方法在对象中治疗癌症。
在第五方面，本发明提供表达癌症相关抗原的细胞在制备用于在对象中治疗癌症的药物中的用途。例如，所述药物用于与特异性靶向所述癌症相关抗原的治疗性T细胞组合来在对象中治疗癌症。在一些实施方案中，其中所述药物还包含特异性靶向所述癌症相关抗原的治疗性T细胞。在一些实施方案中，表达癌症相关抗原的细胞与特异性靶向所述癌症相关抗原的治疗性T细胞独立存在于所述药物中，例如独立地置于不同的容器中。在一些实施方案中，所述药物用于通过本发明第一方面的方法在对象中治疗癌症。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在第六方面，本发明提供表达癌症相关抗原的细胞在制备用于在对象中预防癌症进展或复发的药物中的用途，其中所述对象已经经历施用特异性靶向所述癌症相关抗原的治疗性T细胞的治疗(癌症治疗)。在一些实施方案中，所述药物包含刺激有效量的所述表达癌症相关抗原的细胞。在一些实施方案中，所述药物用于通过本发明第二方面的方法在对象中预防癌症进展或复发。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在第七方面，本发明提供特异性靶向癌症相关抗原的治疗性T细胞在制备用于在对象中治疗癌症的药物中的用途，其中所述药物还包含表达所述癌症相关抗原的细胞。在一些实施方案中，特异性靶向癌症相关抗原的治疗性T细胞与表达癌症相关抗原的细胞独立存在于所述药物中，例如独立地置于不同的容器中。在一些实施方案中，所述药物用于通过本发明第一方面的方法在对象中治疗癌症。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在第八方面，本发明提供表达癌症相关抗原的细胞，其用于与特异性靶向所述癌症相关抗原的治疗性T细胞组合在对象中治疗癌症。在一些实施方案中，所述表达癌症相关抗原的细胞用于通过本发明第一方面的方法在对象中治疗癌症。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在第九方面，本发明提供表达癌症相关抗原的细胞，其用于在对象中预防癌症进展或复发，其中所述对象已经经历使用特异性靶向所述癌症相关抗原的治疗性T细胞的治疗(癌症治疗)。在一些实施方案中，所述表达癌 症相关抗原的细胞用于通过本发明第二方面的方法在对象中预防癌症进展或复发。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在第十方面，本发明提供特异性靶向癌症相关抗原的治疗性T细胞，其用于与表达所述癌症相关抗原的细胞组合在对象中治疗癌症。在一些实施方案中，所述特异性靶向癌症相关抗原的治疗性T细胞用于通过本发明第一方面的方法在对象中治疗癌症。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
在第十一方面，本发明提供药盒，其包含特异性靶向癌症相关抗原的治疗性T细胞和/或表达所述癌症相关抗原的细胞，所述药盒用于通过本发明的方法在对象中治疗癌症或预防癌症进展或复发。在一些实施方案中，表达癌症相关抗原的细胞与特异性靶向所述癌症相关抗原的治疗性T细胞独立存在于所述药盒中，例如独立地置于不同的容器中。所述治疗性T细胞或表达癌症相关抗原的细胞如本文所定义。
本发明所述的细胞或药物中还可以包含“药物可接受赋形剂”或“药物可接受载体”，其是指有助于活性物质给予对象且对象吸收的物质，其可以包括在本发明的药物组合物中而不引起患者的显著毒副作用。药物可接受赋形剂的非限制性例子包括水、NaCl、生理盐水、乳酸化Ringer's、正常蔗糖、正常葡萄糖、粘合剂、填充剂、崩解剂、润滑剂、包衣、甜味剂、香味剂、盐溶液(如Ringer's溶液)、醇、油、明胶、碳水化合物如乳糖、直链淀粉或淀粉、脂肪酸脂、羟甲基纤维素、聚乙烯比咯烷酮和着色剂等。这种制备物可以被灭菌，如果希望，可以与辅助制剂如润滑剂、防腐剂、稳定剂、润湿剂、乳化剂、影响渗透压的盐、缓冲剂、着色剂和/或芳香物质等混合，这些辅助制剂不与本发明的细胞有害反应。本领域技术人员理解其它药物赋形剂也可以用于本发明。
本发明所述癌症相关抗原优选是癌症特异性抗原，包括但不限于CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA  IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。在一具体实施方案中，所述癌症相关抗原是CD19。
本发明所述的癌症的非限制性的例子包括肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本发明的方法或药物组合物治疗的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。在一具体实施方案中，所述癌症是B细胞急性淋巴细胞白血病(B-ALL)。
本发明各个方面中，所述治疗或预防方法可以结合一种或多种选自以下一组针对癌症的疗法：抗体治疗、化学治疗、细胞因子治疗、树突状细胞治疗、基因治疗、激素治疗、激光治疗和放射治疗。
根据本发明的细胞或组合或药物的施用可以以任何方便的方式进行，包括通过注射、输注、植入或移植。本文所述的细胞或组合或药物施用可以通过静脉内、淋巴内、皮内、肿瘤内、髓内、肌内或腹膜内施用。在一个实施方案中，本发明的细胞或组合或药物优选通过静脉内注射施用。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
材料和方法
CRISPR/Cas9介导的B-NDG小鼠产生
CRISPR/Cas9介导的B-NDG小鼠由Beijing Biocytogen Co.,Ltd.生成。简而言之，涉及sgRNA靶向位于Il2rg基因座的第一个和最后一个外显子侧翼的靶区域。对于每个靶位点，通过CRISPR设计工具(http://crispr.mit.edu/)确定4种候选sgRNA并使用UCA试剂盒(北京Biocytogen)筛选靶向活性。选择两种sgRNA注射到小鼠受精卵中。体外转录的Cas9 mRNA和sgRNA以不同浓度混合并共注射至NOD-scid小鼠的受精卵中。注射后，存活的受精卵转移进KM假孕雌性的输卵管。从F0原代修饰小鼠中，进一步产生了Il2rg缺陷的杂合和纯合小鼠(B-NDG小鼠)。所有小鼠均保持在特定的无病原体设施中。
细胞系
将293-T细胞(ATCC，CRL-3216)在补充有10％FBS和100U/ml青霉素/链霉素的DMEM(Hyclone，Logan，Utah，USA)中培养。Raji(ATCC，CCL-86)、Raji-Fluc(军事医学科学院，北京；通过转染编码荧光素和嘌呤霉素抗性的慢病毒载体而衍生自Raji细胞)、CD19-K562和K562细胞(Genomeditech，上海)在补充有10％FBS和100U/ml青霉素/链霉素的RPMI1640中培养。通过将细胞暴露于40Gy钴辐射获得经辐照的Raji-Fluc细胞。所有培养基和抗生素均购自Gibco Life Technologies(Carlsbad,CA,USA)。
基于慢病毒的载体的构建、制备和滴定
抗CD19 CAR包括CD19特异性FMC63 mAb的scFv，CD8的铰链和跨膜区域，4-1BB共刺激结构域和TCR复合物的胞内CD3ζ链。抗CD19 CAR基因被克隆进慢病毒载体pPVLV2(Pharosvaccine Inc.，Gyeonggi，Republic of Korea)中。该载体用于产生CAR-T-19细胞。
通过用CD19编码序列取代抗CD19 CAR编码序列产生aT19慢病毒表达构建体。该载体用于产生aT19细胞。
表达所述抗CD19 CAR或CD19的慢病毒如下生成：在含10％FBS的DMEM中生长的293T细胞用慢病毒载体质粒和pMDLg/pRRE、pRSV-Rev和pMD.G包装质粒使用PEI(Polysciences Inc.，Warrington，PA，USA)转染试剂共转染。转染24小时后，培养基替换为补充有2％FBS、1mM丙酮酸钠和2mM L-谷氨酰胺的DMEM。培养基交换后再24小时后，收集病毒上清液，超滤浓缩并用293T细胞滴定。为了确定功能性转导单位/mL，用连续稀释的浓缩的慢病毒制备物转导293T细胞。72小时后，收集细胞并进行流式细胞术分析。
产生CAR-T-19细胞和aT19细胞
通过收集自健康志愿者的血液获得新鲜的外周血单个核细胞(PBMC)。将PBMC立即在含有90％人AB血清加10％二甲亚砜的培养基中使用或在所述培养基中冷冻保存用于将来使用。PBMC用包被有抗CD3和抗CD28单克隆抗体的顺磁磁珠(Invitrogen，Carlsbad，CA，USA)以3:1的比率刺激24个小时。T细胞活化后，将细胞以1×10 6/mL重悬在IMSF100培养基中，在8μg/ml聚凝胺(SIGMA，圣路易斯，MO，USA)的存在下，与慢病毒载体混合。感染复数(MOI)为0.5-2，并转移到12孔板中。将板在环境温度下使用水平转子离心机在1200g离心2小时，随后转移到37℃，5％CO 2培养箱中。孵育24小时后，收集转导的细胞并用IMSF100培养基洗涤，然后将细胞在补充有500IU/mL IL-2(BMI，韩国)的培养基中孵育。之后每2或3天更换培养基和IL-2(300IU/mL)，培养物维持在37℃/5％CO 2，直至注射到小鼠中。其中CAR-T-19细胞为表达抗CD19 CAR的T细胞。aT19细胞为表达CD19的T细胞。
流式细胞术
为了检测小鼠中的T、B和NK细胞和CAR-T细胞，使用Becton Dickinson(BD)荧光激活细胞分选(FACS)标准按照制造商的方案进行多色流式细胞分析。从右心室采集外周血。通过BD细胞滤器收集和过滤脾细胞。血液中的红细胞和脾细胞通过裂解溶液耗竭。通过用包括PerCP-CD3、FITC-CD4、PE-CD8、FITC-CD19和APC-CD49的抗体单染色或双染色检测白细胞。所有抗体购自Biolegend。
通过流式细胞术进行免疫表型分析。通过用以下特异性抗体染色细胞来监测CAR基因转导的功效：CD45(PerCP-Vio770)、CD3(FITC)、CD4(Vio Green)、CD16(APC)、CD56(APC)、CD19(APC-Vio770)、CD14(Vio Blue；Miltenyi Biotec，Bergisch Gladbach，Germany)。转导的T细胞用生物素-SP(长间隔物)AffiniPure F(ab')片段山羊抗小鼠IgG，F(ab')2片段特异性(Jackson ImmunoResearch Lab)标记，然后用链霉抗生物素蛋白-PE(BD Biosciences)或CARTEST-19(CytoCares Inc.，中国上海)标记，然后通过CytoFLEX流式细胞仪检测。使用CytExpert(2.0)软件分析数据。
从小鼠收获的白细胞和脾细胞用FITC-CD45、PE-CAR、FITC-CD19和APC-CD45RO(BD，NJ)染色，并在FACSCanto II流式细胞仪(BD)中检查。收集脾细胞并通过BD细胞过滤器过滤。通过在裂解溶液(BD)中孵育5分钟来耗尽血液和脾细胞制剂中的红细胞。
动物实验
本研究中的小鼠根据实验动物关怀评估和鉴定协会制定的指导方针进行饲养和操作。研究得到了NIFDC机构动物护理和使用委员会的批准。四周龄的雌性BALB/c、C57BL/6、NOD-scid和B-NDG小鼠获得自NIFDC实验动物资源研究所(中国北京)。在D0，小鼠通过尾静脉注射移植5×10 5个Raji-Fluc细胞，随后在第5天(D5)天或第6天(D6)植入2×10 7个CAR-T-19细胞。在CAR-T记忆实验中，在D10或D11注射不同剂量的Raji-Fluc细胞、经辐照的Raji-Fluc细胞或aT19细胞。在指定的时间点观察所有小鼠的生物发光信号。在指定时间点获得血液样品进行荧光激活细胞分选和定量实时PCR(qPCR)分析。
生物发光成像分析
简言之，使用IVIS-Lumina II成像系统(PerkinElmer，Baltimore，MD)获得小鼠的生物发光成像。通过腹膜内注射戊巴比妥钠(75mg/kg体重)麻醉小鼠。然后在10分钟后使其接受腹膜内注射D-荧光素(75mg/kg体重；PE)。使用Living Image软件(Caliper Life Sciences)分析图像，并将数据表示为总通量(光子/s)。如果产生的最大生物发光值超过1×10 6通量，并且生物发光面积在随后的时间点不再小于1×10 6通量，则将该时间点记录为肿瘤复发的时间。
CAR-T-19细胞的细胞毒性
通过流式细胞术测量CAR-T-19细胞杀死K562、CD19-K562、aT19和Raji-Fluc细胞的能力。简而言之，将靶细胞(K562、CD19-K562、Raji-Fluc或aT19)转移到离心管中并用CFSE染色。接下来，调整靶细胞:CAR-T-19细胞比率并分别以不同比例接种细胞(K562或CD19-K562:CAR-T-19＝1:1、1:3或1:6；aT19或Raji-Fluc:CAR-T-19＝1:0.3、1:1或1:3)。未转导的T细胞记录为NC(阴性对照)。孵育20-24小时后，通过流式细胞术分析CFSE+7-AAD+细胞的百分比。
定量检测CAR-T细胞
通过qPCR检测从小鼠外周血和脾中分离的CAR-T-19细胞中CAR基因的存在。简而言之，使用QIAamp DNA Mini Kit(Qiagen，GER)提取DNA，并在IMPLEN N50超微紫外分光光度计(IMPLEN，N80TOUCH)中定量。将提取的DNA保存在-20℃直至需要。使用4-1BB引物(Invitrogen)和探针(Thermo Fisher Scientific)对DNA(100ng)进行PCR扩增，以检测CAR基因的表达(CD19 4-1BB F引物，5'-TGCCGATTTCCAGAAGAAGAAGAAG-3'；CD19 4-1BB R引物，5'-GCGCTCCTGCTGAACTTC-3'；CD19 4-1BB MGB探针，5'-ACTCTCAGTTCACATCCTC-3'))。
使用StepOnePlus荧光实时定量PCR仪(Thermo Fisher)进行PCR。使用Step One Software v2.3分析结果。
统计
所有图表均使用GraphPad Prism 6.0软件生成所有的统计学比较均采用 非参数单因素方差分析或Student t检验，P值<0.05为统计学显著(*)，P<0.01(**)为非常显著。
实施例1、Raji-B-NDG小鼠模型的产生和表征
具有无效Il2rg的非肥胖糖尿病严重联合免疫缺陷(NOD-scid)小鼠缺乏成熟T细胞、B细胞和天然杀伤(NK)细胞，并且缺乏细胞因子信号传导，导致多种癌症类型更好的植入。在本实施例中，用CRISPR/Cas9基因组编辑技术从NOD-scid小鼠中缺失Il2rg基因，从而制备B-NDG小鼠(图1A)。观察到B-NDG小鼠可存活、可繁殖并且没有显示任何明显的身体异常。与NOD-scid小鼠相比，FACS分析显示B-NDG小鼠的脾和外周血具有较低数量的CD19+B细胞、CD4+和CD8+T细胞(图1B)。NOD-scid小鼠具有高百分比的CD49b+NK细胞(脾脏20.90％，外周血24.10％)，而B-NDG小鼠无功能性NK细胞(脾脏1.45％，外周血5.95％)。因此，通过基因组编辑产生的B-NDG小鼠在表型上与NSG小鼠相似。
快速增长的非霍奇金淋巴瘤细胞系Raji细胞被基因工程化来稳定表达萤火虫荧光素酶报告基因用于生物发光成像(Raji-Fluc)。通过流式细胞术检测Raji-Fluc细胞表面的CD19发光丰度。结果显示，CD19在Raji-Fluc细胞上高表达(图1G)。体外加入Fluc的相应底物后，Fluc的发光强度与Raji-Fluc细胞的数量正相关(R 2＝0.99，图1G)。
为了进一步研究所获得的B-NDG小鼠模型的能力，6周龄的五种不同小鼠品系(包括作为正常对照的CB57/L小鼠、作为部分免疫缺陷组的裸鼠和rag2-/-小鼠、NOD-scid和B-NDG小鼠)通过尾静脉注射移植Raji-Fluc细胞。B-NDG小鼠中在植入后(PI)第四天(D4)检测到生物发光信号，16天后增加1000倍(图1C和D)。流式细胞术分析表明CD19+Raji细胞在B-NDG小鼠的外周血中爆发，与生物发光信号的相关性R 2＝0.90(图1E)。然而，在CB57/L、rag2-/-、裸鼠、甚至NOD-scid小鼠中无法检测到Raji-Fluc细胞。所有这四种小鼠在D30PI存活，而7只B-NDG小鼠在D18PI死亡，另外3只死于D19(图1C、D和F)。这些数据证明B-NDG小鼠中Raji细胞植入效果要好得多，并且可以用作测试治疗剂例如CAR-T细胞的模型。
实施例2、CD19靶向性CAR-T细胞的体外和体内效率
T细胞活化后，从志愿者分离的PBMC用第二代CAR(图2A)进行转导。结果表明，CD19 CAR-T细胞的比例随着培养时间的延长而增加，在D12达到20％，并且细胞的总数在感染后D10大于1×10 8(图2B)。当在与CD19+Raji细胞体外共培养时，培养12天的CD19 CAR-T细胞，在效应细胞/靶细胞比例为大约3:1的情况下，显示50％以上的对Raji细胞的杀灭作用。为了进一步验证CD19 CAR-T细胞的体内抗肿瘤作用，B-NDG小鼠通过尾静脉注射5×10 5Raji-Fluc细胞，随后在5天后植入2×10 7的CD19 CAR-T细胞。CD19 CAR-T治疗四天后(D9)，这些经治疗的小鼠的生物发光信号接近于未接种肿瘤的小鼠(图3A)，表明CD19 CAR-T细胞成功介导了体内肿瘤清除。
实施例3、Raji肿瘤的复发来源
尽管CAR-T细胞很有效，但是B-NDG小鼠最终因肿瘤死亡。为了找到Raji细胞复发的来源，测试了小鼠全部主要器官的生物发光。结果表明Raji细胞在脾脏、心脏和皮肤显著地被CD19 CAR-T细胞清除。例如，脾脏中，CAR-T细胞处理后生物发光减少1000倍(图3A)。然而，CD19 CAR-T在其它器官并不都如此有效，特别是在肝脏和脑中(图3A)。隐藏在这些器官中的不能被CAR-T有效清除的残余Raji细胞可能是肿瘤复发的原因。
实施例4、低剂量活肿瘤细胞的刺激有助于CAR T细胞的免疫记忆的产生
为了测试CAR-T细胞是否因肿瘤微环境而发生改变，导致其无法识别和杀死Raji细胞并导致疾病复发，在D10PI未检测到生物发光的情况下，向CD19 CAR-T处理过的小鼠中重新植入不同量的Raji-Fluc细胞(A1：5×10 4，A2：5×10 5，A3：5×10 6)。未再接种Raji-Fluc细胞的小鼠用作对照(SNT)。在SNT组中，能够持续观察到生物发光，直到全部小鼠死亡。在再植入一天后，全部小鼠快速发展出疾病。令人意想不到的是，再植入9天后，几乎所有的Raji细胞都被清除(图3A)。再植入组的生存优于SNT组(P<0.05，图3A和B)。A1组的三只小鼠存活超过200天。如果某一时间点小鼠的最大生物发光值超过1×10 6通量，并且在随后的时间点生物发光面积不再低于1×10 6通量，则该时间点记录为肿瘤复发时间。与其他各组相比，用低剂量Raji-Fluc细胞再接种可延长无复发时间(图3A和C)。然后分析不同时 间点不同组的平均生物发光强度。低剂量再植入组的信号接近空白对照组的信号(图3D)，而其他组的信号不同程度地增加，表明复发是由CAR的不良持续性而并其能力不足所致，并且低剂量肿瘤细胞的抗原刺激促进CAR-T细胞的免疫记忆的产生。
实施例5、再植入辐射后死亡的肿瘤细胞不起作用
在临床上注射活肿瘤细胞是不可行的，因此测试辐射后死亡肿瘤细胞是否可以刺激CAR-T细胞的免疫记忆。由于当前本领域中多次输注CAR-T细胞是克服其持续性不足的主要策略，设计了重复CAR-T治疗的小鼠模型。如图4所示，在第5天(D5)进行CAR-T细胞治疗后，Raji-B-NDG小鼠在D10注射不同剂量的经辐射的Raji-Fluc细胞(rRaji-Fluc，A1：5×10 4，A2：5×10 5，A3：5×10 6)。三组接种相同剂量rRaji-Fluc的B-NDG小鼠作为经辐射细胞对照组(PA1/PA2/PA3)。无rRaji-FLuc植入的小鼠作为单次处理组(SNT)。双重处理组(DNT)的小鼠在D10注射2×10 7个CAR-T细胞。小鼠中可观察到生物发光信号，直到SNT组全部小鼠死亡。与SNT组相比，再植入rRaji-Fluc细胞不能延长小鼠的整体存活期和无复发存活期(图4B和C)。SNT组与DNT组无显著性差异，说明双重处理无效。A1/A2/A3/SNT组小鼠外周血中CAR-T细胞的数量在相似的时间达到峰值(图4D)。由于两次注射CAR-T细胞，DNT组的峰值时间晚于其他组。A1组的CAR-T细胞减少速率慢于A2/A3/SNT组，表明低剂量rRaji-Fluc细胞的刺激倾向于促进CAR-T细胞的维持。
实施例6、使用表达CD19的T细胞(aT19)的再刺激
CAR基因构建体包含源自CD19特异性FMC63 mAb的scFv、CD8的铰链和跨膜区域、和4-1BB胞质结构域，以及TCR复合物的胞内CD3ζ链(图5A)。pEF1α是CAR基因启动子。通过用人CD19替换CAR转基因产生aT19构建体(图5B)。用CAR-19或CD19构建体(aT19)转导从获自健康志愿者的PBMC分离的T细胞。表1显示了实验中使用的CAR-T-19、活Raji-Fluc、经辐照的Raji-Fluc和aT19细胞的表型和数量。接下来，进行细胞毒性测定以检查CAR-T-19细胞的杀伤能力。在与CD19+Raji或aT19细胞进行体外共培养后，CAR-T-19细胞以>3:1的效应物/靶标比例杀死超过 50％的Raji-Fluc细胞或aT19，而当T细胞与对照K562细胞共培养时几乎没有观察到细胞毒性作用(图5C)。
表1.实验中使用的活Raji-Fluc、经辐照的Raji-Fluc和aT19细胞的数量，以及CAR-T-19表型和数量
Figure PCTCN2019084773-appb-000004
CAR-T-19治疗然后施用活Raji-Fluc细胞可延长Raji-B-NDG小鼠的存活
阳性组和NC组中的小鼠单独接受肿瘤细胞或仅接受PBS，并分别记录为P组或N组(n＝3/组)。NT组中的小鼠在没有刺激的情况下接受CAR-T-19细胞(n＝5/组)。A组被指定为免疫记忆测试组；该组中的小鼠接受三种不同剂量的再刺激细胞中的一种(A1组，5×10 4细胞；A2组，1.5×10 5细胞；和A3组，5×10 5细胞)(n＝5/组)。PA1、2和3组分别用作每组A的对照组(n＝3/组)。这些组列于表2中。图6A示出肿瘤细胞、CAR-T细胞和再刺激细胞的使用时间线。
表2.在再刺激实验中使用的Raji-Fluc细胞的数量
Figure PCTCN2019084773-appb-000005
在注射后(PI)D4获得的体内成像结果显示，接受Raji-Fluc细胞的小鼠的总通量(>2.2×10 6)远高于未接受Raji-Fluc细胞的小鼠(<1.6×10 6；NC组)，表明小鼠模型构建成功(图6B)。在PI的D5，小鼠接受(或不接受)2×10 7个CAR-T-19细胞。D9上的成像显示接受CAR-T-19细胞的所有小鼠(除了P组中的那些)的总通量显著降低(图6B)，表明CAR-T-19细胞有效杀死Raji-Fluc细胞。
接下来，为了研究Raji-Fluc细胞是否刺激残留的CAR-T-19细胞的生长，产生记忆细胞，并延长CAR-T-19治疗的效果，小鼠在PI的D10接受了随后的Raji-Fluc细胞注射。如图6B所示，P组中的所有小鼠均死于PI的D18-23。PA1/2/3组中的所有小鼠分别死于D31-33/D27-31/D28。由于PA3组中的小鼠接受与P组小鼠相同数量的肿瘤细胞，因此存活时间与D20-24相似。N组中的一只小鼠以不明原因死于D71；另外两只幸存了200多天。NT组中的所有5只小鼠均死于D82。对于A1组小鼠，荧光信号从D11到D24逐渐下降，并且D34没有检测到Raji-Fluc细胞(图6B)；该组中的三只小鼠在D200无肿瘤。在A2组中，三只小鼠死于D96，而另外两只小鼠在没有肿瘤的情况下存活至D100(图6B)。在A3组中，一只小鼠在D40死于不明原因，三只因肿瘤复发在D56、D80和D86死亡，并且另一只死于D96。总之，这些结果表明，用肿瘤细胞再刺激的小鼠存活的时间长于没用肿瘤细胞再刺激的小鼠(图6C，P<0.05)。此外，用低剂量的Raji-Fluc细胞重新接种导致无复发存活率显著增加(图6D)。最后，检查了不同时间点不同组的平均生物发光强度。由低剂量再刺激组产生的信号与由空白对照组产生的信号非常相似(图6B)，而由其他组产生的信号不同程度地增加，表明用低剂量肿瘤细胞的再刺激扩增CAR-T-19细胞群并清除Raji-Fluc细胞。
用CAR-T-19细胞随后用经辐照的Raji-Fluc细胞对Raji-B-NDG小鼠进行连续治疗并不能延长生存期
给患者注射活肿瘤细胞是不可行的。因此，接下来研究经辐照死亡的肿瘤细胞是否以与活细胞相似的方式增加CAR-T-19细胞的功效。这组实验使用与活细胞实验相同的小鼠组。施用的细胞的组和数量显示在表3中。时间线见图7A。如前所述，在PI的不同时间进行生物发光成像。发现用经 辐照的死亡Raji-Fluc细胞再刺激CAR-T-19细胞不会增加总体存活率和无复发存活率(图7B和C)。
表3.用于再刺激的经辐照的Raji-Fluc细胞的数量
Figure PCTCN2019084773-appb-000006
另外，在D0/D5/D10/D15/D20上测量了从小鼠内眦获得的血液的CAR拷贝数(代表CAR+细胞数)。如图7E所示，A1、A2、A3和NT组中CAR-T-19细胞的数量同时达到峰值。此外，在D10用辐射的肿瘤细胞再刺激后，CAR拷贝数没有增加(图7E)。这些结果表明，用辐射的死亡肿瘤细胞再刺激不会增加残留CAR-T-19细胞的生长或促进记忆CAR-T-19细胞的产生。
用CAR-T-19细胞处理小鼠，然后用aT19细胞再刺激，增加CAR+细胞的数量和CAR+记忆细胞的百分比，并延长生存期
Raji-Fluc细胞是具有高增殖能力的肿瘤细胞。因此，如果患者接受刺激剂量的Raji细胞，则存在这些细胞将形成肿瘤的风险。这显然是不可接受的。因此，使用携带人CD19基因的T细胞(aT19细胞)代替Raji-Fluc细胞来再刺激CAR-T-19细胞。注射时间和使用的细胞数列于表4中。阳性/NC组(P和N组)中的小鼠分别接受肿瘤细胞或仅接受PBS。NT组中的小鼠接受单剂量的CAR-T-19细胞，没有随后剂量的刺激细胞。A1组用Raji-Fluc细胞(n＝4)再刺激，而aT19组用aT19细胞(n＝6)再刺激(图8A)。此外，在D10、D20和D30上从内眦取血(图8D)。结果显示，aT19组小鼠的存活时间明显长于NT组小鼠；aT19组中的三只小鼠存活至D160(图8C)。
表4.用于再刺激的Raji-Fluc或aT19细胞的剂量
Figure PCTCN2019084773-appb-000007
为了阐明延长的存活和延迟的肿瘤复发是由于CAR-T-19细胞的增殖和随后在抗原阳性细胞再刺激后产生记忆CAR-T-19细胞，使用qPCR来测量CAR拷贝数并使用流式细胞术测量D10、D20和D30外周血和脾中CAR+和CD45RO+细胞的百分比。QPCR显示NT组小鼠血液中的CAR拷贝数随时间下降(从1175拷贝到295拷贝)，而aT19组小鼠血液中的CAR拷贝数随着时间的推移而增加(从1224拷贝到6824拷贝)，尤其是在D10后(图8D)。正如预期的那样，NT组小鼠血液中CAR+细胞百分比降低(从3.28％降至0.29％)，而aT19组小鼠血液中CAR+细胞百分比增加(从9.6％增至13.0％)(数据未显示)。除CAR+细胞外，还测量了CD45RO+(记忆)细胞的百分比。如图8F所示，NT组血液中CAR+CD45RO+细胞的百分比从0.44％(D20)略微下降至0.37％(D30)，而aT19组血液中的百分比从0.34％(D20)上升至0.86％(D30)，证实在aT19细胞再刺激后产生记忆CAR-T-19细胞。此外，检查了小鼠中CAR+细胞、CD45RO+细胞和总通量(肿瘤负荷)之间的关系。结果显示，总通量与两种类型细胞的数量呈负相关，表明CAR拷贝数的检测可用于估计残留肿瘤负荷。
讨论
过去十年中，CAR-T细胞疗法取得了进展；然而，许多障碍尚待克服。其中，延长CAR-T细胞在体内的功效和减少CAR-T治疗的副作用是最重要的。CAR-T细胞的功效可以通过优化胞外识别 19,20和CAR介导的细胞内信号转导 21(通过调整CAR+细胞的比例和类型实现) 22以及同时使用靶向药物 23等策略来延长。通过调整CAR-T细胞的剂量和同时使用免疫抑制药物， 可以降低与CAR-T治疗相关的毒性和副作用 24。对疫苗采取了类似的方法，这些方法不断优化，以便在更长的时间内提供有效的保护，同时减少不良反应 25。最常见的疫苗接种方案涉及多次免疫或甚至使用不同的免疫原来增强免疫应答。以HIV疫苗策略为例，初级和加强免疫分别包含病毒抗原和核酸，分别增加抗体保护的持续时间和增加免疫激活 26。然而，对于CAR-T细胞疗法，多次接种增加了成本和毒性但降低了功效。
在本申请中，令人惊奇地发现，通过在CAR-T细胞施用后，使用表达肿瘤抗原的细胞(例如aT19细胞)进行再刺激，可以通过增加细胞有效的时间和减少其副作用来优化CAR-T治疗(例如CAR-T-19治疗)。
肿瘤抗原的丧失或较短的有效性意味着肿瘤倾向于在CAR-T细胞治疗后复发。肿瘤抗原的丧失是常见的免疫逃逸策略，这在ALL中是常见的。潜在的机制涉及表型谱系和选择型剪接的转换 27。此外，必须将CAR-T细胞的数量维持在长时间控制肿瘤所需的最低水平。有很多复杂的原因导致难以维持CAR-T细胞的数量；这些包括针对CAR的免疫反应、细胞衰老诱导的增殖能力丧失和活化诱导的细胞死亡。通过优化用于产生CAR的结构和条件，可以减少对CAR的免疫应答。由于T细胞活化和增殖需要持续的免疫刺激，携带肿瘤抗原的细胞是诱导CAR-T细胞增殖的最佳刺激信号。在本申请中，在D5给小鼠注射CAR-T细胞，并然后将它们暴露于大剂量的肿瘤(抗原阳性)细胞。CAR-T细胞迅速增殖并杀死肿瘤细胞直至D9，此时血液中可检测到很少的肿瘤细胞。然而，当它们未暴露于抗原介导的刺激时，T细胞的增殖能力降低。因此，发现CAR-T细胞的数量随着时间的推移而下降，并最终不能抑制残留癌细胞的增殖。SNT组小鼠血液中CAR+细胞百分比从D20至D30逐渐下降。然而，如果在癌细胞几乎完全消除后注射第二“抗原”(即aT19细胞)，则再刺激CAR-T细胞并保持其增殖能力。此外，本申请的结果表明，在D30不仅CAR+细胞数量显著高于D20而且CD45RO+记忆细胞百分比显著高于D20，这进一步巩固了CAR-T介导的免疫力。因此，aT19细胞起到类似于加强免疫的作用。接受连续治疗的小鼠的无复发生存期和总生存期优于接受单一治疗的小鼠(单独使用CAR-T细胞)，这表明连续免疫可能是一种更安全，更有效的延长CAR-T细胞在临床环境中的有效时间的策略。
CAR-T治疗可能导致严重病例的细胞因子释放综合征(CRS；通常称为 “细胞因子风暴”)，这可能危及生命 3,28,29,30。这是CAR-T疗法除了脱靶效应最严重的副作用之一。CAR-T细胞可以被肿瘤细胞激活，并且免疫系统又可以被CAR-T细胞释放的细胞因子激活。如果释放大量炎症因子，这可能导致发烧、低血压、呼吸窘迫、多器官功能障碍、神经调节障碍和其他症状 28。CRS通常在CAR-T注射后的前2周内发生，尽管潜在的机制尚不清楚。使用抗细胞因子单克隆抗体或糖皮质激素的早期干预可降低CRS的风险 31。有证据表明，CRS的严重程度与接种的CAR-T细胞数和肿瘤负荷正相关 4,22,32,33,34。在这里，通过用CAR-T细胞然后用aT19细胞的连续“免疫”，在小鼠中产生持续的免疫应答。这可以减少每次接种所需的CAR-T细胞数量和随时间推移所需的接种次数，从而降低CRS的可能性和/或严重性。
本申请数据表明aT19细胞具有临床实用性。这是因为这些细胞来自与CAR-T-19细胞相同的来源。临床上，自体PBMC可用于产生这些细胞，这可有效避免移植物对抗宿主疾病。此外，CAR-T细胞通过CD19分子而不是其他免疫信号识别aT19细胞，从而确保特异性。最后，通过用CD19基因取代CAR抗体的Fab片段构建aT19转导片段；这将消除体内正常组织中的任何脱靶效应。例如，CAR-T-Her2可以攻击肺粘膜上皮并引起肺水肿，这在严重的情况下可能是致命的 35。因此，从临床角度来看，使用aT19细胞的连续“免疫”应该是可行的。在本申请中，因为在D9没有检测到肿瘤，在D10给小鼠注射了aT19细胞。此时，靶细胞的丢失可能导致CAR-T细胞攻击宿主组织。然而，CAR-T-19细胞的增殖率可能由于抗原特异性刺激的丧失而降低，而非特异性攻击宿主的T细胞可以连续增殖。因此，在D10注射aT19细胞以维持CAR-T-19细胞。
临床上，可以根据CAR-T细胞的数量调整aT19施用的时间。在这里，通过qPCR测量血液中的CAR拷贝数来估计CAR-T细胞的数量。结果与流式细胞术提供的结果一致。此外，血液样品中CAR+细胞的数量与荧光值(总通量)(其代表体内残留肿瘤)之间存在负相关。然而，人体中的循环系统比小鼠更长且更复杂，并且临床中给予的CAR-T的剂量远大于给予小鼠的剂量。因此，在CAR-T细胞治疗后的第一周内，人体内的癌细胞可能无法完全清除，并且血液中的CAR拷贝数将继续增加。随后，当CAR+拷贝的数量开始下降时，CAR-T细胞的数量逐渐下降并且它们被新生成的T细胞 替换。此时应考虑用aT19细胞进行免疫。连续监测CAR+拷贝数，然后及时接种aT19细胞可能是在这种情况下临床应用aT19细胞的可行方法。
总之，用CAR-T细胞处理过的小鼠注射Raji-Fluc细胞、辐射的Raji-Fluc细胞或aT19细胞。结果显示，用活Raji-Fluc细胞和aT19细胞再刺激CAR-T细胞延长了无复发存活率和总体存活率，而用辐射的(死亡)Raji-Fluc细胞再刺激则没有延长无复发存活率和总体存活率。aT19的连续治疗增加了血液中CAR+和CD45RO+细胞的百分比，证实了记忆CAR-T细胞的产生。
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Claims (42)

  1. 一种在对象中治疗癌症的方法，包括以下步骤：
    (a)给所述对象施用特异性靶向癌症相关抗原的治疗性T细胞；和
    (b)给所述对象施用表达所述癌症相关抗原的细胞。
  2. 权利要求1的方法，所述治疗性T细胞施用一或多次，优选一次。
  3. 权利要求1或2的方法，表达所述癌症相关抗原的细胞施用一或多次，优选一次。
  4. 权利要求1-3中任一项的方法，其中在施用所述治疗性T细胞之后施用所述表达癌症相关抗原的细胞。
  5. 权利要求4的方法，其中在施用所述治疗性T细胞1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天或20天后施用表达所述癌症相关抗原的细胞。
  6. 权利要求1-5中任一项的方法，其中在施用所述治疗性T细胞使得癌症细胞负荷降低后，施用表达所述癌症相关抗原的细胞；或者在施用所述治疗性T细胞后，当所述治疗性T细胞在所述对象体内的量下降时，施用表达所述癌症相关抗原的细胞。
  7. 权利要求6的方法，其中在癌症细胞负荷降低至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％后，施用表达所述癌症相关抗原的细胞。
  8. 权利要求1-7中任一项的方法，其中在癌症通过施用所述治疗性T细胞而完全缓解之后，施用表达所述癌症相关抗原的细胞。
  9. 权利要求1-8中任一项的方法，其中所述治疗性T细胞以治疗有效量施用，例如施用约10 4至约10 9个所述治疗性T细胞。
  10. 权利要求1-9中任一项的方法，其中所述表达癌症相关抗原的细胞以刺激有效量施用，例如施用约10 3至约10 6个所述表达癌症相关抗原的细胞。
  11. 权利要求1-10中任一项的方法，所述治疗性T细胞是包含外源T细胞受体(TCR)的T细胞(TCR-T细胞)或包含嵌合抗原受体(CAR)的T细胞 (CAR-T细胞)。
  12. 权利要求11的方法，所述外源TCR特异性结合癌症相关抗原，所述CAR包含针对癌症相关抗原的细胞外抗原结合结构域。
  13. 权利要求11或12的方法，所述CAR包括特异性结合癌症相关抗原的细胞外抗原结合结构域如scFv、CD8α铰链和跨膜结构域、CD3ζ信号转导结构域、和4-1BB共刺激结构域。
  14. 权利要求1-13中任一项的方法，所述表达癌症相关抗原的细胞是活细胞，优选地，所述细胞不是癌症细胞。
  15. 权利要求14的方法，所述癌症相关抗原表达于所述活细胞的表面。
  16. 权利要求1-15中任一项的方法，所述表达癌症相关抗原的细胞衍生自免疫细胞。
  17. 权利要求1-16中任一项的方法，所述治疗性T细胞和/或所述表达癌症相关抗原的细胞衍生自对象的自体细胞。
  18. 权利要求1-16中任一项的方法，所述治疗性T细胞和/或所述表达癌症相关抗原的细胞衍生自异体细胞，例如源自与所述对象HLA相容的供体。
  19. 一种在对象中预防癌症进展或复发的方法，其中所述对象已经经历使用特异性靶向癌症相关抗原的治疗性T细胞的癌症治疗，所述方法包括给所述对象施用表达癌症相关抗原的细胞。
  20. 权利要求19的方法，其中表达所述癌症相关抗原的细胞施用一或多次，优选一次。
  21. 权利要求19或20的方法，其中在用所述治疗性T细胞治疗1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天或20天后施用表达所述癌症相关抗原的细胞。
  22. 权利要求19-21任一项所述的方法，其中在所述治疗性T细胞使得癌症细胞负荷降低后，施用表达所述癌症相关抗原的细胞。
  23. 权利要求22的方法，其中在癌症细胞负荷降低至少大约5％、至少大约10％、至少大约20％、至少大约30％、至少大约40％、至少大约50％、至少大约60％、至少大约70％、至少大约80％、至少大约90％后，施用表达所述癌症相关抗原的细胞。
  24. 权利要求19-23任一项所述的方法，在癌症通过用所述治疗性T细胞治疗而完全缓解之后，施用表达所述癌症相关抗原的细胞。
  25. 权利要求19-24中任一项的方法，其中所述表达癌症相关抗原的细胞以刺激有效量施用，例如施用约10 3至约10 6个所述表达癌症相关抗原的细胞。
  26. 权利要求19-25中任一项的方法，所述治疗性T细胞是包含外源T细胞受体(TCR)的T细胞(TCR-T细胞)或包含嵌合抗原受体(CAR)的T细胞(CAR-T细胞)。
  27. 权利要求26的方法，所述外源TCR特异性结合癌症相关抗原，所述CAR包含针对癌症相关抗原的细胞外抗原结合结构域。
  28. 权利要求26或27的方法，所述CAR包括特异性结合癌症相关抗原的细胞外抗原结合结构域如scFv、CD8α铰链和跨膜结构域、CD3ζ信号转导结构域、和4-1BB共刺激结构域。
  29. 权利要求19-28中任一项的方法，所述表达癌症相关抗原的细胞是活细胞，优选地，所述细胞不是癌症细胞。
  30. 权利要求29的方法，所述癌症相关抗原表达于所述活细胞的表面。
  31. 权利要求19-30中任一项的方法，所述表达癌症相关抗原的细胞衍生自免疫细胞。
  32. 权利要求19-31中任一项的方法，所述治疗性T细胞和/或所述表达癌症相关抗原的细胞衍生自对象的自体细胞。
  33. 权利要求19-31中任一项的方法，所述治疗性T细胞和/或所述表达癌症相关抗原的细胞衍生自异体细胞，例如源自与所述对象HLA相容的供体。
  34. 一种用于在对象中治疗癌症的组合，其包含特异性靶向癌症相关抗原的治疗性T细胞和表达所述癌症相关抗原的细胞。
  35. 权利要求34的组合，其用于通过权利要求1-18中任一项的方法在对象中治疗癌症。
  36. 权利要求34或35的组合，其包含治疗有效量的所述治疗性T细胞和刺激有效量的所述表达癌症相关抗原的细胞。
  37. 权利要求34-36中任一项的组合在制备用于在对象中治疗癌症的药物中的用途，例如，所述药物用于根据权利要求1-18中任一项的方法在对 象中治疗癌症。
  38. 表达癌症相关抗原的细胞在制备用于在对象中治疗癌症的药物中的用途，任选地，所述药物还包含特异性靶向所述癌症相关抗原的治疗性T细胞。
  39. 表达癌症相关抗原的细胞在制备用于在对象中预防癌症进展或复发的药物中的用途，其中所述对象已经经历使用特异性靶向所述癌症相关抗原的治疗性T细胞的癌症治疗。
  40. 一种药盒，其包含特异性靶向癌症相关抗原的治疗性T细胞和/或表达所述癌症相关抗原的细胞，所述药盒用于通过权利要求1-18中任一项的方法在对象中治疗癌症或通过权利要求19-33中任一项的方法在对象中预防癌症进展或复发。
  41. 前述权利要求中任一项的方法、组合、用途、药物或药盒，其中所述癌症相关抗原选自CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和 FCRL5(UNIPROT Q68SN8)，优选地，所述癌症相关抗原是CD19。
  42. 前述权利要求中任一项的方法、组合、用途、药物或药盒，其中所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤、骨肉瘤、骨癌、胰腺癌、皮肤癌、前列腺癌、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合，优选地，所述癌症是B细胞急性淋巴细胞白血病(B-ALL)。
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