WO2019062817A1 - 可诱导分泌抗cd47抗体的工程化免疫细胞 - Google Patents

可诱导分泌抗cd47抗体的工程化免疫细胞 Download PDF

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WO2019062817A1
WO2019062817A1 PCT/CN2018/108022 CN2018108022W WO2019062817A1 WO 2019062817 A1 WO2019062817 A1 WO 2019062817A1 CN 2018108022 W CN2018108022 W CN 2018108022W WO 2019062817 A1 WO2019062817 A1 WO 2019062817A1
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cells
car
cell
antibody
tumor
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PCT/CN2018/108022
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French (fr)
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张永亮
刘丽萍
曹卫
马琳
马安云
何佳平
沈连军
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亘喜生物科技（上海）有限公司
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Priority to US16/651,867 priority Critical patent/US20200255803A1/en
Priority to CN201880062110.7A priority patent/CN111133101B/zh
Priority to EP18860318.7A priority patent/EP3690033A4/en
Publication of WO2019062817A1 publication Critical patent/WO2019062817A1/zh

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Definitions

  • the invention belongs to the field of tumor immune cell therapy, and in particular to an engineered immune cell capable of inducing secretion of an anti-CD47 antibody.
  • Cellular immunotherapy is an emerging and highly effective tumor treatment model, and is a new type of treatment for autoimmune and anti-cancer. It is a method for in vitro culture and amplification of immune cells collected from a patient using biotechnology and biological agents to be returned to a patient to stimulate and enhance the body's own immune function, thereby achieving the purpose of treating tumors.
  • CAR-T chimeric antigen receptor gene-modified T cells
  • the design of CAR has undergone the following process:
  • the first generation of CAR has only one intracellular signaling component, CD3 ⁇ or Fc ⁇ RI molecules, which can only cause transient T cell proliferation and less cytokine secretion due to only one activation domain in the cell.
  • CD3 ⁇ or Fc ⁇ RI molecules which can only cause transient T cell proliferation and less cytokine secretion due to only one activation domain in the cell.
  • it does not provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical results.
  • the second generation of CAR introduced a costimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, ICOS, which has greatly improved function compared with the first generation of CAR, further enhancing the persistence of CAR-T cells and tumor cells. The ability to kill.
  • a costimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, ICOS, which has greatly improved function compared with the first generation of CAR, further enhancing the persistence of CAR-T cells and tumor cells. The ability to kill.
  • some new immune co-stimulatory molecules such as CD27 and CD134 were connected in series, and developed into three generations and four generations of CAR.
  • the second-generation CAR is now the most widely used in clinical trials of hematological tumors.
  • CAR-T cells have shown unprecedented efficacy in the treatment of hematological malignancies, such as complete remission (CR) of 90% for advanced relapsed refractory acute lymphoblastic leukemia (ALL), for chronic lymphoblastic leukemia (CR of CLL) and some B-cell lymphomas reached more than 50%.
  • CAR-T has great potential for the treatment of leukemia and lymphoma, it is not effective in treating many solid tumors and some hematomas.
  • CAR-T cell therapy still has problems such as off-target effect, toxic side effects, short duration in vivo, and high recurrence rate in the treatment of hematological tumors.
  • the safety and efficacy of CAR-T cells in the treatment of solid tumors has been confirmed, but the efficacy needs to be improved.
  • CD47 is a potential target for the treatment of tumors, and current research has focused on the use of antibodies targeting CD47 for tumor therapy.
  • CD47 is ubiquitously expressed in normal tissues, systemic infusion of antibodies can cause many off-target toxic side effects, such as anemia and neurotoxicity. Therefore, antibodies targeting CD47 are less used to treat tumors expressing CD47.
  • an engineered immune cell e.g., a CAR-T cell
  • an engineered immune cell e.g., CAR-T cell
  • an engineered immune cell is provided, the engineered immune cell being a T cell or an NK cell, and the immune cell cell has the following characteristics:
  • said immune cell expresses a chimeric antigen receptor CAR or a foreign TCR, said CAR targeting a marker of a tumor cell, said exogenous TCR targeting a marker of a tumor cell;
  • the immune cell induces secretion of an anti-CD47 antibody when the CAR is activated and/or the exogenous TCR is activated.
  • the engineered immune cells are selected from the group consisting of:
  • CAR-T cells chimeric antigen receptor T cells
  • CAR-NK cell a chimeric antigen receptor NK cell
  • TCR Exogenous T cell receptor (TCR) T cells
  • a chimeric antigen receptor T cell (CAR-T cell) is provided, the CAR-T cell having the following characteristics:
  • said cell expresses a chimeric antigen receptor CAR, said CAR targeting a marker of a tumor cell;
  • the anti-CD47 antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the anti-CD47 antibody is a partially or fully humanized antibody.
  • the anti-CD47 antibody is in a single-stranded or double-stranded form.
  • the anti-CD47 antibody comprises a plurality of (2, 3, or 4) single chain antibodies in tandem.
  • a linker peptide La between single-chain antibodies between two adjacent single-chain antibodies is provided.
  • the linker peptide La has a length of 5 to 25, preferably 10 to 20 amino acids.
  • the linker peptide is flexible.
  • the "activation" refers to the binding of the CAR or exogenous TCR to a marker of a tumor cell.
  • the "marker of a tumor” refers to a specific antigen of a tumor.
  • the chimeric antigen receptor CAR or exogenous TCR is localized to the cell membrane of the engineered immune cell.
  • the chimeric antigen receptor CAR is localized to the cell membrane of the CAR-T cell.
  • the structure of the CAR is as shown in Formula I:
  • L1 is a no or signal peptide sequence
  • scFv is an antigen binding domain
  • H1 is a no or hinge zone
  • TM is a transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • the L1 is selected from the group consisting of a signal peptide of a protein of the group: CD8, GM-CSF, CD4, CD137, or a combination thereof.
  • the sequence of L is as shown in positions 1-22 of SEQ ID NO.: 1.
  • the scFv is an antibody single chain variable region sequence that targets a tumor antigen.
  • the scFv is an antibody single-chain variable region sequence that is selected from the group consisting of: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, mesothelin (MSLN) ), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands, LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin 18.2, or a combination thereof.
  • the scFv is an antibody single chain variable region sequence that targets CD19.
  • the scFv is FMC63 and the sequence is shown in positions 23-270 of SEQ ID NO.: 1.
  • the scFv is an antibody single chain variable region sequence of a targeting or MSLN.
  • the scFv is P4 and the sequence is as shown in positions 22-279 of SEQ ID NO.: 5.
  • the H is selected from the hinge region of the lower panel of proteins: CD8, CD28, CD137, or a combination thereof.
  • said H1 is a hinge region derived from CD28, and preferably the sequence of H1 is as shown at positions 271-309 of SEQ ID NO.: 1.
  • the TM is selected from the group consisting of transmembrane regions of proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 , CD 137, CD 154, or a combination thereof.
  • the TM is a CD28-derived transmembrane region, preferably the sequence of TM is as shown at positions 310-336 of SEQ ID NO.: 1.
  • the C is selected from the group consisting of a costimulatory signal molecule of the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10 , CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, or a combination thereof.
  • a costimulatory signal molecule of the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10 , CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, or a combination thereof.
  • C is a CD28-derived costimulatory signal molecule, preferably the sequence of C is as shown in positions 337-377 of SEQ ID NO.: 1.
  • sequence of CD3 ⁇ is as shown in positions 378-489 of SEQ ID NO.: 1.
  • the CAR targets CD19 and the structure is L-FMC63-CD28-CD3 ⁇ .
  • the CAR targets MSLN and has the structure L-P4-CD28-CD3 ⁇ .
  • sequence of the CAR is as shown in SEQ ID NO.: 1 or 5.
  • the anti-CD47 antibody is an anti-CD47 scFv.
  • the structure of the anti-CD47 scFv is as shown in Formula II:
  • L2 is a no- or signal peptide sequence
  • VH is a heavy chain variable region of an anti-CD47 antibody
  • X is no or a linker peptide
  • VL is a light chain variable region of an anti-CD47 antibody
  • H2 is a hinge region free of immunoglobulin
  • G is a no or Fc fragment.
  • the L2 is selected from the group consisting of a signal peptide of a protein of the group: CD8, GM-CSF, CD4, CD137, or a combination thereof.
  • the sequence of L2 is shown as position 1-21. of SEQ ID NO.: 2.
  • sequence of the VH is as shown in positions 22-139 of SEQ ID NO.: 2.
  • sequence of VL is as shown at positions 155-261 of SEQ ID NO.: 2.
  • the X has a length of from 2 to 50, preferably from 3 to 30 amino acids.
  • the X is (G4S) N and N is a positive integer from 1-8.
  • the X is (G4S) 3 .
  • sequence of X is as shown at positions 140-154 of SEQ ID NO.: 2.
  • the H2 is selected from the hinge region of a protein of the lower group: IgG1, IgG2, IgG3, IgG4, or a combination thereof.
  • the H2 is selected from the group consisting of IgG1.
  • amino acid sequence of the anti-CD47 scFv is set forth in SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6.
  • step (B) comprising (B1) introducing a first expression cassette expressing the CAR or exogenous TCR into the immune cell; and (B2) inducing secretion of an anti-CD47 antibody.
  • a second expression cassette is introduced into the immune cell; wherein said step (B1) can be performed before, after, simultaneously, or alternately with step (B2).
  • step (B) comprising (B1) introducing a first expression cassette expressing the CAR into the T cell; and (B2) a second expression cassette inducing secretion of an anti-CD47 antibody Introducing the T cells; wherein the step (B1) can be performed before, after, simultaneously with, or alternately with the step (B2).
  • the first expression cassette contains a nucleic acid sequence encoding the chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the second expression cassette has the structure of formula III from 5'-3':
  • Each "-" is independently a bond or nucleotide linkage sequence
  • Z1 is an inducible promoter
  • Z2 is a nucleic acid sequence encoding an anti-CD47 antibody.
  • said Z1 is a NFAT-inducible promoter, preferably a NFAT-IL2 mixed promoter.
  • said Z1 contains 4, 5 or 6 NFAT binding domains and an IL-2 promoter (preferably a fragment of the IL-2 minimal promoter) in this order from 5'-3'.
  • sequence of Z1 is as shown in positions 1-297 of SEQ ID NO.: 3.
  • sequence of Z2 is shown as positions 361-1080 of SEQ ID NO.: 3.
  • sequence of the second expression cassette is as shown in SEQ ID NO.: 3.
  • step (B) when the T cell to be engineered in step (A) has expressed a certain CAR, then in step (B), the (B2) second expression cassette is introduced into the T cell.
  • the transcription directions of the first expression cassette and the second expression cassette are the same ( ⁇ ), opposite ( ⁇ ), and opposite ( ⁇ ).
  • first expression cassette and the second expression cassette are located on the same or different vectors.
  • first expression cassette and the second expression cassette are located on the same carrier.
  • the vector is a viral vector.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.
  • the vector is a FUW lentiviral vector.
  • a formulation comprising the engineered immune cells of the first aspect of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.
  • a formulation comprising the CAR-T cell of the first aspect of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the dosage form of the formulation includes an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 - 1 ⁇ 10 8 cells / ml, preferably 1 ⁇ 10 4 - 1 ⁇ 10 7 cells / ml. .
  • an engineered immune cell according to the first aspect of the invention for the manufacture of a medicament or formulation for the prevention and/or treatment of cancer or a tumor.
  • a CAR-T cell according to the first aspect of the invention for the preparation of a medicament or preparation for preventing and/or treating cancer or a tumor.
  • the tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.
  • the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • DLBCL diffuse large B cell lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer, peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, or a combination thereof.
  • gastric cancer gastric cancer
  • peritoneal metastasis liver cancer
  • leukemia kidney tumor
  • lung cancer small intestine cancer
  • bone cancer prostate cancer
  • colorectal cancer breast cancer
  • colorectal cancer Cervical cancer
  • ovarian cancer lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, or a combination thereof.
  • NSCLC non-small cell lung cancer
  • the tumor is a tumor with high expression of CD47.
  • the tumor is selected from the group consisting of B cell lymphoma, non-Hodgkin's lymphoma, ovarian cancer, or a combination thereof.
  • kits for the preparation of the engineered immune cells of the first aspect of the invention comprising a container, and the container:
  • a first nucleic acid sequence comprising a first expression cassette for expressing the CAR or an exogenous TCR;
  • kits for the preparation of the CAR-T cell of the first aspect of the invention comprising a container, and located within the container:
  • a first nucleic acid sequence comprising a first expression cassette for expressing the CAR
  • first and second nucleic acid sequences are independent or linked.
  • first and second nucleic acid sequences are located in the same or different containers.
  • first and second nucleic acid sequences are on the same or different vectors.
  • first and second nucleic acid sequences are located on the same vector.
  • the vector is a viral vector, preferably the viral vector comprises first and second nucleic acid sequences in tandem.
  • Figure 1 is a schematic view showing the structure of CAR in Example 1, wherein A is the structure of CAR which targets CD19, and B is the structure of CAR which targets MSLN.
  • L is a signal peptide.
  • FIG. 2 is a schematic view showing the structure of an expression cassette which can induce secretion of aCD47scFv in Example 1.
  • IL-2 TATA is the IL-2 mini promoter and HA is a tag.
  • Figure 3 is a schematic view showing the structure of an expression cassette which can induce secretion of aCD47scFv-FC in Example 1.
  • Figure 4 shows the effective killing of target cells by MSLN CAR-T.
  • A is the expression of MSLN CAR
  • B is the target cell NCI-H226 with high expression of MSLN antigen
  • C is the RTCA killing experiment
  • MSLN CAR-T effectively kills the target cells.
  • NCI-H226;D secretes a large amount of IFN- ⁇ after activation of MSLN CAR-T cell antigen.
  • Figure 5 shows the expression supernatant assay of anti-CD47scFV single chain antibody/anti-CD47 scFV-FC antibody.
  • A is a schematic diagram of the vector gene expression cassette; B is the expression of anti-CD47scFV single chain antibody in 293T cells; C is the expression of anti-CD47scFV-FC antibody in Jurkat T cells.
  • EF-1 ⁇ is a constitutive promoter.
  • FIG. 6 shows the induction of downstream gene expression by MSLN CAR-T binding to antigen.
  • A is a schematic diagram of the structure of induced expression of CAR gene;
  • B Jurkat T cells electroporate to A expression vector, stably express MSLN CAR, bind to high expression MSLN antigen K562 cells, C, induce expression of secreted luciferase;
  • D peripheral blood separation T After the cell virus infection expression vector is packaged with the virus, the MSLN CAR is stably expressed, and binds to the high expression MSLN antigen K562 cell, and E, induces expression of secreted luciferase.
  • Figure 7 is a schematic diagram showing the expression of the iCD47scFV secreting gene expression induced by MSLN CAR-T antigen activation.
  • Figure 8 shows that anti-CD47scFV single-chain antibody promotes phagocytosis of tumor cells by bone marrow-derived macrophages.
  • A, B is a phagocytic flow cytological analysis and statistical analysis of Nalm6 by bone marrow-derived macrophages.
  • aCD47scFV supernatant group Compared with the control group (**P ⁇ 0.01);
  • C D bone marrow-derived macrophages phagocytosis of K562, flow cytological analysis and statistical analysis aCD47scFV supernatant group compared with the control group (*P ⁇ 0.05) .
  • Figure 9 shows that anti-CD47 scFV single chain antibody synergistically promotes killing of tumor K562 by macrophages, MSLN CAR-T.
  • the engineered immune cells of the present invention are representatively described in detail by taking CAR-T cells as an example.
  • the engineered immune cells of the present invention are not limited to CAR-T cells as described above and below, and the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described above and below.
  • NK cells are equivalent to T cells (or T cells can replace NK cells);
  • TCR is equivalent to CAR (or CAR can be replaced with TCR) ).
  • the present inventors have extensively and intensively studied, and after a large number of screenings, the CAR and anti-CD47 antibodies were first combined, and a CAR-T cell capable of inducing secretion of an anti-CD47 antibody was unexpectedly found.
  • the present invention can kill CD47-positive tumor cells by using anti-CD47 antibody without causing toxic side effects.
  • the CAR-T cells of the present invention initiate transcriptional translation of an anti-CD47 antibody only upon activation of the CAR to achieve a function of specific secretion only in the tumor microenvironment, and do not secrete CD47 antibodies in normal tissues or blood, thereby enabling Avoid systemic off-target toxic side effects and do not interfere with normal tissues in the body.
  • the CAR-T cells of the invention can induce the secretion of anti-CD47 antibody, relieve the inhibition of macrophages by CD47-positive tumor cells, reversely promote macrophages to attack tumor cells, and synergize with CAR to better exert anti-tumor effect.
  • the killing effect of tumor cells is significantly enhanced, and it can simultaneously kill tumor cells expressing CD-targeted antigens and CD47-positive tumor cells, preventing immune escape of tumor cells, and being difficult to off target and relapse.
  • the present invention has been completed on this basis.
  • administering refers to physically introducing a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or Other routes of parenteral administration, for example by injection or infusion.
  • the term "antibody” shall include, but is not limited to, an immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. , or an antigen binding portion thereof.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three constant domains CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region comprises a constant domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • antigen binding domain refers to a Fab fragment, Fab' fragment, F(ab') 2 fragment, or a single Fv fragment having antigen binding activity.
  • the Fv antibody contains the antibody heavy chain variable region, the light chain variable region, but no constant region, and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the antigen binding domain is typically a scFv (single-chain variable fragment).
  • the single chain antibody is preferably a sequence of one amino acid strand encoded by one nucleotide chain.
  • the scFv comprises an antibody that specifically recognizes an antigen highly expressed by the tumor, preferably a single chain antibody or an Fv antibody.
  • the anti-CD47 antibody is an scFv antibody that targets CD47.
  • anti-CD47 scFV "CD47 scFV” and “anti-CD47 antibody” are used interchangeably and are scFvs targeting CD47, including aCD47 scFV, aCD47 scFV-FC and the like.
  • sequence of the anti-CD47 antibody is set forth in SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6.
  • amino acid sequence of the aCD47 scFV is set forth in SEQ ID NO.: 2.
  • amino acid sequence of the aCD47 scFV-FC is set forth in SEQ ID NO.: 4.
  • the anti-CD47 antibody is a humanized antibody, and the amino acid sequence is set forth in SEQ ID NO.: 6.
  • a chimeric antigen receptor includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes an optional signal peptide and a target-specific binding element (also referred to as an antigen binding domain).
  • the intracellular domain includes a costimulatory molecule and a purine chain portion. When CAR is expressed in T cells, the extracellular domain recognizes a specific antigen and then transduces the signal through the intracellular domain, causing activation and proliferation of cells, cytolysis toxicity, and secretion of cytokines such as IL-2 and IFN- ⁇ .
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a sputum chain.
  • the antigen binding domain is fused to an intracellular domain that is combined with a CD28 signaling domain and a CD3 ⁇ signal domain.
  • the CAR of the invention targets CD19 and specifically binds to CD19.
  • the structure of the CAR of the present invention is L-FMC63-CD28-CD3 ⁇ .
  • the sequence of the CAR of the present invention is as shown in SEQ ID NO.: 1.
  • the CAR of the invention targets MSLN and is capable of specifically binding to MSLN.
  • the structure of the CAR that targets MSLN is L-P4-CD28-CD3 ⁇ , preferably, the amino acid sequence of the CAR is as shown in SEQ ID NO.: 5.
  • TCR Exogenous T cell receptor
  • an exogenous T cell receptor is a gene transfer technique for cloning the alpha and beta chains of TCR from tumor-reactive T cells by genetic engineering, lentivirus or The retrovirus is a vector and is exogenously transferred into the TCR in T cells.
  • Exogenous TCR-modified T cells can specifically recognize and kill tumor cells, and by optimizing the affinity of TCR with tumor-specific antigens, the affinity of T cells to tumors can be improved, and the anti-tumor effect can be improved.
  • CAR-T cell As used herein, the terms “CAR-T cell”, “CAR-T”, “CAR-T cell of the invention” all refer to the CAR-T cell of the first aspect of the invention.
  • the CAR-T cells of the present invention are useful for treating tumors with high expression of CD47, such as B cell lymphoma, non-Hodgkin's lymphoma, ovarian cancer and the like.
  • CAR-T cells have the following advantages over other T cell-based treatments: (1) the process of CAR-T cells is not restricted by MHC; (2) in view of the fact that many tumor cells express the same tumor antigen, targeting a certain tumor Once the CAR gene construction of the antigen is completed, it can be widely used; (3) CAR can use the tumor protein antigen and the glycolipid non-protein antigen to expand the target range of the tumor antigen; (4) use the patient's own body Cells reduce the risk of rejection; (5) CAR-T cells have an immunological memory function and can survive in vivo for a long time.
  • CAR-NK cells Chimeric antigen receptor NK cells
  • CAR-NK cell As used herein, the terms “CAR-NK cell”, “CAR-NK”, “CAR-NK cell of the invention” all refer to the CAR-NK cell of the first aspect of the invention.
  • the CAR-NK cells of the present invention can be used for the treatment of tumors with high expression of CD47, such as B cell lymphoma, non-Hodgkin's lymphoma, ovarian cancer and the like.
  • Natural killer (NK) cells are a major class of immune effector cells that protect the body from viral infections and tumor cells by non-antigen-specific pathways. New functions may be acquired by engineering (genetically modified) NK cells, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxic effects.
  • CAR-NK cells Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) direct killing of tumor cells by releasing perforin and granzyme, but no killing effect on normal cells of the body; (2) they release A very small number of cytokines reduce the risk of cytokine storms; (3) very easy to expand and develop into "off the shelf” products in vitro. In addition, it is similar to CAR-T cell therapy.
  • CD47 is a member of the Ig superfamily, consisting of an extracellular amino-terminal Ig-like variable domain (ligand binding region), five hydrophobic transmembrane segments, and a carboxy-terminal intracellular tail region.
  • CD47 is widely expressed on the surface of different tissue cells, such as hematopoietic cells (erythrocytes, lymphocytes, platelets, etc.), non-hematopoietic cells (placenta, liver, brain cells, etc.) and tumor cells.
  • CD47 is highly expressed in leukemia stem cells, such as AML, chronic myeloid leukemia, and T-cell acute lymphoblastic leukemia.
  • CD47 expression is found in various tumor tissues, including multiple myeloma, bladder cancer, rectal cancer, Melanoma and the like. Although normal tissues have CD47 expression, the expression level is significantly lower than that of tumor tissues.
  • CD47 is highly expressed in many tumor cells, and CD47 is highly expressed in tumor cells to escape macrophage phagocytosis. As a self-signal, CD47 evades the phagocytosis of macrophages by the expression of anti-phagocytic signals. In lymphocytes, CD47 binds to its specific ligand SIRP ⁇ to form a CD7-SIRP ⁇ signal complex, which can emit anti-phagocytic signals, inhibit phagocytosis of phagocytic cells, cause loopholes in the immune system, and promote tumor development.
  • CD47 in peripheral blood and germinal center-like B cells of patients with B-cell lymphoma were significantly higher than those of normal B cells.
  • different pathological histological types of non-Hodgkin's lymphoma (NHL) CD47 expression such as diffuse large B-cell lymphoma (DLBCL), follicular cell lymphoma (FL), marginal area Lymphoma (MZL), mantle cell lymphoma (FCL), etc.
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular cell lymphoma
  • MZL marginal area Lymphoma
  • FCL mantle cell lymphoma
  • CD47 is a potential target for the treatment of tumors.
  • the current research focuses on the use of antibodies targeting CD47 for tumor therapy.
  • CD47 antibody therapy exerts tumor killing effects through DC cells and CD8+T.
  • DC cells cooperate with CD47 antibody and pro-phagocytic molecules to phagocytose tumor cells and present tumor-associated antigens to CD8+T, thereby exerting specific killing effect of CD8+T on tumors.
  • CD47 is ubiquitously expressed in normal tissues, systemic infusion of antibodies can cause many off-target toxic side effects, such as anemia and neurotoxicity.
  • the inventors developed a chimeric antigen receptor T cell that is induced to express CD47 scFV when specifically activated by tumor antigens, especially for solid tumors, which can directly deliver CD47 antibodies to the tumor microenvironment.
  • the tumor cells are inhibited from inhibiting macrophages, thereby exerting the phagocytosis of macrophages, thereby achieving an anti-tumor effect.
  • NFAT nuclear factor of activated T cells
  • NFAT Activated T cell nuclear factor
  • the inventors designed an expression vector in which the promoter region contains 4, 5 or 6 regions capable of binding to NFAT, followed by a fragment of the IL-2 minimal promoter, and the antibody sequence of CD47 was placed.
  • the promoter region contains 4, 5 or 6 regions capable of binding to NFAT, followed by a fragment of the IL-2 minimal promoter, and the antibody sequence of CD47 was placed.
  • the anti-CD47 antibody is not secreted, and only after activation by the tumor-specific antigen will dephosphorylation of NFAT be activated, and CD47 is regulated after entry into the nucleus.
  • the secretion of the antibody reaches the specific secretion of anti-CD47 antibody in the tumor microenvironment, thereby relieving the inhibition of tumor cells on macrophages, exerting anti-tumor activity, and avoiding systemic off-target toxicity.
  • RTCA Real Time Cellular Analysis
  • RTCA technology is based on the principle of electrical impedance, which is used to detect the biological properties of adherent cells. For the suspended cells added to the well, it does not cause contact with the bottom electrode of the test plate or weak contact with myopia, so it does not cause electrical impedance changes.
  • an "expression cassette” or “expression cassette of the invention” includes a first expression cassette and a second expression cassette.
  • Expression cassette of the invention As described in the fifth aspect of the invention, the first expression cassette comprises a nucleic acid sequence encoding the CAR.
  • the second expression cassette has the structure of formula A from 5'-3'.
  • the CAR When the CAR is activated by a tumor-specific antigen, the second expression cassette expresses an anti-CD47 antibody; when the CAR-T cell of the invention is in a resting state, when the CAR does not bind to the specific antigen, the second expression cassette does not express an anti-expression CD47 antibody.
  • the first expression cassette and the second expression cassette further comprise a promoter and/or a terminator, respectively, wherein the promoter of the second expression cassette is an inducible promoter, preferably an NFAT-inducible promoter More preferably, a fragment containing 4, 5 or 6 NFAT binding domains and an IL-2 minimal promoter.
  • the invention also provides a vector comprising an expression cassette of the invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells.
  • Lentiviral vectors have the advantage over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • the expression cassette or nucleic acid sequence of the invention is operably linked to a promoter and incorporated into an expression vector.
  • This vector is suitable for replication and integration of eukaryotic cells.
  • a typical cloning vector comprises a transcriptional and translational terminator, an initial sequence and a promoter that can be used to modulate expression of a desired nucleic acid sequence.
  • the expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5, 580, 859, 5, 589, 466, incorporated herein by reference.
  • the expression cassette or nucleic acid sequence can be cloned into many types of vectors.
  • the expression cassette or nucleic acid sequence can be cloned into such vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector comprises an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (eg, WO01/96584; WO01/29058; and the United States) Patent No. 6,326, 193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to a subject cell in vivo or ex vivo.
  • retroviral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • a lentiviral vector is used.
  • promoter elements can regulate the frequency of transcription initiation.
  • these are located in the 30-110 bp region upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between the promoter elements is often flexible to maintain the promoter function when the element is inverted or moved relative to the other.
  • tk thymidine kinase
  • the interval between promoter elements can be increased by 50 bp, and the activity begins to decrease.
  • a single element can function cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoter such as, but not limited to, actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention.
  • an inducible promoter provides a molecular switch capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or shutting down expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cell may also comprise either or both of a selectable marker gene or reporter gene to facilitate identification and selection of the expression cell from a population of cells sought to be transfected or infected by the viral vector.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • reporter genes were used to identify potentially transfected cells and to assess the functionality of regulatory sequences.
  • the reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and which encodes a polypeptide whose expression is clearly indicated by some readily detectable properties such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is determined at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (eg, Ui-Tei et al, 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and can be prepared using known techniques or commercially available.
  • a construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter.
  • Such a promoter region can be ligated to a reporter gene and used to assess the ability of the agent to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell by any method in the art, for example, a mammalian (e.g., human T cell), bacterial, yeast or insect cell.
  • a mammalian e.g., human T cell
  • bacterial e.g., bacterial
  • yeast e.g., insect
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors.
  • Viral vectors particularly retroviral vectors, have become the most widely used method of inserting genes into mammals, such as human cells.
  • Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids. Platinum.
  • An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane sac).
  • an exemplary delivery tool is a liposome. It is contemplated to use a lipid formulation to introduce the nucleic acid into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid.
  • the nucleic acid associated with the lipid can be encapsulated into the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached via a linker molecule associated with both the liposome and the oligonucleotide
  • a linker molecule associated with both the liposome and the oligonucleotide
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets that occur naturally in the cytoplasm and in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides a CAR-T cell comprising the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the formulation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 - 1 ⁇ 10 8 cells / ml, more preferably 1 ⁇ 10 4 - 1 ⁇ 10 7 cells / ml.
  • the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine ; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (for example, aluminum hydroxide); and a preservative.
  • the formulations of the invention are preferably formulated for intravenous administration.
  • the invention encompasses therapeutic applications of lentiviral vector (LV) transduced cells (e.g., T cells) comprising an expression cassette of the invention.
  • LV lentiviral vector
  • Transduced T cells can target tumor cell markers and antibodies that specifically secrete anti-CD47, synergistically activate macrophages, and simultaneously induce immune responses of T cells and macrophages, thereby significantly improving their killing efficiency against tumor cells. .
  • the invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the step of administering to the mammal a CAR-T cell of the invention.
  • the invention encompasses a type of cell therapy that separates patient autologous T cells (or heterologous donors), activates and genetically engineers to produce CAR-T cells, which are then injected into the same patient.
  • a CAR-T can treat all cancers that express the antigen.
  • CAR-T cells replicate in vivo, producing long-lasting persistence that leads to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion for an extended amount of time.
  • the CAR-mediated immune response can be part of a step of adoptive immunotherapy in which CAR-modified T cells induce an immune response specific for the antigen binding domain in the CAR.
  • anti-CD19 CAR-T cells elicit a specific immune response against cells expressing CD19.
  • Anti-MSLN CAR-T cells elicit a specific immune response against cells expressing MSLN.
  • Treatable cancers include tumors that have not been vascularized or have not been vascularized, as well as vascularized tumors.
  • Cancer can include non-solid tumors (such as hematological tumors such as leukemias and lymphomas) or can include solid tumors.
  • Types of cancer treated with the CAR of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcoma, carcinoma, and melanoma. Also included are adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancer is a cancer of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocyte-monocyte type).
  • monocyte and erythroleukemia monocyte and erythroleukemia
  • chronic leukemia such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia
  • polycythemia vera lymphoma
  • Hodgkin's disease non Hodgkin's lymphoma (painless and high-grade forms)
  • multiple myeloma Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • a solid tumor is an abnormal mass of tissue that usually does not contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, mucinous sarcoma, liposarcoma mesothelioma, lymphoid malignancy, pancreatic cancer, ovarian cancer.
  • the CAR-T cells of the invention can also be used as a vaccine type for ex vivo immunity and/or in vivo therapy in mammals.
  • the mammal is a human.
  • cells are isolated from a mammal, preferably a human, and genetically modified (i.e., transduced or transfected in vitro) with a vector containing an expression cassette of the invention.
  • the CAR-T cells of the invention can be administered to a mammalian recipient to provide a therapeutic benefit.
  • Mammalian recipients can be human, and CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.
  • cells activated and expanded as described herein can be used to treat and prevent diseases produced in individuals without an immune response. Accordingly, the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
  • the CAR-T cells of the invention may be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a population of target cells as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition of the present invention can be administered in a form suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's condition - although appropriate dosages may be determined by clinical trials.
  • a pharmaceutical composition comprising a T cell as described herein may be at a dose of from 10 4 to 10 9 cells/kg body weight, preferably from 10 5 to 10 6 cells/kg body weight (including all integers in those ranges) Value) application. T cell compositions can also be administered multiple times in these doses.
  • Cells can be administered by using injection techniques well known in immunotherapy (see, eg, Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988).
  • Optimal dosages and treatment regimens for a particular patient can be readily determined by a person skilled in the medical arts by monitoring the patient's signs of disease and thus modulating the treatment.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.
  • a T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or infected sites.
  • cells activated and expanded using the methods described herein or other methods known in the art to extend T cells to therapeutic levels are combined with any number of related therapeutic modalities (eg, prior Administering to the patient, concurrently or afterwards, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies Or other immunotherapeutic agents.
  • the cell composition of the invention is administered to a bone marrow transplant, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • XRT external beam radiation therapy
  • cyclophosphamide eg, before, simultaneously or after
  • the subject may undergo standard treatment of high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an injection of the expanded immune cells of the invention after transplantation.
  • the expanded cells are administered prior to or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratios administered by a human can be carried out according to practices accepted in the art.
  • from 1 x 10 5 to 1 x 10 10 modified T cells of the invention can be administered to a patient by, for example, intravenous reinfusion for each treatment or each course of treatment.
  • the present invention can kill CD47-positive tumor cells by using an anti-CD47 antibody without causing side effects.
  • the CAR-T cells of the present invention initiate transcriptional translation of an anti-CD47 antibody only upon activation of the CAR to achieve a function of specific secretion only in the tumor microenvironment, and do not secrete CD47 antibodies in normal tissues or blood, thereby enabling Avoid systemic off-target toxic side effects, do not interfere with normal tissues in the body, and have low safety and side effects.
  • the CAR-T cells of the invention can induce the secretion of anti-CD47 antibodies, relieve the inhibition of macrophages by CD47-positive tumor cells, reversely promote macrophages to attack tumor cells, and synergize with CAR to better exert anti-tumor
  • the effect of the killing effect on the tumor cells is significantly enhanced, and can simultaneously kill the tumor cells expressing the CAR-targeted antigen and the CD47-positive tumor cells, preventing the immune escape of the tumor cells, and being difficult to off target and relapse.
  • CAR-T cells can directly deliver anti-CD47 antibodies to the tumor microenvironment, and release the inhibitory effect of tumor cells on macrophages, thereby exerting the phagocytosis of macrophages, thereby achieving anti-tumor effects.
  • the anti-CD47 antibody of the present invention has an Fc fragment, which can bind to an Fc receptor on the surface of an NK cell to activate NK cells, thereby exerting a killing action of NK cells, thereby achieving a better antitumor effect.
  • the Fc fragment also enhances the stability of the scFV of the invention.
  • the anti-CD47 antibody of the present invention also includes a humanized CD47 antibody, which has lower immunogenicity and less toxic side effects.
  • Mononuclear cells were isolated from cord blood, subjected to density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich), and enriched with T cells (EasySep human T cell enrichment kit, Stemcell Technologies) using coupled anti-CD3/anti-CD28
  • the magnetic beads activate culture and expansion of T cells; the medium uses X-vivo15 (containing 5% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 300 IU/ml rhIL2); all cells are placed at 37 ° C, 5 Culture in a %CO2 incubator.
  • Jurkat T cell human T lymphocyte leukemia cell line, TIB-152
  • Nalm6 cells human acute lymphocytic leukemia cell line, CRL-3273
  • Raji-ffluc cell line obtained after screening for Raji cells using lentivirus luciferase
  • K562-ffluc cells human erythroleukemia cell line, ATCC-CCL243;
  • 293T cells human renal epithelial cell line, ATCC-CRL3216;
  • K562 cells expressing CD19 and 293T cells were infected with a lentiviral vector expressing a CD19 molecule, and the resulting cell line was selected.
  • K562 cells expressing siRNA and 293T cells were infected with a lentiviral vector expressing a MSLN molecule, and the resulting cell line was selected.
  • Jurkat T, Nalm6, Raji cells, Raji-ffluc, K562, K562 cells expressing CD19, K562 cells expressing MSLN were cultured in RPMI1640 medium, 293T cells, CD19-expressing 293T cells, and MSLN-expressing 293T cells were cultured in DMEM. Base culture. All media were supplemented with 10% (v/v) fetal bovine serum and 100 U/ml of oxymycin and streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate. All cells were cultured at 37 ° C in a 5% CO 2 incubator.
  • Example 1 CAR structure and iCD47scFv structure design and transduction
  • CD19CAR structure using second-generation CD19 CARs, including scFv from FMC63, from the hinge and transmembrane region of CD28, the intracellular segment is CD28 and CD3 ⁇ , the structural schematic is shown in Figure 1A, and the amino acid sequence is as shown in SEQ ID NO.: Show.
  • a CD19-targeting CAR-T cell wherein the CAR-T cell stably expresses a CAR gene.
  • CAR is an artificially designed amino acid sequence comprising a signal peptide, a scFv, a hinge region, a transmembrane region, and an intracellular signal region, which are ligated in sequence.
  • the vector in which the CAR gene is expressed may be DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon or other gene transfer system.
  • the CD19 CAR gene was cloned into the FUW lentiviral vector backbone, placed under the EF1 ⁇ promoter to form Fuw-EF1 ⁇ -CD19CAR, and the three plasmids Fuw-EF1 ⁇ -CD19CAR, pMD2.G and psPAX2 (addgene) were transferred using Lipofectamine3000.
  • the lentiviral expression vector was prepared in 293T; the viral supernatant was collected at 48 h and 72 h, and concentrated (Merck Millipore); the concentrated virus was used to infect T cells.
  • MSLNCAR structure using second-generation MSLN CARs, including scFv from P4, hinge and transmembrane region from CD28, intracellular segment is CD28 and CD3 ⁇ , the structural schematic is shown in Figure 1B, and the amino acid sequence is as shown in SEQ ID NO.: Shown, wherein scFV can underline the CAR-T scFV antigen recognition sequence that targets MSLN.
  • the present invention devises an expression cassette which can induce secretion of anti-CD47 scFv (signal peptide selected from CD8), and the structure is schematically shown in Figure 2 (aCD47 scFv) or Figure 3 (aCD47 scFv-FC), aCD47 scFv and aCD47 scFv-FC amino acids.
  • the sequences are shown in SEQ ID NO.: 2 and SEQ ID NO.: 4, respectively.
  • the nucleic acid sequence of the expression cassette NFAT-IL-2-aCD47 scFv which can induce secretion of aCD47scFv (signal peptide selected from CD8) is shown in SEQ ID NO.: 3.
  • An expression cassette for inducible secretion of aCD47scFv-FC (signal peptide selected from CD8) can be obtained by substituting the aCD47scFv coding sequence (positions 361 to 1080) of the sequence of SEQ ID NO.: 3 with the aCD47scFv-FC coding sequence.
  • the expression cassette of the anti-CD47 antibody fragment placed under the NFAT-IL-2 promoter was cloned into the FUW lentiviral vector backbone containing the CD19 CAR or MSLN CAR gene to form Fuw-EF1 ⁇ -CD19CAR-NFAT-IL-2-CD47scFv Or Fuw-EF1 ⁇ -MSLNCAR-NFAT-IL-2-CD47scFv, and the three plasmids of pMD2.G and psPAX2 (Addgene) were transferred into 293T cells using Lipofectamine3000 to prepare lentiviral expression vector; virus supernatant was collected at 48h and 72h. It is concentrated and concentrated (Merck Millipore); the concentrated virus can be used to infect T cells.
  • the isolated and purified primary T cells were infected with the lentiviral expression vector containing MSLN-CAR and MSLN-CAR-iCD47scFv after three days of activation, transferred to a cell culture flask, and placed in a 37 ° C, 5% CO 2 incubator. to cultivate.
  • the positive rate of CAR of T cells was measured using MSLN (Thermo Fisher Scientific, Thermo Fisher Scientific) on the 3rd and 7th day after infection, and half of the medium was replaced every 2-3 days.
  • the CAR-T cells obtained after the culture were MSLN CAR-T cells and iCD47scFv-MSLN CAR-T cells, respectively.
  • the experimental results of MSLN CAR-T cells are shown in Fig. 4A, and MSLN CAR-T cells can express MSLN CAR well.
  • iCD47scFv-MSLN CAR-T cells also expressed MSLN CAR well.
  • CD19 CAR-T cells and iCD47scFv-CD19 CAR-T cells were prepared according to the same experimental procedure as described above. As a result, CD19 CAR-T cells and iCD47scFv-CD19 CAR-T cells were well expressed for CD19 CAR, and CD19 CAR-T cells and iCD47scFv-CD19 CAR-T cells were successfully prepared.
  • RTCA Real-time unlabeled cell analysis
  • RTCA technology is based on the principle of electrical impedance, which is used to detect the biological properties of adherent cells. For the suspended cells added to the well, it does not cause contact with the bottom electrode of the test plate or weak contact with myopia, so it does not cause electrical impedance changes. Therefore, CAR-T cell-mediated killing of monolayer cancer cells can be directly monitored by RTCA technology.
  • MSLN-CAR MSLN-positive CAR-T cells
  • NT T cells
  • iCD47scFv-MSLN CAR-T cells can also rapidly and effectively kill MSLN positive tumor cells.
  • the experimental method was the same as above 3.1, in which MSLN CAR-T cells were replaced with CD19 CAR-T cells, and mesothelioma cells were replaced with CD19-positive tumor cells.
  • CD19 CAR-T cells can rapidly and effectively kill CD19-positive tumor cells compared with T cells (NT) which were not transfected with CAR structure in the control group.
  • the MSLN CAR-T cells/tumor cells NCI-H226 cells obtained in Example 3 were co-cultured for 42 hours, and after centrifugation, the release levels of cytokine IFN- ⁇ were measured and detected by Elisa kit (Biolegend).
  • CD19 CAR-T cells were replaced, and mesothelioma cells were replaced with CD19-positive tumor cells.
  • the release of IFN- ⁇ was significantly higher in the CD19 CAR-T cell group than in the control group.
  • the expression cassette (Fig. 5A) containing the constitutive promoter EF-1 ⁇ and aCD47 scFV (SEQ ID NO.: 2) or aCD47scFV-FC (SEQ ID NO.: 4) was cloned into p-fuw-EF -1 alpha lentiviral expression vector.
  • Example 1 According to the method of 1.3 in Example 1, three plasmids of pMD2.G and psPAX2 (Addgene) were transfected into 293T cells using Lipofectamine 3000 to prepare a lentiviral expression vector; virus supernatants were collected at 48 h and 72 h, and concentrated by super-ionization (Merck Millipore) The concentrated virus can be used to infect 293T cells to obtain a cell line stably expressing aCD47 scFV or aCD47 scFV-FC.
  • aCD47 scFV SEQ ID NO. 2 2
  • aCD47 scFV expression vector was well expressed in eukaryotic cell 293T and reached a high level of 494 ng/ml (Fig. 5B).
  • the vector expressing aCD47scFV-FC (SEQ ID NO.: 4) was introduced into Jurkat T cells by a Lonza electroporator. The cell supernatant was collected after 18 hours. Secretion of aCD47-scFV-FC was also detected in the cell supernatant by ELISA, and the expression abundance was 10.5 ng/ml (Fig. 5C).
  • the present invention hopes that CAR-T cells can directly deliver anti-CD47 antibodies to the tumor microenvironment, and the tumor cells can be inhibited from inhibiting macrophages, thereby exerting the phagocytic effect of macrophages.
  • the design of the present invention (Fig. 6A) after the CAR-T cells recognizing the MSLN reach the tumor site, the MSLN tumor antigen is bound, the downstream NFAT transcription factor is activated, and the antibody secretion program is started.
  • the lentiviral vector of Figure 6A was transfected into Jurkat T cells by electroporation, as shown in Figure 6B, and the transfected Jurkat T cells expressed MSLN CAR. 4 hours after transfection, 5 ⁇ 10 5 Jurkat T cells were plated in round-bottomed 96-well plates, while adding 2.5x10 5 K562 or K562 cells overexpressing MSLN, and Jurkat T cells co-cultured. At the same time, a separate medium was set as a negative control, and a T cell activator (PMA/Inomycin) was used as a positive stimulation control.
  • PMA/Inomycin T cell activator
  • MSLN CAR can induce the secretion and expression of downstream genes regulated by NFAT after binding to the specific antigen MSLN.
  • the procedure of 6.1 was repeated except that the luciferase gene was replaced with aCD47scFV (SEQ ID NO.: 2) or aCD47scFV-FC (SEQ ID NO.: 4) to thereby obtain a construct containing the structure shown in FIG. T cells; and the binding reaction of the CD47 antigen to the antibody is used to detect the presence or absence of an antibody against CD47 in the supernatant.
  • aCD47scFV SEQ ID NO.: 2
  • aCD47scFV-FC SEQ ID NO.: 4
  • Example 7 aCD47 scFV secretory supernatant promotes macrophage phagocytosis
  • the lentivirus-infected 293T cell secretory supernatant (containing aCD47 scFV) was added to the macrophage/tumor cell co-culture system, and then the giant was detected.
  • Bone marrow-derived macrophage culture The femur of C57BL/6 mice was taken, the ends were cut with scissors, and the syringe was inserted into the bone marrow of the femur from one end of the femur. The bone marrow-derived cell mixture is collected, centrifuged, and red blood cell lysate is added to resuspend the red blood cells. After washing twice with PBS, the cells were resuspended in macrophage medium (10% fetal bovine serum DMEM medium + 20% M-CSF L929 cell culture supernatant) and plated in non-adherent cell culture 6-well plates ( 1 ⁇ 10 6 / hole). After 8 days of cell culture, the cells were differentiated into F4/80 staining-positive macrophages.
  • macrophage medium 10% fetal bovine serum DMEM medium + 20% M-CSF L929 cell culture supernatant
  • Tumor phagocytosis test Tulm target cells Nalm6, K562 cells were fluorescently labeled with CFSE (1uM), and then plated with spare bone marrow-derived macrophages after culture in a ratio of 1:1 (5 ⁇ 10 4 : 5 ⁇ 10 4 ) to 96 wells. Culture in the plate. After 4 hours of co-cultivation, the percentage of macrophages (F4/80+CFSE+) that phagocytized target cells was analyzed by flow cytometry.
  • Example 8 aCD47scFV promotes the synergistic tumoricidal effect of macrophages and MSLN-positive CAR-T
  • K562 cells stably transfected with firefly luciferase were overexpressed with MSLN antigen, and luciferase was used as an indicator of the activity of the target cells for killing experiments.
  • Luciferase-positive K562 cells were incubated with effector cells (MSLNCAR-T/macrophages) and aCD47scFV supernatant to study the synergistic killing effect of macrophages and CAR-T cells in the presence of aCD47.
  • the experimental design was as follows. The K562 target cells positive for MLSN antigen were subjected to different experimental treatments as follows:
  • the luciferase substrate was added by centrifugation to measure the number of viable cells. It was found that macrophages can synergistically promote the killing of target cell MSLN antigen-positive K562 cells by MSLN-positive CAR-T cells in the presence of aCD47 scFV cell supernatant (Fig. 9).
  • the present invention also investigated the effect of aCD47 scFV-FC in the same manner as aCD47 scFV, in which aCD47 scFV was replaced with aCD47 scFV-FC. As a result, it was found that the function and effect of aCD47 scFV-FC were almost the same as those of aCD47 scFV.
  • MSLN CAR-T cells and macrophage cells which can induce the secretion of aCD47scFv or aCD47scFV-FC prepared in Example 6 are co-cultured with the above luciferase-positive K562 cells, and the experimental method is the same as above.

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Abstract

提供了一种在CAR和/或外源TCR被激活时可诱导分泌抗CD47抗体的免疫细胞及其用途、含有所述免疫细胞的制剂，还提供了所述免疫细胞的制备方法以及用于所述制备方法的试剂盒。

Description

可诱导分泌抗CD47抗体的工程化免疫细胞 技术领域
本发明属于肿瘤免疫细胞治疗领域，具体地，涉及一种可诱导分泌抗CD47抗体的工程化免疫细胞。
背景技术
细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式，是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养和扩增后回输到病人体内的方法，来激发、增强机体自身免疫功能，从而达到治疗肿瘤的目的。
近年来，嵌合抗原受体基因修饰T(CAR-T)细胞作为“活的药物”在血液肿瘤治疗中取得了令人振奋的效果，成为肿瘤治疗新的发展方向。CAR的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效。第二代CAR在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CAR相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CAR基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CAR。现在血液肿瘤的临床试验中应用最多的是第二代CAR。
CAR-T细胞在血液系统恶性肿瘤的治疗中显示出前所未有的疗效，如对晚期复发难治性急性淋巴细胞白血病(ALL)治疗的完全缓解(CR)可达到90％，对慢性淋巴细白血病(CLL)和部分B细胞淋巴瘤的CR达到50％以上。虽然CAR-T在治疗白血病和淋巴瘤上潜力很大，但在治疗很多实体瘤和一些血液瘤上效果欠佳。目前CAR-T细胞疗法在血液肿瘤的治疗过程中尚存在脱靶效应、毒副作用、体内持续时间短、复发率高等问题。CAR-T细胞在治疗实体瘤方面，其安全性和有效性述已得到证实，但疗效还有待提高。
CD47是治疗肿瘤的一个潜在靶点，目前研究主要集中在使用靶向CD47的抗体来进行肿瘤治疗。但是，由于CD47普遍表达于正常组织中，全身性的输注抗体会带来很多的脱靶毒副作用，比如贫血、神经毒性等。因此较少使用靶向CD47的抗体来治疗表达CD47的肿瘤。
综上所述，本领域仍然需要进一步的研究，开发一种能更有效、特异性好、副作用小地治疗肿瘤的工程化免疫细胞。
发明内容
本发明的目的是提供一种能更有效、特异性好、副作用小地治疗肿瘤的工程化免疫细 胞(如CAR-T细胞)。
本发明的又一目的是提供一种可诱导分泌抗CD47抗体的工程化免疫细胞(如CAR-T细胞)及其制法和应用。
本发明的第一方面，提供了一种工程化的免疫细胞，所述工程化的免疫细胞为T细胞或NK细胞，并且所述的免疫细胞细胞具有以下特征：
(a)所述免疫细胞表达嵌合抗原受体CAR或外源TCR，所述CAR靶向肿瘤细胞的标志物，所述外源TCR靶向肿瘤细胞的标志物；和
(b)当所述CAR被激活和/或所述外源TCR被激活时，所述免疫细胞诱导分泌抗CD47抗体。
在另一优选例中，所述的工程化的免疫细胞选自下组：
(i)嵌合抗原受体T细胞(CAR-T细胞)；
(ii)嵌合抗原受体NK细胞(CAR-NK细胞)；或
(iii)外源T细胞受体(TCR)T细胞(TCR-T细胞)。
在另一优选例中，提供了一种嵌合抗原受体T细胞(CAR-T细胞)，所述CAR-T细胞具有以下特征：
(a)所述细胞表达嵌合抗原受体CAR，所述CAR靶向肿瘤细胞的标志物；和
(b)当所述CAR被激活时，所述CAR-T细胞诱导分泌抗CD47抗体。
在另一优选例中，所述抗CD47抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述抗CD47抗体是部分或全人源化的抗体。
在另一优选例中，所述的抗CD47抗体为单链或双链形式。
在另一优选例中，所述的抗CD47抗体包括串联的多个(2、3、或4个)单链抗体。
在另一优选例中，在串联的多个(2、3、或4个)单链抗体中，设有位于两个相邻的单链抗体之间单链抗体间的连接肽La。
在另一优选例中，所述连接肽La的长度为5-25，较佳地10-20个氨基酸。
在另一优选例中，所述的连接肽为柔性的。
在另一优选例中，所述“激活”指所述CAR或外源TCR与肿瘤细胞的标志物结合。
在另一优选例中，所述“肿瘤的标志物”指肿瘤的特异性抗原。
在另一优选例中，所述的嵌合抗原受体CAR或外源TCR定位于所述工程化免疫细胞的细胞膜。
在另一优选例中，所述的嵌合抗原受体CAR定位于所述CAR-T细胞的细胞膜。
在另一优选例中，所述CAR的结构如式I所示：
L1-scFv-H1-TM-C-CD3ζ (I)
式中，
L1为无或信号肽序列；
scFv为抗原结合结构域；
H1为无或铰链区；
TM为跨膜结构域；
C为共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列；
所述“-”连接肽或肽键。
在另一优选例中，所述L1分别选自下组的蛋白的信号肽：CD8、GM-CSF、CD4、CD137、或其组合。优选地L的序列如SEQ ID NO.:1的第1-22位所示。
在另一优选例中，所述scFv为靶向肿瘤抗原的抗体单链可变区序列。
在另一优选例中，所述scFv为靶向选自下组抗原的抗体单链可变区序列：CD19、CD20、CD22、CD123、CD47、CD138、CD33、CD30、间皮素(mesothelin,MSLN)、EGFR、GPC3、BCMA、ErbB2、NKG2D配体(ligands)、LMP1、EpCAM、VEGFR-1、Lewis-Y、ROR1、Claudin18.2、或其组合。
在另一优选例中，所述scFv为靶向CD19的抗体单链可变区序列。
在另一优选例中，所述scFv为FMC63，序列如SEQ ID NO.:1第23-270位所示。
在另一优选例中，所述scFv为靶向或MSLN的抗体单链可变区序列。
在另一优选例中，所述scFv为P4，序列如SEQ ID NO.:5中第22-279位所示。
在另一优选例中，所述H选自下组的蛋白的铰链区：CD8、CD28、CD137、或其组合。
在另一优选例中，所述的H1为CD28来源的铰链区，优选地H1的序列如SEQ ID NO.:1的第271-309位所示。
在另一优选例中，所述TM选自下组的蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述TM为CD28来源的跨膜区，优选地TM的序列如SEQ ID NO.:1的第310-336位所示。
在另一优选例中，所述C选自下组的蛋白的共刺激信号分子：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、或其组合。
在另一优选例中，C为CD28来源的共刺激信号分子，优选地C的序列如SEQ ID NO.:1的第337-377位所示。
在另一优选例中，CD3ζ的序列如SEQ ID NO.:1的第378-489位所示。
在另一优选例中，所述CAR靶向CD19，结构为L-FMC63-CD28-CD3ζ。
在另一优选例中，所述CAR靶向MSLN，结构为L-P4-CD28-CD3ζ。
在另一优选例中，所述CAR的序列如SEQ ID NO.:1或5所示。
在另一优选例中，所述抗CD47抗体为抗CD47 scFv。
在另一优选例中，所述抗CD47 scFv的结构如下式II所示：
L2-VH-X-VL-H2-G (II)
其中，
L2为无或信号肽序列；
VH为抗CD47抗体的重链可变区；
X为无或连接肽；
VL为抗CD47抗体的轻链可变区；
H2为无或免疫球蛋白的铰链区；
G为无或Fc片段。
在另一优选例中，所述L2分别选自下组的蛋白的信号肽：CD8、GM-CSF、CD4、CD137、或其组合。优选地L2的序列如SEQ ID NO.:2的第1-21位所示。
在另一优选例中，所述VH的序列如SEQ ID NO.:2的第22-139位所示。
在另一优选例中，所述VL的序列如SEQ ID NO.:2的第155-261位所示。
在另一优选例中，所述X的长度为2-50个，较佳地3-30个氨基酸。
在另一优选例中，所述X为(G4S) N，N为1-8的正整数。
在另一优选例中，所述X为(G4S) 3。
在另一优选例中，所述X的序列如SEQ ID NO.:2的第140-154位所示。
在另一优选例中，所述H2选自下组的蛋白的铰链区：IgG1、IgG2、IgG3、IgG4或其组合。
在另一优选例中，所述H2选自IgG1。
在另一优选例中，所述抗CD47 scFv的氨基酸序列如SEQ ID NO.:2、SEQ ID NO.:4或SEQ ID NO.:6所示。
本发明的第二方面，提供了一种制备本发明第一方面所述的工程化的免疫细胞的方法，包括以下步骤：
(A)提供一待改造的免疫细胞；和
(B)对所述的免疫细胞进行改造，从而使得所述的免疫细胞表达所述的CAR或外源TCR，且在所述CAR被激活和/或所述外源TCR被激活时，所述免疫细胞诱导分泌抗CD47抗体，从而获得本发明第一方面所述的工程化的免疫细胞。
在另一优选例中，在步骤(B)中，包括(B1)将表达所述CAR或外源TCR的第一表达盒导入所述免疫细胞；和(B2)将可诱导分泌抗CD47抗体的第二表达盒导入所述免疫细胞；其中所述的步骤(B1)可在步骤(B2)之前、之后、同时、或交替进行。
在另一优选例中，提供了一种制备本发明第一方面所述的CAR-T细胞的方法，包括以下步骤：
(A)提供一待改造的T细胞；和
(B)对所述的T细胞进行改造，从而使得所述的T细胞表达所述的CAR，且在所述CAR激活时分泌所述抗CD47抗体，从而获得本发明第一方面所述的CAR-T细胞。
在另一优选例中，在步骤(B)中，包括(B1)将表达所述CAR的第一表达盒导入所述T细胞；和(B2)将可诱导分泌抗CD47抗体的第二表达盒导入所述T细胞；其中所述的步骤(B1)可在步骤(B2)之前、之后、同时、或交替进行。
在另一优选例中，所述的第一表达盒含有编码所述的嵌合抗原受体(CAR)的核酸序列。
在另一优选例中，所述第二表达盒从5’-3’具有式III结构：
Z1-Z2 (III)
式中，
各“-”独立地为键或核苷酸连接序列；
Z1为诱导型启动子；
Z2为编码抗CD47抗体的核酸序列。
在另一优选例中，所述Z1为NFAT诱导型启动子，较佳地为NFAT-IL2混合启动子。
在另一优选例中，所述Z1从5’-3’依次含有4、5或6个NFAT结合域和IL-2启动子(优选IL-2最小启动子的片段)。
在另一优选例中，所述Z1的序列如SEQ ID NO.:3的第1-297位所示。
在另一优选例中，所述Z2的序列如SEQ ID NO.:3的第361-1080位所示。
在另一优选例中，所述第二表达盒的序列如SEQ ID NO.:3所示。
在另一优选例中，当步骤(A)中的待改造的T细胞已经表达某一CAR时，则在步骤(B)中，包括(B2)第二表达盒导入所述T细胞。
在另一优选例中，所述的第一表达盒和第二表达盒的转录方向是同向的(→→)、相向的(→←)、相背的(←→)。
在另一优选例中，所述的第一表达盒、第二表达盒位于相同或不同的载体上。
在另一优选例中，所述的第一表达盒、第二表达盒位于同一载体。
在另一优选例中，所述的载体为病毒载体。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、其他基因转移系统、或其组合。
在另一优选例中，所述的载体为FUW慢病毒载体。
本发明的第三方面，提供了一种制剂，所述制剂含有本发明第一方面所述的工程化的免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，提供了一种制剂，所述制剂含有本发明第一方面所述的CAR-T细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂的剂型包括注射剂。
在另一优选例中，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
本发明的第四方面，提供了如本发明第一方面所述的工程化的免疫细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，提供了如本发明第一方面所述的CAR-T细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、或其组合。
在另一优选例中，所述肿瘤为CD47高表达的肿瘤。
在另一优选例中，所述肿瘤为选自下组：B细胞淋巴瘤、非霍奇金淋巴瘤、卵巢癌、或其组合。
本发明的第五方面，提供了一种用于制备本发明第一方面所述的工程化的免疫细胞的试剂盒，所述试剂盒含有容器，以及位于容器内的：
(1)第一核酸序列，所述第一核酸序列含有用于表达所述CAR或外源TCR的第一表达盒；和
(2)第二核酸序列，所述第二核酸序列含有用于可诱导分泌抗CD47抗体的第二表达盒。
在另一优选例中，提供了一种用于制备本发明第一方面所述的CAR-T细胞的试剂盒，所述试剂盒含有容器，以及位于容器内的：
(1)第一核酸序列，所述第一核酸序列含有用于表达所述CAR的第一表达盒；和
(2)第二核酸序列，所述第二核酸序列含有用于可诱导分泌抗CD47抗体的第二表达盒。
在另一优选例中，所述的第一、第二核酸序列为独立的或相连的。
在另一优选例中，所述的第一、第二核酸序列位于相同或不同的容器内。
在另一优选例中，所述的第一、第二核酸序列位于相同或不同的载体上。
在另一优选例中，所述的第一、第二核酸序列位于同一载体。
在另一优选例中，所述的载体为病毒载体，较佳地所述病毒载体含有串联形式的第一和第二核酸序列。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了实施例1中CAR的结构示意图，其中A为靶向CD19的CAR的结构，B为靶向MSLN的CAR的结构。图中，L为信号肽。
图2显示了实施例1中可诱导aCD47scFv分泌的表达盒的结构示意图。其中IL-2 TATA为IL-2 mini启动子，HA为标签。
图3显示了实施例1中可诱导aCD47scFv-FC分泌的表达盒的结构示意图。
图4显示了MSLN CAR-T对靶细胞的有效杀伤其中，A为MSLN CAR的表达；B为高表达MSLN抗原的靶细胞NCI-H226；C为RTCA杀伤实验,MSLN CAR-T有效杀伤靶细胞NCI-H226；D为MSLN CAR-T细胞抗原激活后分泌大量IFN-γ。
图5显示了抗CD47scFV单链抗体/抗CD47scFV-FC抗体的表达上清检测。A为载体基因表达框示意图；B为抗CD47scFV单链抗体在293T细胞中的表达；C是抗CD47scFV-FC抗体在Jurkat T细胞中的表达。EF-1α为组成型启动子。
图6显示了MSLN CAR-T结合抗原后诱导下游基因的表达。A为诱导表达CAR基因结构示意图；B,Jurkat T细胞电转A表达载体后，稳定表达MSLN CAR，与高表达MSLN抗原K562细胞结合，C，诱导表达分泌型荧光素酶；D,外周血分离T细胞病毒感染表达载体包装病毒后，稳定表达MSLN CAR，与高表达MSLN抗原K562细胞结合，E，诱导表达分泌型荧光素酶。
图7显示了MSLN CAR-T抗原活化诱导iCD47scFV分泌基因表达框示意图。
图8显示了抗CD47scFV单链抗体可促进骨髓来源巨噬细胞对肿瘤细胞的吞噬作用其中，A，B为骨髓来源巨噬细胞对Nalm6的吞噬流式细胞学分析和统计学分析aCD47scFV上清组与对照组相比(**P<0.01)；C，D骨髓来源巨噬细胞对K562的吞噬,流式细胞学分析和统计学分析aCD47scFV上清组与对照组相比(*P<0.05)。
图9显示了抗CD47scFV单链抗体协同促进巨噬细胞，MSLN CAR-T对肿瘤K562的杀伤。
具体实施方式
本发明以CAR-T细胞为例，代表性地对本发明的工程化的免疫细胞进行详细说明。本发明的工程化的免疫细胞不限于上下文所述的CAR-T细胞，本发明的工程化的免疫细胞具 有与上下文所述的CAR-T细胞相同或类似的技术特征和有益效果。具体地，当免疫细胞表达嵌合抗原受体CAR时，NK细胞等同于T细胞(或T细胞可替换NK细胞)；当免疫细胞为T细胞时，TCR等同于CAR(或CAR可替换为TCR)。
本发明人经过广泛而深入地研究，经过大量的筛选，首次将CAR和抗CD47抗体结合，意外地发现一种可诱导分泌抗CD47抗体的CAR-T细胞。实验表明，本发明可以利用抗CD47抗体杀伤CD47阳性肿瘤细胞，而不引起毒副作用。本发明的CAR-T细胞仅在CAR激活时启动抗CD47抗体的转录翻译，以达到仅在肿瘤微环境中特异性分泌的功能，而在正常的组织或者血液中并不分泌CD47抗体，因此能避免全身性脱靶毒副作用，不干扰体内正常组织。本发明CAR-T细胞可诱导分泌抗CD47抗体，解除CD47阳性肿瘤细胞对于巨噬细胞的抑制，反向促进巨噬细胞去攻击肿瘤细胞，并与CAR协同作用更好的发挥抗肿瘤作用，对肿瘤细胞的杀伤效果显著性增强，能同时杀伤表达CAR靶向的抗原的肿瘤细胞和CD47阳性肿瘤细胞，防止肿瘤细胞的免疫逃逸，不易脱靶和复发。在此基础上完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
抗体
如本文所用，术语“抗体”(Ab)应包括但不限于免疫球蛋白，其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链，或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区，其散布有更保守的称为框架区(FR)的区域。每个VH和VL包含三个CDR和四个FR，从氨基末端到羧基末端按照以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
抗原结合结构域
如本文所用，“抗原结合结构域”“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab’片段，F(ab’) 2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述scFv包含特异性识别肿瘤高表达的抗原的抗体，较佳地为单链抗体或Fv抗体。
本发明中，抗CD47抗体为靶向CD47的scFv抗体。在本发明中“抗CD47 scFV”、“CD47 scFV”与“抗CD47抗体”可互换使用，均为靶向CD47的scFv，包括aCD47 scFV、aCD47 scFV-FC等。优选地，抗CD47抗体的序列如SEQ ID NO.:2、SEQ ID NO.:4或SEQ ID NO.:6所示。
在另一优选例中，所述aCD47 scFV的氨基酸序列如SEQ ID NO.:2所示。
Figure PCTCN2018108022-appb-000001
在另一优选例中，所述aCD47 scFV-FC的氨基酸序列如SEQ ID NO.:4所示。
Figure PCTCN2018108022-appb-000002
在另一优选例中，所述抗CD47抗体为人源化的抗体，氨基酸序列如SEQ ID NO.:6所示。
Figure PCTCN2018108022-appb-000003
嵌合抗原受体(CAR)
如本文所用，嵌合免疫抗原受体(Chimeric antigen receptor，CAR)包括细胞外结构域、任选的铰链区、跨膜结构域、和细胞内结构域。胞外结构域包括任选的信号肽和靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激分子和ζ链部分。CAR在T细胞中表达时，胞外段可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子如IL-2和IFN-γ等，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与CD28信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
在一个实施方式中，本发明CAR靶向CD19，能与CD19特异性结合。在另一优选例中，本发明CAR的结构为L-FMC63-CD28-CD3ζ。优选地，本发明CAR的序列如SEQ ID NO.:1所示。
在一个实施方式中，本发明CAR靶向MSLN，能与MSLN特异性结合。
在另一优选例中，所述靶向MSLN的CAR的结构为L-P4-CD28-CD3ζ，较佳地，所述CAR的氨基酸序列如SEQ ID NO.:5所示。
外源T细胞抗原受体(T cell receptor,TCR)
如本文所用，外源T细胞抗原受体(T cell receptor,TCR)为通过基因转移技术从肿瘤反应性T细胞中克隆出TCR的α链和β链，通过基因工程的手段，以慢病毒或逆转录病毒为载体，外源性转入到T细胞内的TCR。
外源TCR修饰的T细胞能够特异性识别和杀伤肿瘤细胞，并通过优化TCR与肿瘤性特异性抗原的亲和力，可以提高T细胞与肿瘤的亲和力，提高抗肿瘤效果。
嵌合抗原受体T细胞(CAR-T细胞)
如本文所用，术语“CAR-T细胞”、“CAR-T”、“本发明CAR-T细胞”均指本发明第一方面所述的CAR-T细胞。本发明CAR-T细胞可用于治疗CD47高表达的肿瘤，如B细胞淋巴瘤、非霍奇金淋巴瘤、卵巢癌等。
CAR-T细胞较其它基于T细胞的治疗方式存在以下优势：(1)CAR-T细胞的作用过程不受MHC的限制；(2)鉴于很多肿瘤细胞表达相同的肿瘤抗原，针对某一种肿瘤抗原的CAR基因构建一旦完成，便可以被广泛利用；(3)CAR既可以利用肿瘤蛋白质抗原，又可利用糖脂类非蛋白质抗原，扩大了肿瘤抗原的靶点范围；(4)使用患者自体细胞降低了排异反应的风险；(5)CAR-T细胞具有免疫记忆功能，可以长期在体内存活。
嵌合抗原受体NK细胞(CAR-NK细胞)
如本文所用，术语“CAR-NK细胞”、“CAR-NK”、“本发明CAR-NK细胞”均指本发明 第一方面所述的CAR-NK细胞。本发明CAR-NK细胞可用于治疗CD47高表达的肿瘤，如B细胞淋巴瘤、非霍奇金淋巴瘤、卵巢癌等。
自然杀伤(NK)细胞是一类主要的免疫效应细胞，通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能，包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与自体CAR-T细胞相比，CAR-NK细胞还具有一下优点，例如：(1)通过释放穿孔素和颗粒酶直接杀伤肿瘤细胞，而对机体正常的细胞没有杀伤作用；(2)它们释放很少量的细胞因子从而降低了细胞因子风暴的危险；(3)体外极易扩增及发展为“现成的”产品。除此之外，与CAR-T细胞治疗类似。
CD47
CD47是Ig超家族成员，由一个胞外氨基端Ig样可变结构域(配体结合区)、5个疏水跨膜片段和一个羧基端胞内尾区。CD47广泛表达于不同组织细胞表面，如造血细胞(红细胞、淋巴细胞、血小板等)，非造血细胞(胎盘、肝、脑细胞等)及肿瘤细胞。CD47在白血病干细胞中存在高表达，如AML、慢性髓系白血病急变期和T细胞急性淋巴白血病等，在多种肿瘤组织中均发现CD47的表达，包括多发性骨髓瘤、膀胱癌、直肠癌、黑色素瘤等。正常组织虽有CD47表达，但表达水平却显著低于肿瘤组织。
CD47在很多肿瘤细胞中高表达，肿瘤细胞中高表达CD47用于逃避巨噬细胞吞噬。CD47作为自我信号，肿瘤细胞通过抗吞噬信号的表达，逃避了巨噬细胞的吞噬。在淋巴细胞中，CD47与其特异性配体SIRPα相结合形成CD7-SIRPα信号复合体，能够发出抗吞噬信号，抑制吞噬细胞的吞噬，造成免疫系统见识漏洞，促进肿瘤的发展。
B细胞淋巴瘤患者的外周血及生发中心样B细胞中CD47的表达水平都明显高于正常B细胞。同时，研究还发现不同病理组织学类型的非霍奇金淋巴瘤(NHL)中CD47均有表达，如弥漫大B细胞性淋巴瘤(DLBCL)、滤泡细胞性淋巴瘤(FL)、边缘区淋巴瘤(MZL)、套细胞性淋巴瘤(FCL)等。
CD47是治疗肿瘤的一个潜在靶点，目前研究主要集中在使用靶向CD47的抗体来进行肿瘤治疗，CD47抗体治疗是通过DC细胞和CD8+T发挥肿瘤杀伤效应的。DC细胞通过CD47抗体和亲吞噬分子协同作用，吞噬肿瘤细胞，并提呈肿瘤相关抗原给CD8+T，进而发挥CD8+T对肿瘤的特异性杀伤作用。但是，由于CD47普遍表达于正常组织中，全身性的输注抗体会带来很多的脱靶毒副作用，比如贫血、神经毒性等。为此，发明人开发了一种嵌合抗原受体T细胞，被肿瘤抗原特异性激活时才被诱导表达分泌CD47 scFV，尤其对于实体肿瘤，CAR-T细胞可直接递送CD47抗体到肿瘤微环境中，解除肿瘤细胞对于巨噬细胞的抑制作用，从而发挥巨噬细胞的吞噬作用，从而达到抗肿瘤的效果。
活化T细胞核因子(nuclear factor of activated T cells，NFAT)
活化T细胞核因子(NFAT)，是一类转录因子家族，在免疫反应中对诱导基因转录有重要的作用。在静止细胞中，NFAT存在于胞质中，且处于无活性的磷酸化状态，称为NF-ATp，对DNA具低亲和力。当肿瘤抗原被CAR-T细胞特异性识别时，T细胞被特异性激活，介导Ca 2+内流，从而激活钙调磷酸酶的活性，诱导NFAT的去磷酸化，使得NFAT活化且入核，入核后与相关基因的启动子结合，诱导基因的表达。
本发明中，发明人设计一种表达载体，启动子区域含有4、5或6个能与NFAT结合的区域，后面再连一段IL-2最小启动子的片段，同时将CD47的抗体序列置于该启动子区域后，当CAR-T细胞处于静息状态时，不分泌抗CD47抗体，只有在被肿瘤特异性抗原激活以后，才会会导致NFAT的去磷酸化而活化，入核后调控CD47抗体的分泌，达到在肿瘤微环境特异性分泌抗CD47抗体的作用，从而解除肿瘤细胞对巨噬细胞的抑制作用，发挥抗肿瘤活性，同时也避免了全身性脱靶毒性。
实时无标记细胞分析技术
采用(Real Time Cellular Analysis，RTCA)，在无需任何标记的情况下，可实现动态检测免疫细胞杀伤检测及最佳效靶比评估。RTCA技术基于电阻抗原理，针对贴壁细胞生物学表性进行检测，而对于加入孔内的悬浮细胞，因其不与检测板底面电极接触或近视微弱接触，所以不会引起电阻抗变化。
表达盒
如本文所用，“表达盒”或“本发明表达盒”包括第一表达盒和第二表达盒。本发明表达盒如本发明第五方面所述，第一表达盒包含编码所述CAR的核酸序列。所述第二表达盒从5’-3’具有式A结构。在所述CAR被肿瘤特异性抗原激活时，第二表达盒表达抗CD47抗体；在本发明CAR-T细胞处于静息状态时，CAR未与特异性抗原结合时，第二表达盒不表达抗CD47抗体。
在一个实施方式中，所述第一表达盒和第二表达盒分别还包括启动子和/或终止子，其中第二表达盒的启动子为诱导型启动子，优选地为NFAT诱导型启动子，更优选地，含有4、5或6个NFAT结合域和IL-2最小启动子的片段。
载体
本发明还提供了含有本发明表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常通过可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸 序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。
所述表达盒或核酸序列可被克隆入许多类型的载体。例如，该表达盒或核酸序可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物(如人T细胞)、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。例如见美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作 为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明第一方面所述的CAR-T细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括含本发明表达盒的慢病毒载体(LV)转导的细胞(例如，T细胞)进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物及特异性分泌抗CD47的抗体，协同激活巨噬细胞，同时引起T细胞及巨噬细胞的免疫应答，从而显著提高其对肿瘤细胞的杀伤效率。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗CD19 CAR-T细胞引起抗表达CD19的细胞的特异性免疫应答。抗MSLN CAR-T细胞引起抗表达MSLN的细胞的特异性免疫应答。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶 性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌。
本发明的CAR-T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩展细胞，ii)将本发明表达盒引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用含本发明表达盒的载体进行基因修饰(即，体外转导或转染)。本发明CAR-T细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
通常地，如本文所述活化和扩展的细胞可用于治疗和预防无免疫应答的个体中产生的疾病。因此，本发明提供了治疗癌症的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可 以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 5个至1×10 10个本发明经修饰的T细胞，通过例如静脉回输的方式，施用于患者。
本发明的主要优点
(1)本发明可以利用抗CD47抗体杀伤CD47阳性肿瘤细胞，而不引毒副作用。本发明的CAR-T细胞仅在CAR激活时启动抗CD47抗体的转录翻译，以达到仅在肿瘤微环境中特异性分泌的功能，而在正常的组织或者血液中并不分泌CD47抗体，因此能避免全身性脱靶毒副作用，不干扰体内正常组织，安全、毒副作用小。
(2)本发明CAR-T细胞可诱导分泌抗CD47抗体，解除CD47阳性肿瘤细胞对于巨噬细胞的抑制，反向促进巨噬细胞去攻击肿瘤细胞，并与CAR协同作用更好的发挥抗肿瘤作用，对肿瘤细胞的杀伤效果显著性增强，能同时杀伤表达CAR靶向的抗原的肿瘤细胞和CD47阳性肿瘤细胞，防止肿瘤细胞的免疫逃逸，不易脱靶和复发。
(3)对于实体肿瘤，CAR-T细胞可直接递送抗CD47抗体到肿瘤微环境中，解除肿瘤细胞对于巨噬细胞的抑制作用，从而发挥巨噬细胞的吞噬作用，从而达到抗肿瘤的效果。
(4)本发明中的抗CD47抗体带有Fc片段，可以与NK细胞表面Fc受体结合而激活NK细胞，发挥NK细胞的杀伤作用，从而达到更佳的抗肿瘤效果。Fc片段还可提高本发明scFV的稳定性。本发明的抗CD47抗体还包括人源化的CD47抗体，免疫源性更低，毒副作用小。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
材料与方法
1.从供体血液中分离外周血单个核细胞PBMC和扩增T细胞
从脐带血中分离单核细胞，使用Histopaque-1077(Sigma-Aldrich)进行密度梯度离心，并富集T细胞(EasySep human T cell enrichment kit，Stemcell Technologies)，使用偶联anti-CD3/anti-CD28的磁珠激活培养和扩增T细胞；培养基使用X-vivo15(含5％FBS，2mM L-谷氨酰胺，1mM丙酮酸钠，300IU/ml rhIL2)；所有细胞均置于37℃，5％CO2恒温培养箱中培养。
2.细胞培养
Jurkat T细胞(人T淋巴细胞白血病细胞系，
Figure PCTCN2018108022-appb-000004
TIB-152)
Nalm6细胞(人急性淋巴细胞性白血病细胞系，
Figure PCTCN2018108022-appb-000005
CRL-3273)
Raji细胞(Burkitt’s淋巴瘤细胞，ATCC-CCL86)；
Raji-ffluc细胞系(使用萤火虫荧光素酶的慢病毒感染Raji细胞筛选后得到)；
K562-ffluc细胞(人红白血病细胞系，ATCC-CCL243)；
293T细胞(人肾上皮细胞系，ATCC-CRL3216)；
表达CD19的K562细胞、293T细胞为用表达CD19分子的慢病毒载体感染后筛选所得细胞系。
表达MSLN的K562细胞、293T细胞为用表达MSLN分子的慢病毒载体感染后筛选所得细胞系。
其中，Jurkat T，Nalm6,Raji细胞、Raji-ffluc、K562、表达CD19的K562细胞、表达MSLN的K562细胞使用RPMI1640培养基培养，293T细胞、表达CD19的293T细胞、表达MSLN的293T细胞使用DMEM培养基培养。所有培养基均添加10％(v/v)胎牛血清和100U/ml的亲霉素和链霉素，2mM L-谷氨酰胺，1mM丙酮酸钠。所有细胞均置于37℃，5％CO 2恒温培养箱中培养。
实施例1 CAR结构与iCD47scFv结构设计与转导
1.1 靶向CD19的CAR结构设计(简称CD19CAR)
CD19CAR结构：使用二代CD19 CARs，包含来自FMC63的scFv，来自CD28的铰链和跨膜区，胞内段是CD28和CD3ζ，结构示意图如图1A所示，氨基酸序列如SEQ ID NO.:1所示。
Figure PCTCN2018108022-appb-000006
靶向CD19的CAR-T细胞，其中所述CAR-T细胞稳定表达CAR基因。CAR为人工设计的氨基酸序列，包括依次连接的信号肽、scFv、铰链区、跨膜区和胞内信号区。其中CAR基因表达的载体可以是DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子或其他基因转移系统。
将CD19 CAR基因克隆至FUW慢病毒载体骨架中，置于EF1α的启动子下，形成Fuw-EF1α-CD19CAR，将Fuw-EF1α-CD19CAR、pMD2.G和psPAX2(addgene)三个质粒使用Lipofectamine3000转入293T中制备慢病毒表达载体；在48h和72h收集病毒上清，超离进行浓缩(Merck Millipore)；浓缩后的病毒即可用于感染T细胞。
1.2 靶向MSLN的CAR结构设计(简称MSLN-CAR)
MSLNCAR结构：使用二代MSLN CARs，包含来自P4的scFv，来自CD28的铰链和跨膜区，胞内段是CD28和CD3ζ，结构示意图如图1B所示，氨基酸序列如SEQ ID NO.:5所示，其中scFV可以下划线表示靶向MSLN的CAR-T scFV抗原识别序列。
Figure PCTCN2018108022-appb-000007
Figure PCTCN2018108022-appb-000008
1.3 可诱导CD47scFv表达载体的结构设计(简称iCD47scFv)
本发明设计了一种可诱导抗CD47scFv(信号肽选自CD8)分泌的表达盒，结构示意图如图2(aCD47 scFv)或图3(aCD47 scFv-FC)，aCD47 scFv和aCD47 scFv-FC的氨基酸序列分别如SEQ ID NO.:2和SEQ ID NO.:4所示。
可诱导aCD47scFv(信号肽选自CD8)分泌的表达盒NFAT-IL-2-aCD47 scFv的核酸序列如SEQ ID NO.:3所示。将SEQ ID NO.:3所示序列的aCD47scFv编码序列(第361-1080位)替换为aCD47scFv-FC编码序列，即可得到可诱导aCD47scFv-FC(信号肽选自CD8)分泌的表达盒。
1 ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt
61 ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt
121 ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt
181 tttgacaccc ccataatatt tttccagaat taacagtata aattgcatct cttgttcaag
241 agttccctat cactctcttt aatcactact cacagtaacc tcaactcctg ccacaatatg
301 gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg
361 gaggtgcagc tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc
421 tcctgtgcag cctctggatt cactttcagt ggctatggca tgtcttgggt tcgccagact
481 ccagacaaga ggctggagtg ggtcgcaacc attactagtg gtggtactta cacctactat
541 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caccctgtac
601 ctgcaaatag acagtctgaa gtctgaggat acagccatat atttctgtgc aagatccctc
661 gcgggaaatg ctatggacta ctggggtcaa gggaccagcg tcaccgtctc ctcaggtggc
721 ggtggttctg gtggcggtgg ttctggtggc ggtggttctg atattgtgat gactcagtct
781 ccagccaccc tgtctgtgac tccaggagat agagtctctc tttcctgcag ggccagccag
841 actattagcg actacttaca ctggtatcaa caaaaatcac atgagtctcc aaggcttctc
901 atcaaatttg cttcccaatc catttctgga atcccctcca ggttcagtgg cagtggatca
961 ggctcagatt tcactctcag tatcaacagt gtggaacctg aagatgttgg agtgtattac
1021 tgtcaaaatg gtcacggctt tcctcggacg ttcggtggag ggaccaagct ggaaataaaa(SEQ ID NO.:3)
将置于NFAT-IL-2启动子下的抗CD47抗体片段的表达盒克隆至含有CD19 CAR或MSLN CAR基因的FUW慢病毒载体骨架中，形成Fuw-EF1α-CD19CAR-NFAT-IL-2-CD47scFv或Fuw-EF1α-MSLNCAR-NFAT-IL-2-CD47scFv，将其与pMD2.G和psPAX2(Addgene)三个质粒使用Lipofectamine3000转入293T细胞中制备慢病毒表达载体；在48h和72h收集病毒上清，超离进行浓缩(Merck Millipore)；浓缩后的病毒即可用于感染T细胞。
实施例2 CAR-T细胞制备
分离纯化的原代T细胞在激活三天后，使用包含MSLN-CAR和MSLN-CAR-iCD47scFv的慢病毒表达载体进行感染，转移至细胞培养瓶，置于37℃，5％CO 2恒温培养箱中培养。感染后第3天和7天使用MSLN(赛默飞世尔科技，Thermo Fisher Scientific)检测T细胞的CAR阳性率，每隔2-3天更换一半培养基。培养后获得的CAR-T细胞分别为MSLN CAR-T细胞和iCD47scFv-MSLN CAR-T细胞。其中，MSLN CAR-T细胞的实验结果如图4A所示，MSLN CAR-T细胞可以很好地表达MSLN CAR。
同样地，iCD47scFv-MSLN CAR-T细胞也可以很好地表达MSLN CAR。
按照上述相同的实验方法制备CD19 CAR-T细胞和iCD47scFv-CD19CAR-T细胞。结果发现，CD19 CAR-T细胞和iCD47scFv-CD19CAR-T细胞可以很好地表达CD19 CAR，成功制备得到CD19 CAR-T细胞和iCD47scFv-CD19CAR-T细胞。
实施例3 CAR-T细胞体外杀伤
3.1 MSLN CAR-T细胞的体外杀伤
采用实时无标记细胞分析技术(Real Time Cellular Analysis，RTCA)，在无需任何标记的情况下，可实现动态检测免疫细胞杀伤检测及最佳效靶比评估。RTCA技术基于电阻抗原理，针对贴壁细胞生物学表性进行检测，而对于加入孔内的悬浮细胞，因其不与检测板底面电极接触或近视微弱接触，所以不会引起电阻抗变化。因此，CAR-T细胞介导的单层癌细胞杀伤可直接利用RTCA技术进行定量监测。将实施例2中制备的CAR-T细胞作为效应细胞，与MSLN阳性的间皮瘤细胞NCI-H226(图4B)作为靶细胞。按照E:T＝5:1比例共培养，通过连续检测CAR-T细胞对肿瘤细胞的杀伤，分析得出CAR-T细胞对肿瘤的瞬时杀伤以及长时程杀伤能力。
结果表明，与对照组没有转染CAR结构的T细胞(NT)相比MSLN阳性CAR-T细胞(MSLN-CAR)可快速有效的杀伤MSLN阳性肿瘤细胞(图4C)。
同样地，iCD47scFv-MSLN CAR-T细胞也可以快速有效的杀伤MSLN阳性肿瘤细胞。
3.2 CD19 CAR-T细胞的体外杀伤
实验方法同上述3.1，其中将MSLN CAR-T细胞替换为CD19 CAR-T细胞，间皮瘤细胞替换为CD19阳性肿瘤细胞。结果发现，与对照组没有转染CAR结构的T细胞(NT)相比，CD19 CAR-T细胞可快速有效的杀伤CD19阳性肿瘤细胞。
实施例4 CAR-T细胞因子释放检测
将实施例3中获得的MSLN CAR-T细胞/肿瘤细胞NCI-H226细胞共培养42小时上清，收集离心后，检测细胞因子IFN-γ的释放水平，采用Elisa试剂盒(Biolegend)进行检测。
结果表明，MSLN-CAR细胞组中，IFN-γ的释放显著高于对照组(图4D)。
同样地，iCD47scFv-MSLN CAR-T细胞的IFN-γ的释放也显著高于对照组。
按照上述方法，将替换为CD19 CAR-T细胞，间皮瘤细胞替换为CD19阳性肿瘤细胞， 结果发现CD19 CAR-T细胞组中，IFN-γ的释放显著高于对照组。
实施例5 aCD47 scFV载体的构建表达
将含有组成型启动子EF-1α和aCD47 scFV(SEQ ID NO.:2)或aCD47scFV-FC(SEQ ID NO.:4)的编码核酸序列的表达盒(图5A)克隆到p-fuw-EF-1α慢病毒表达载体。按照实施例1中1.3的方法，pMD2.G和psPAX2(Addgene)三个质粒使用Lipofectamine3000转入293T细胞中制备慢病毒表达载体；在48h和72h收集病毒上清，超离进行浓缩(Merck Millipore)；浓缩后的病毒即可用于感染293T细胞，得到稳定表达aCD47 scFV或aCD47 scFV-FC的细胞株。
病毒感染293FT细胞后，通过ELISA方法检测稳定转染细胞上清中aCD47 scFV(SEQ ID NO.:2)的表达以及含量。结果发现aCD47 scFV表达载体可以很好的在真核细胞293T中表达，可以达到494ng/ml的高水平(图5B)。
同样,将表达aCD47scFV-FC(SEQ ID NO.:4)的载体，通过Lonza电转仪，将质粒载体导入Jurkat T细胞中。18小时后收集细胞上清。ELISA法在细胞上清中也检测到aCD47-scFV-FC的分泌，表达丰度为10.5ng/ml(图5C)。
实施例6 T细胞诱导表达系统检测
本发明希望CAR-T细胞可直接递送抗CD47抗体到肿瘤微环境中，解除肿瘤细胞对于巨噬细胞的抑制作用，从而发挥巨噬细胞的吞噬效应。为此，在本发明的设计中(如图6A)，让识别MSLN的CAR-T细胞到达肿瘤部位后，结合MSLN肿瘤抗原，激活下游NFAT转录因子上调，开始启动抗体分泌程序。
6.1 使用一个分泌型的荧光素酶作为报告基因
[根据细则91更正 10.12.2018]　
将图6A的慢病毒载体通过电转的方法转染Jurkat T细胞，如图6B，转染后的Jurkat T细胞表达MSLN CAR。转染4小时后，5×10 5Jurkat T细胞铺到圆底96孔板中，同时加入2.5x10 5K562或MSLN过表达的K562细胞，与Jurkat T细胞共培养。同时，设置单独培养基作为阴性对照，T细胞激活剂(PMA/Inomycin)作为阳性刺激对照。MSLN+Jurkat T细胞刺激24hrs后，收集细胞培养上清,离心后，取出10ul检测上清，使用New England Biolabs公司Gaussia Lucifrease活性检测试剂盒检测分泌型荧光素酶的活性。与对照组只加入培养基组比较，加入不表达MSLN抗原的K562，不能刺激MSLN CAR阳性Jurkat T细胞分泌荧光素酶，而加入表达MSLN抗原的K562细胞可显著刺激MSLN CAR阳性Jurkat T细胞产生荧光素酶(图6C)。该结果表明,MSLN CAR阳性的Jurkat T细胞结合MSLN抗原后，激活转录因子NFAT，呈抗原特异性诱导NFAT下游报告基因Gaussia Luciferase的表达。
[根据细则91更正 10.12.2018]　
于此同时，用图6A慢病毒质粒转染293FT细胞，收获慢病毒，感染外周血分离的T细胞，制备MSLN CAR阳性T细胞。感染MSLN CAR的阳性T细胞通过免疫磁珠富集后,达到85.8％的阳性率(图6D)。同样的，MSLN高表达的K562细胞，显著促进分泌型荧光素酶 的表达，而单纯K562没有刺激作用(图6E)。
以上两个实验结果表明，在实验体系中，MSLN CAR结合特异抗原MSLN后,可诱导NFAT调控的下游基因的分泌表达。
6.2 构建可诱导分泌aCD47scFv或aCD47scFV-FC的MSLN CAR-T细胞
重复6.1的操作，不同点在于，用aCD47scFV(SEQ ID NO.:2)或aCD47scFV-FC(SEQ ID NO.:4)替换荧光素酶基因，从而制得含有图7所示结构的构建物的T细胞；并且采用CD47抗原与抗体的结合反应来检测上清中是否存在抗CD47的抗体。
实验结果表明，在MSLN-CAR-T被激活的情况下，在上清中检测到aCD47scFV或aCD47scFV-FC；而在MSLN-CAR-T未被激活的情况下，在上清中几乎检测不到aCD47scFV或aCD47scFV-FC。这提示，在本发明的T细胞中，CD47抗体可以在CAR被激活(或诱导活化)的情况下有效地表达。
实施例7 aCD47 scFV分泌上清促进巨噬细胞吞噬
为验证转染细胞分泌的aCD47 scFV单链抗体是否有协同抗肿瘤作用，将慢病毒感染的293T细胞分泌上清(含aCD47 scFV)加入到巨噬细胞/肿瘤细胞共培养体系中，然后检测巨噬细胞对肿瘤的吞噬效应。
骨髓来源巨噬细胞培养：取C57BL/6小鼠的股骨，末端用剪刀剪开，插入注射器从股骨一端冲出股骨内的骨髓。收集骨髓来源的细胞混合液，离心后，加入红细胞裂解液重悬破除红细胞。PBS洗涤两遍后，用巨噬细胞培养基(10％胎牛血清DMEM培养基+20％含M-CSF L929细胞培养上清)重悬,铺到非贴壁细胞培养6孔板中培养(1×10 6/孔)。细胞培养8天后，分化为F4/80染色阳性的巨噬细胞。
肿瘤吞噬试验：肿瘤靶细胞Nalm6,K562细胞用CFSE(1uM)荧光标记后，与培养后备用骨髓来源的巨噬细胞按1：1比例(5×10 4:5×10 4)铺到96孔板中培养。共培养4小时后，吞噬靶细胞的巨噬细胞(F4/80+CFSE+)百分比通过流式细胞计数分析。在本发明的实验体系中，293细胞培养上清作为对照，100ul含aCD47 scFV分泌培养上清为处理组，aCD47抗体(5ug/ml)为阳性对照组。
结果如图8所示，含分泌aCD47 scFV单链抗体的293T细胞培养上清可促进骨髓来源的巨噬细胞对肿瘤细胞Nalm6(A,B)和K562(C,D)的吞噬。
此外，还利用相同的实验方法研究了aCD47 scFV-FC的效果，结果发现，aCD47 scFV-FC对巨噬细胞的功能和效果与aCD47 scFV几乎相同。
实施例8 aCD47scFV促进巨噬细胞和MSLN阳性CAR-T的协同杀肿瘤效应
为观察在293T细胞来源的aCD47scFV存在下，MSLN CAR-T细胞和巨噬细胞是否会发挥协同杀伤MSLN阳性肿瘤细胞效应。将稳定转染表达荧光素酶(firefly luciferase)的K562细胞过表达MSLN抗原，荧光素酶作为杀伤实验靶细胞活性的指示。将荧光素酶阳性 K562细胞与效应细胞(MSLNCAR-T/巨噬细胞)和aCD47scFV上清共同孵育培养，研究aCD47存在下巨噬细胞和CAR-T细胞的协同杀伤肿瘤细胞效应。实验设计如下,MLSN抗原阳性的K562靶细胞接受不同的实验处理，分别如下：
实验组1.293T细胞培养上清(对照组)
实验组2.巨噬细胞+293T细胞培养上清
实验组3.巨噬细胞+aCD47scFV细胞上清
实验组4.MSLN CAR-T细胞+293T细胞培养上清
实验组5.MSLN CAR-T细胞+aCD47scFV细胞上清
实验组6.MSLN CAR-T细胞+巨噬细胞+细胞培养上清
实验组7.MSLN CAR-T细胞+巨噬细胞+aCD47 scFV细胞上清
共培养5小时后，离心加入荧光素酶底物，检测存活细胞的数量。结果发现在aCD47 scFV细胞上清存在下，巨噬细胞可协同促进MSLN阳性CAR-T细胞对靶细胞MSLN抗原阳性K562细胞的杀伤(图9)。
此外，本发明还研究了aCD47 scFV-FC的效果，实验方法同aCD47 scFV，其中将aCD47 scFV替换为aCD47 scFV-FC。结果发现，aCD47 scFV-FC的功能和效果与aCD47 scFV几乎相同。
本发明还将实施例6中制备的可诱导分泌aCD47scFv或aCD47scFV-FC的MSLN CAR-T细胞和巨噬细胞细胞(实验组8)与上述荧光素酶阳性K562细胞共培养，实验方法同上。
结果发现，实验组8的结果与实验组7的结果几乎相同，荧光素酶活性略低于实验组7。
结果表明，实验组7和8对靶细胞MSLN抗原阳性K562细胞的杀伤效果显著高于其它实验组，并且实验组8杀伤效果最好。表明抗CD47抗体(如aCD47 scFV和aCD47scFV-FC)和靶向MSLN的CAR具有协同作用，能更有效地杀伤肿瘤细胞。并且本发明CAR-T细胞在靶向MSLN CAR被激活时诱导分泌抗CD47抗体，因此更安全，毒副作用更小。
实施例9
实验方法同实施例6、7和8，制备得到可诱导分泌aCD47scFv的CD19 CAR-T细胞，并验证其对靶细胞的杀伤效果。
结果表明，可诱导分泌aCD47scFv的CD19 CAR-T细胞的CAR与抗原结合后可有效表达aCD47scFV或aCD47scFV-FC，并且能更有效地杀伤肿瘤细胞，毒副作用更小，更安全。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种工程化的免疫细胞，其特征在于，所述工程化的免疫细胞为T细胞或NK细胞，并且所述的免疫细胞细胞具有以下特征：
    (a)所述免疫细胞表达嵌合抗原受体CAR或外源TCR，所述CAR靶向肿瘤细胞的标志物，所述外源TCR靶向肿瘤细胞的标志物；和
    (b)当所述CAR被激活和/或所述外源TCR被激活时，所述免疫细胞诱导分泌抗CD47抗体。
  2. 如权利要求1所述的免疫细胞，其特征在于，所述的工程化的免疫细胞选自下组：
    (i)嵌合抗原受体T细胞(CAR-T细胞)；
    (ii)嵌合抗原受体NK细胞(CAR-NK细胞)；或
    (iii)外源T细胞受体(TCR)T细胞(TCR-T细胞)。
  3. 如权利要求1所述的免疫细胞，其特征在于，所述CAR的结构如式I所示：
    L1-scFv-H1-TM-C-CD3ζ (I)
    式中，
    L1为无或信号肽序列；
    scFv为抗原结合结构域；
    H1为无或铰链区；
    TM为跨膜结构域；
    C为共刺激信号分子；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    所述“-”连接肽或肽键。
  4. 如权利要求1所述的免疫细胞，其特征在于，所述抗CD47抗体为抗CD47scFv，所述抗CD47scFv的结构如下式II所示：
    L2-VH-X-VL-H2-G (II)
    其中，
    L2为无或信号肽序列；
    VH为抗CD47抗体的重链可变区；
    X为无或连接肽；
    VL为抗CD47抗体的轻链可变区；
    H2为无或免疫球蛋白的铰链区；
    G为无或Fc片段。
  5. 如权利要求1所述的免疫细胞，其特征在于，所述抗CD47抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
  6. 如权利要求1所述的免疫细胞，其特征在于，所述抗CD47抗体是部分或全人源化 的抗体。
  7. 如权利要求1所述的免疫细胞，其特征在于，所述的抗CD47抗体为单链或双链形式。
  8. 如权利要求4所述的免疫细胞，其特征在于，所述抗CD47scFv的氨基酸序列如SEQ ID NO.:2、SEQ ID NO.:4或SEQ ID NO.:6所示。
  9. 一种制备权利要求1所述的工程化的免疫细胞的方法，其特征在于，包括以下步骤：
    (A)提供一待改造的免疫细胞；和
    (B)对所述的免疫细胞进行改造，从而使得所述的免疫细胞表达所述的CAR或外源TCR，且在所述CAR被激活和/或所述外源TCR被激活时，所述免疫细胞诱导分泌抗CD47抗体，从而获得权利要求1所述的工程化的免疫细胞。
  10. 如权利要求9所述的方法，其特征在于，在步骤(B)中，包括(B1)将表达所述CAR或外源TCR的第一表达盒导入所述免疫细胞；和(B2)将可诱导分泌抗CD47抗体的第二表达盒导入所述免疫细胞；所述的步骤(B1)可在步骤(B2)之前、之后、同时、或交替进行；其中，
    所述第二表达盒从5’-3’具有式III结构：
    Z1-Z2 (III)
    式中，
    各“-”独立地为键或核苷酸连接序列；
    Z1为诱导型启动子；
    Z2为编码抗CD47抗体的核酸序列。
  11. 一种制剂，其特征在于，所述制剂含有权利要求1所述的工程化的免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  12. 如权利要求1所述的工程化的免疫细胞的用途，其特征在于，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
  13. 一种用于制备权利要求1所述的工程化的免疫细胞的试剂盒，其特征在于，所述试剂盒含有容器，以及位于容器内的：
    (1)第一核酸序列，所述第一核酸序列含有用于表达所述CAR或外源TCR的第一表达盒；和
    (2)第二核酸序列，所述第二核酸序列含有用于可诱导分泌抗CD47抗体的第二表达盒。
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