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WO2024199197A1 - 功能增强型工程化免疫细胞及其制备和应用 - Google Patents

功能增强型工程化免疫细胞及其制备和应用 Download PDF

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WO2024199197A1
WO2024199197A1 PCT/CN2024/083660 CN2024083660W WO2024199197A1 WO 2024199197 A1 WO2024199197 A1 WO 2024199197A1 CN 2024083660 W CN2024083660 W CN 2024083660W WO 2024199197 A1 WO2024199197 A1 WO 2024199197A1
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cells
car
cell
tgfβrii
engineered immune
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PCT/CN2024/083660
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English (en)
French (fr)
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张曦
黄智宏
燕妮
陈少沛
朱晓娜
韩德平
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广州百吉生物制药有限公司
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Definitions

  • the present invention belongs to the field of tumor immunity and cell therapy, and specifically relates to function-enhanced engineered immune cells and their preparation and application.
  • T cells are an important type of lymphocyte involved in cellular immunity. With the help of antigen-presenting cells, they can specifically recognize and kill tumor cells. However, tumor cells can also hinder the specific recognition of T cells by reducing or losing antigen epitopes, building an immunosuppressive microenvironment, and evolving different heterogeneous tumors (i.e., the differences in genotype and phenotype between the same malignant tumor in different patients or between tumor cells in different parts of the same patient), thereby escaping the body's immune response.
  • the CAR molecule is an artificially designed and constructed receptor molecule, which is composed of a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory domain, an intracellular signal transduction domain, etc. It has the functions of specifically recognizing tumor surface antigens, activating T cell killing activity, and stimulating T cell proliferation.
  • the patient's own T cells express CAR molecules to obtain CAR-T cells. After being re-infused into the patient's body, CAR-T cells can specifically and efficiently recognize and kill tumor cells through CAR molecules, achieving the effect of cancer treatment.
  • the first-generation CAR-T cells are composed of only single-chain antibodies as extracellular antigen binding domains and CD3 ⁇ as intracellular signal transduction domains.
  • the first-generation CAR-T cells cannot fully activate the activity of T cells and have poor therapeutic effects.
  • the second-generation CAR-T cells introduced a co-stimulatory domain on the basis of the first-generation CAR-T cells, which improved the in vitro proliferation ability and cytokine release level of T cells.
  • the third-generation CAR-T cells added a co-stimulatory domain on the basis of the second-generation CAR-T cells.
  • the new generation of CAR-T cells co-express other auxiliary factors on the basis of the second-generation CAR-T cells, such as the co-expression of IL-12 or the intracellular domains of certain cytokine receptors, which helps to improve tumor killing activity and safety.
  • CAR-T cells have disadvantages such as low proliferation ability, increased exhaustion, low persistence, and high toxic side effects.
  • Clinical studies have shown that in the treatment of solid cancers, due to the inhibitory tumor immune microenvironment, poor survival of immune cells and many other factors, anti-tumor drugs "fail” and cause tumor immune escape. Therefore, expressing auxiliary factors to promote the immune cell's ability to kill tumors while promoting its proliferation, prolonging survival time and improving memory ability is the key to improving the therapeutic effect of immune cells.
  • the purpose of the present invention is to provide an engineered immune cell (such as CAR-T cells, TIL cells) that is more efficient and has better therapeutic effects for malignant tumors (especially solid tumors).
  • an engineered immune cell such as CAR-T cells, TIL cells
  • Another object of the present invention is to provide an engineered immune cell (such as CAR-T cell, TIL cell) that regulates IL-7 and TGF ⁇ signaling pathways, and a preparation method and application thereof.
  • an engineered immune cell such as CAR-T cell, TIL cell
  • an engineered immune cell wherein the engineered immune cell is a T cell, a TIL cell, or a NK cell, and the immune cell has the following characteristics:
  • the immune cells express exogenous TGF ⁇ RII dominant inhibitory mutant protein or shRNA targeting endogenous TGF ⁇ RII transcripts.
  • the T cells include ⁇ T cells, ⁇ T cells, NKT cells, MAIT cells, or a combination thereof.
  • the engineered immune cells are selected from the following group:
  • CAR-NK cells chimeric antigen receptor NK cells
  • the engineered immune cells are TIL cells.
  • the engineered immune cells are T cells from patients who have undergone radiotherapy and/or chemotherapy.
  • the engineered immune cells are autologous TIL cells from patients who have undergone radiotherapy and/or chemotherapy.
  • the engineered immune cells are autologous or allogeneic.
  • the IL-7 protein and/or TGF ⁇ RII dominant inhibitory mutant may be constitutively expressed or inducibly expressed.
  • the IL-7 protein is membrane-bound or secreted.
  • the TGF ⁇ RII dominant inhibitory mutant is membrane-bound.
  • the immune cells express a chimeric antigen receptor (CAR), wherein the CAR targets surface markers of tumor cells.
  • CAR chimeric antigen receptor
  • the CAR is constitutively expressed or inducibly expressed.
  • the structure of the IL-7 protein is as shown in Formula Z: AH-TM-D (Z)
  • A is IL-7 protein, or an active fragment or a mutant thereof
  • H is none or hinge region
  • TM is none or transmembrane domain
  • D is the absence or degradation domain (including the wild type, or its mutant/modified form); the "-" is a connecting peptide or peptide bond.
  • the A is a wild-type or mutant IL-7 protein of a human or non-human mammal.
  • amino acid sequence of A is shown in SEQ ID No: 1.
  • the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, IgG, or a combination thereof.
  • the H is the hinge region of the CD8 protein.
  • the H is the hinge region of IgG protein Fc.
  • amino acid sequence of H is shown in SEQ ID No: 2.
  • the TM is a transmembrane region of a protein selected from the following group: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or their mutants/modified forms, or their combinations.
  • a protein selected from the following group: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or their mutants/modified forms, or their combinations.
  • the TM is the transmembrane region of CD8.
  • amino acid sequence of the TM is shown in SEQ ID No: 3.
  • the D is an oxygen-dependent degradation domain (ODD) or a partial sequence thereof;
  • amino acid sequence of ODD is shown in SEQ ID No:35.
  • the structures of the IL-7 protein and the TGF ⁇ RII dominant inhibitory mutant are as shown in Formula I: Z0-Z1-Z2-D (I)
  • Z0 and Z2 are IL-7 protein, and the other is a dominant inhibitory mutant of TGF ⁇ RII;
  • Z1 is none or a connecting peptide
  • D is the absence or degradation domain (including the wild type, or its mutant/modified form);
  • the "-" means nothing, a connecting peptide or a peptide bond.
  • Z0 is IL-7 protein
  • Z2 is a dominant inhibitory mutant of TGF ⁇ RII.
  • the coding sequence of the IL-7 protein and the coding sequence of the dominant inhibitory mutant of TGF ⁇ RII are connected through an internal ribosome entry site (IRES), a coding sequence of a 2A connecting peptide, or a coding sequence of a flexible connecting peptide.
  • IRS internal ribosome entry site
  • the connecting peptide is selected from the following group: a 2A connecting peptide, or a flexible connecting peptide.
  • the 2A connecting peptide is P2A (SEQ ID No: 4) or T2A (SEQ ID No: 5).
  • the flexible connecting peptide is a connecting peptide composed of one or more G4S, such as 4 ⁇ GGGGS (SEQ ID No: 6).
  • nucleotide sequence of IRES is shown as SEQ ID No:7.
  • amino acid sequence of the IL-7 protein (Z0) is as shown in SEQ ID No: 1 or SEQ ID No: 8.
  • amino acid sequence of the TGF ⁇ RII dominant inhibitory mutant (Z2) is shown in SEQ ID No: 9 or SEQ ID No: 34.
  • the shRNA targeting endogenous TGF ⁇ RII transcript inhibits the expression of endogenous TGF ⁇ RII transcript.
  • the TGF ⁇ RII dominant inhibitory mutant protein can be replaced by a shRNA expression frame targeting the endogenous TGF ⁇ RII transcript (SEQ ID No: 10).
  • the shRNA expression frame consists of a promoter and a shRNA coding sequence.
  • the promoter of the shRNA expression frame is U6 promoter (SEQ ID No: 11).
  • the binding site of the shRNA is located within 703-738 nt of the open reading frame of the TGF ⁇ RII gene (SEQ ID No: 12).
  • nucleotide sequence bound by the shRNA is shown as SEQ ID No:13, SEQ ID No:14, SEQ ID No:15, SEQ ID No:16 or SEQ ID No:17.
  • nucleotide sequence of the shRNA expression frame is shown as SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21 or SEQ ID No: 22.
  • the cell is a chimeric antigen receptor T cell (CAR-T cell), and the CAR-T cell has one or more of the following characteristics:
  • the CAR-T cells express exogenous TGF ⁇ RII dominant inhibitory mutant protein or shRNA targeting endogenous TGF ⁇ RII transcripts;
  • the CAR-T cell expresses a chimeric antigen receptor CAR.
  • the CAR-T cells when the CAR-T cells are exposed to an inducer or induction condition, the CAR-T cells induce expression of CAR molecules and/or IL-7 and/or TGF ⁇ RII dominant inhibitory mutant proteins.
  • CAR in the CAR-T cells, CAR, IL-7 and TGF ⁇ RII dominant inhibitory mutant proteins are expressed in series.
  • CAR in the CAR-T cells, CAR, IL-7, TGF ⁇ RII dominant inhibitory mutant protein and shRNA targeting endogenous TGF ⁇ RII are expressed independently.
  • the "activation" refers to the binding of the CAR to the surface markers of tumor cells.
  • the "tumor surface marker” refers to a specific antigen on the surface of the tumor.
  • the chimeric antigen receptor CAR is located on the cell membrane of the engineered immune cell.
  • the chimeric antigen receptor CAR is located on the cell membrane of the CAR-T cell.
  • the IL-7 protein and/or TGF ⁇ RII dominant inhibitory mutant protein are localized on the cell membrane of the CAR-T cell.
  • the CAR targets surface markers of tumor cells, and the markers are selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133, CD44, CD166, CD276, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands, LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin18.2, CEA, TAG-72, TROP2, CD147, CLDN6.
  • the markers are selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133, CD44, CD166, CD276, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands, LMP1, EpCAM
  • the structure of the CAR is as shown in Formula II: L-scFv1-H-TM-C-CD3 ⁇ -D (II)
  • L is none or a signal peptide sequence
  • scFv1 is an antigen binding domain (such as an antibody or its active fragment) that targets a surface marker of a tumor cell;
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is the co-stimulatory signaling domain
  • CD3 ⁇ is a cytoplasmic signal transduction sequence derived from CD3 ⁇ (including wild type, or mutant/modified form thereof);
  • D is the absence or degradation domain (including the wild type, or its mutant/modified form);
  • the "-" is a connecting peptide or a peptide bond.
  • the antigen binding domain is selected from the following group: single-chain antibody scFv, single-domain antibody VHH, the extracellular domain of a receptor protein, or a combination thereof.
  • the surface marker of the tumor cells is CD133 (SEQ ID No: 23) or CEA (SEQ ID No: 24).
  • the L is respectively selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modified forms thereof, or a combination thereof.
  • amino acid sequence of L is shown in SEQ ID No:25.
  • the H is selected from the hinge region of the following proteins: CD8, CD28, CD137, IgG, or a combination thereof.
  • the H is selected from the following group: hinge region of IgG protein Fc or CD8.
  • amino acid sequence of H is as shown in SEQ ID No: 26 or SEQ ID No: 27.
  • the TM is selected from the transmembrane region of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or their mutants/modified forms, or their combinations.
  • the TM is the transmembrane region of CD8.
  • amino acid sequence of the TM is shown in SEQ ID No: 3.
  • the C is selected from the co-stimulatory domain of a protein in 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, OX40L, or its mutant/modified form, or a combination thereof.
  • a protein in 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, OX40L, or its mutant/modified form, or a combination thereof.
  • C is a co-stimulatory domain derived from 4-1BB.
  • amino acid sequence of C is shown in SEQ ID No:28.
  • amino acid sequence of CD3 ⁇ is shown in SEQ ID No:29.
  • amino acid sequence of the scFv1 is shown as SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 32.
  • the CAR-T cell in addition to the first CAR shown in Formula II, the CAR-T cell further contains a second CAR for targeting a second antigen, and the structure of the second CAR is shown in Formula III: L-scFv2-H-TM-C-CD3 ⁇ -D (III)
  • L is none or a signal peptide sequence
  • scFv2 is an antigen binding domain (such as an antibody or an active fragment thereof) that targets a surface marker of a second tumor cell;
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is the co-stimulatory domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ or a mutant/modified form thereof;
  • D is the absence or degradation domain (including the wild type, or its mutant/modified form);
  • the "-" is a connecting peptide or a peptide bond.
  • the antigen binding domain is selected from the following group: single-chain antibody scFv, single-domain antibody VHH, the extracellular domain of a receptor protein, or a combination thereof.
  • the scFv2 in Formula III and the scFv1 in Formula II target different antigens.
  • the scFv2 is a single-chain variable region of an antibody or a single-domain antibody sequence and an active fragment thereof that targets a tumor antigen.
  • the scFv2 is an antibody single-chain variable region or a single-domain antibody sequence and its active fragment targeting an antigen selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133, CD44, CD166, CD276, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands, LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin18.2, TROP2, CEA, TAG-72, CD147, CLDN6 or a combination thereof.
  • an antigen selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD24, CD133, CD44, CD166, CD276, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC
  • first CAR shown in Formula II and the second CAR shown in Formula III can be combined into one to form a CAR shown in Formula IVa or IVb: L-scFv1-scFv2-H-TM-C-CD3 ⁇ -D (IVa) L-scFv2-scFv1-H-TM-C-CD3 ⁇ -D (IVb)
  • L is none or a signal peptide sequence
  • scFv1 is an antibody or an active fragment thereof targeting a surface marker of a first tumor cell
  • scFv2 is an antibody or an active fragment thereof targeting a surface marker of a second tumor cell
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is the co-stimulatory domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ or a mutant/modified form thereof;
  • D is the absence or degradation domain (including the wild type, or its mutant/modified form);
  • the "-" is a connecting peptide or a peptide bond.
  • a method for preparing the engineered immune cell according to the first aspect of the present invention comprising the following steps:
  • step (B) comprising:
  • the steps (B1) and (B2) can be performed in any order or in combination.
  • the step (B1) can be performed before, after, simultaneously with, or alternately with the step (B2).
  • the step (B) further includes (B3) introducing a third expression cassette expressing the chimeric antigen receptor CAR into the immune cell; wherein the steps (B1), (B2), and (B3) can be performed in any order or combined.
  • the "combining" refers to combining two or three of the first expression cassette, the second expression cassette, and the optional third expression cassette into one expression cassette, and introducing the resultant into immune cells.
  • a method for preparing the CAR-immune cell comprising the following steps:
  • the immune cells are T cells, TIL cells, or NK cells.
  • step (B) in step (B), it includes: introducing a first expression cassette expressing IL-7, a second expression cassette expressing a dominant inhibitory mutant of TGF ⁇ RII or a shRNA targeting endogenous TGF ⁇ RII transcript, and an optional third expression cassette expressing the CAR into the immune cells; wherein the introduction steps can be performed in any order or in combination.
  • any two expression cassettes among the first, second and third expression cassettes taking the first expression cassette and the second expression cassette as an example
  • their transcription directions are in the same direction ( ⁇ ), in opposite directions ( ⁇ ) or in opposite directions ( ⁇ ).
  • first expression cassette, the second expression cassette and the third expression cassette are located on the same or different vectors.
  • first expression cassette, the second expression cassette and the third expression cassette are located in the same vector.
  • a connecting peptide or an IRES element is also included between the two proteins.
  • the connecting peptide is P2A or T2A or 4 ⁇ GGGGS.
  • the vector is a viral vector, and preferably the viral vector contains the first, second and third expression cassettes in tandem.
  • the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.
  • the vector is a pCDH series lentiviral vector.
  • a preparation which contains the engineered immune cells as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the preparation contains the CAR-T cells described in the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the preparation is a liquid preparation.
  • the dosage form of the preparation includes injection.
  • the concentration of the engineered immune cells (such as CAR-T cells) in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • a use of the engineered immune cells as described in the first aspect of the present invention for preparing a drug or preparation for preventing and/or treating cancer.
  • the use of the CAR-T cells and TIL cells as described in the first aspect of the present invention is provided for preparing a drug or preparation for preventing and/or treating cancer or tumors.
  • the preparation contains CAR-T cells and TIL cells, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the tumor comprises a solid tumor.
  • the tumor is selected from the following group: colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer or liver cancer.
  • a kit for preparing the engineered immune cells according to the first aspect of the present invention comprising a container, and:
  • a first nucleic acid sequence comprising a first expression cassette for expressing IL-7;
  • a second nucleic acid sequence comprising a second expression cassette for expressing a dominant inhibitory mutant of TGF ⁇ RII or a shRNA targeting an endogenous TGF ⁇ RII transcript.
  • kit for preparing the engineered immune cells according to the first aspect of the present invention comprises a container, and:
  • a first nucleic acid sequence wherein the first nucleic acid sequence comprises a first expression cassette for expressing IL-7;
  • a second nucleic acid sequence comprising a second expression cassette for expressing a dominant inhibitory mutant of TGF ⁇ RII or a shRNA targeting an endogenous TGF ⁇ RII transcript;
  • first, second and third nucleic acid sequences are independent or connected.
  • first, second and third nucleic acid sequences are located in the same or different containers.
  • first, second and third nucleic acid sequences are located on the same or different vectors.
  • first, second and third nucleic acid sequences are located in the same vector.
  • a coding sequence for expressing a connecting peptide or an IRES element is also included between two adjacent expression cassettes.
  • the connecting peptide is P2A or T2A or 4 ⁇ GGGGS.
  • the vector is a viral vector, and preferably the viral vector contains the first, second and third nucleic acid sequences in tandem.
  • Figure 1 shows the structural design of the cofactor.
  • FIG2 shows the structural designs of the second-generation CAR and the fourth-generation CAR targeting CEA.
  • FIG3 shows the structural design of the second-generation CAR and the fourth-generation CAR targeting CD133.
  • FIG4 shows the expression rate of each auxiliary factor in enhanced TIL cells detected by flow cytometry.
  • FIG5 shows the secretion level of IL-7 in enhanced TIL cells.
  • Figure 6 shows the CAR molecule expression rate of CEA CAR-T cells detected by flow cytometry.
  • Figure 7 shows the flow cytometry detection of dnTGF ⁇ RII expression rate in CEA CAR-T cells.
  • Figure 8 shows the detection of IL-7 secretion levels of CEA CAR-T cells.
  • Figure 9 shows the CAR molecule expression rate of CD133 CAR-T cells detected by flow cytometry.
  • Figure 10 shows the expression rate of dnTGF ⁇ RII in CD133 CAR-T cells detected by flow cytometry.
  • Figure 11 shows the detection of IL-7 secretion levels of CD133 CAR-T cells.
  • FIG. 12 shows the detection of the killing effect of enhanced TIL cells on liver cancer cells.
  • FIG. 13 shows the IFN- ⁇ release level of enhanced TIL cells.
  • Figure 14 shows the detection of the killing effect of CEA CAR-T cells jointly expressing IL-7 and dnTGF ⁇ RII on HT55.
  • Figure 15 shows the detection of the killing effect of CD133 CAR-T cells jointly expressing IL-7 and dnTGF ⁇ RII on SW620.
  • Figure 16 shows the in vivo tumor suppression effect of CD133 CAR-T cells co-expressing IL-7 and dnTGF ⁇ RII.
  • FIG17 shows the killing effect of CAR-T cells jointly expressing IL-7 and dnTGF ⁇ RII fusion protein on various solid tumor cells.
  • FIG. 18 shows the in vitro proliferation ability of CAR-T cells that jointly express IL-7 and dnTGF ⁇ RII fusion protein.
  • the inventors After extensive and in-depth research and a large number of screenings, the inventors have for the first time simultaneously regulated the IL-7 and TGF ⁇ pathways, that is, upregulated the IL-7 pathway and inhibited the TGF ⁇ pathway, significantly improving the activation degree of the IL-7 signal of the engineered immune cells and significantly improving the resistance to the tumor microenvironment.
  • the engineered immune cells of the present invention taking TIL and CAR-T cells as examples) not only significantly improve the proliferation ability of immune cells, reduce exhaustion, and enhance resistance to the immunosuppressive microenvironment, but also unexpectedly reduce side effects compared to immune cells expressing IL-7 ⁇ CCL19.
  • the present invention was completed on this basis.
  • the present invention takes TIL and CAR-T cells as examples, and representatively describes the engineered immune cells of the present invention in detail.
  • the engineered immune cells of the present invention are not limited to the TIL and CAR-T cells described in the context, and the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the TIL and CAR-T cells described in the context.
  • immune cells express chimeric antigen receptor CAR
  • NK cells or TIL cells are equivalent to T cells (ie, T cells can be replaced by NK cells or TIL cells).
  • administer refers to introducing the 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, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.
  • antibody shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof.
  • 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 one constant domain, CL.
  • VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain regions that interact with the antigen. Used binding domain.
  • Antibodies also include single-domain antibodies (also called nanobodies).
  • Single-domain antibodies are antibodies that are naturally missing light chains and exist in the peripheral blood of animals such as alpacas, camels, and sharks.
  • the antibody only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.
  • VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen.
  • antigen binding domain and “single-chain antibody fragment” all refer to Fab fragments, Fab' fragments, F(ab') 2 fragments, single Fv fragments, or VHH fragments of single-domain antibodies that have antigen binding activity.
  • Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding.
  • the antigen binding domain is usually scFv (single-chain variable fragment).
  • a single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain.
  • the immune cells of the present invention may also contain additional antibodies that specifically recognize antigens highly expressed in tumors, preferably single-chain antibodies, Fv antibodies, or single-domain antibodies.
  • 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 domain (also referred to as an antigen binding domain).
  • the intracellular domain includes a co-stimulatory domain and a CD3 ⁇ chain portion.
  • the extracellular segment can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity, and secretion of cytokines such as IL-2 and IFN- ⁇ , etc., affecting tumor cells, causing tumor cells to not grow, be caused to die or be affected in other ways, and causing the patient's tumor load to be reduced or eliminated.
  • the antigen binding domain is preferably fused with an intracellular domain from one or more of the co-stimulatory molecules and the CD3 ⁇ chain.
  • the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB signaling domain and a CD3 ⁇ signaling domain.
  • CAR-T cells As used herein, the terms “CAR-T cells”, “CAR-T”, and “CAR-T cells of the present invention” all refer to the CAR-T cells described in the first aspect of the present invention.
  • CAR-T cells have the following advantages over other T cell-based treatments: (1) (1) The use process is not restricted by MHC; (2) Since many tumor cells express the same tumor antigens, once the CAR gene construction targeting a certain tumor antigen is completed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) The use of the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • the construct for activating the IL-7 pathway, the construct for inhibiting the TGF ⁇ pathway, and the CAR molecule construct can be combined in various ways, representative methods include (but are not limited to): 2A peptide, flexible connecting peptide, IRES element, multiple promoters, co-transfection, co-transduction and the like.
  • CAR-NK cell As used herein, the terms “CAR-NK cell”, “CAR-NK”, and “CAR-NK cell of the present invention” all refer to the CAR-NK cell described in the first aspect of the present invention.
  • Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways.
  • Engineered (genetically modified) NK cells may acquire new functions, 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) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are very easy to expand in vitro and develop into "ready-made” products. Other than that, it is similar to CAR-T cell therapy.
  • tumor infiltrating lymphocytes (TIL) therapy can also effectively identify and kill tumor cells to achieve the effect of cancer treatment.
  • TIL is an infiltrating immune cell that is isolated from tumor tissue and responds to tumor cells. These immune cells have the ability to recognize and attack tumor cells, and TIL cells are currently used to treat cancer patients. Compared with other immunotherapies, such as CAR-T cell therapy, TCR-T cell therapy, and PD-1/PD-L1 immune checkpoint inhibitors, TIL therapy has the advantages of stronger specificity, higher safety, and low recurrence rate after treatment. However, the number of TIL cells in tumor tissue is small and their activity is usually suppressed by the immune microenvironment. In clinical treatment, a very large number of TIL cells must be prepared to obtain the ideal anti-tumor effect. Therefore, TIL therapy needs to be further optimized.
  • IL-7 Human interleukin-7
  • IL-7 Human interleukin-7
  • the therapeutic effect of engineered immune cells is improved by activating the IL-7 pathway.
  • Representative methods for activating the IL-7 pathway include (but are not limited to): expressing secretory or membrane-bound IL-7.
  • IL-7 can be fused with any structure that can be anchored on the cell membrane to express, thereby achieving the expression of membrane-bound IL-7.
  • IL-7 includes wild-type and mutant IL-7 of humans and other mammals. It should be understood that the term also includes IL-7 analogs or IL-7 derivative proteins, as long as they have or retain the basic functions of wild-type IL-7.
  • TGF- ⁇ Transforming growth factor- ⁇
  • TGF- ⁇ is a protein superfamily composed of structurally related multifunctional cytokines, including TGF- ⁇ 1/2/3 subtypes, activins, bone morphogenetic proteins, and growth differentiation factors.
  • TGF- ⁇ superfamily ligands regulate cell functions through seven type I TGF- ⁇ superfamily receptors (activin-like kinases-1 to 7) and five type II TGF- ⁇ superfamily receptors (T ⁇ RII, ActRII, ActRIIB, AMHRII, and BMPRII), playing an important role in embryonic development, tissue repair, and homeostasis of skeletal muscle, cardiovascular, nervous, endocrine, and immune systems.
  • TGF- ⁇ is a tumor-promoting factor that can induce epithelial-mesenchymal transition (EMT) of tumor cells and promote tumor cell invasion and metastasis.
  • EMT epithelial-mesenchymal transition
  • TGF- ⁇ is a key regulator of T cell responses and plays an important role in regulating immune responses. It can regulate the functions of almost every innate and adaptive immune cell, including dendritic cells, B cells, NK cells, innate lymphocytes, and granulocytes. In the late stage of tumors, most tumor cells can secrete TGF- ⁇ . Once the concentration of TGF- ⁇ increases, it can block the differentiation of immature T cells into Th1 cells and promote their transformation into Treg subsets, while inhibiting the antigen presentation function of dendritic cells, thereby leading to the immune escape of tumor cells.
  • representative methods of inhibiting the TGF ⁇ pathway include but are not limited to: overexpressing TGF ⁇ The dominant inhibitory mutant of the receptor (dnTGF ⁇ RII) or the antibody of TGF ⁇ , knocking down the TGF ⁇ receptor by RNAi, knocking out the TGF ⁇ receptor by gene knockout (TGF ⁇ receptor includes TGF ⁇ RI and TGF ⁇ RII).
  • RNAi is to use shRNA targeting endogenous TGF ⁇ RII transcripts.
  • dnTGF ⁇ RII can be replaced by a TGF ⁇ R inhibitor (membrane-bound/secreted), an inhibitor of related proteins in the TGF ⁇ R signaling pathway/a means of interfering with the expression of related genes, knocking out or knocking down the TGF ⁇ receptor TGF ⁇ RII in immune cells, or overexpressing its dominant inhibitory mutant dnTGF ⁇ RII, or overexpressing a membrane-bound TGF ⁇ antibody.
  • TGF ⁇ R inhibitor membrane-bound/secreted
  • TGF ⁇ RII dominant suppressor mutant dnTGF ⁇ RII
  • TGF ⁇ initiates the transmission of related signals by binding to TGF ⁇ RI and TGF ⁇ RII.
  • TGF ⁇ RI and TGF ⁇ RII can be spatially close to each other, and the intracellular domain of TGF ⁇ RII activates and phosphorylates the intracellular domain of TGF ⁇ RI, causing TGF ⁇ RI to exhibit kinase activity, thereby phosphorylating the transcription factor Smad and entering the nucleus to regulate the expression of downstream genes.
  • dnTGF ⁇ RII is a mutant of the intracellular domain of TGF ⁇ RII that can still form a complex with TGF ⁇ and TGF ⁇ RI through the extracellular domain, but cannot phosphorylate the intracellular domain of TGF ⁇ RI. Therefore, overexpressed dnTGF ⁇ RII can prevent endogenous TGF ⁇ RII from binding to TGF ⁇ and TGF ⁇ RI or activating downstream Smads through competition, thereby achieving the effect of blocking the TGF ⁇ signaling pathway.
  • the signaling pathway of endogenous TGF ⁇ RII can be effectively blocked, thereby reducing the inhibitory effect of TGF ⁇ on immune cells, maintaining the activity of immune cells in the tumor microenvironment, and effectively improving the effect of tumor treatment.
  • expression cassette or "expression cassette of the present invention” includes a first expression cassette, a second expression cassette and a third expression cassette.
  • the expression cassette of the present invention is as described in the fifth aspect of the present invention, wherein the first expression cassette expresses an exogenous IL-7 protein, the second expression cassette expresses an exogenous TGF ⁇ RII dominant inhibitory mutant protein, and the optional third expression cassette expresses the CAR.
  • the IL-7 protein, the TGF ⁇ RII dominant inhibitory mutant protein and the CAR molecule can be independently expressed constitutively or inducibly.
  • the first expression cassette expresses the IL-7 protein
  • the second expression cassette expresses the TGF ⁇ RII dominant inhibitory mutant protein
  • the third The expression cassette expresses the CAR molecule.
  • the first expression cassette does not express the IL-7 protein
  • the second expression cassette does not express the TGF ⁇ RII dominant inhibitory mutant protein
  • the third expression cassette does not express the CAR molecule.
  • the first expression cassette, the second expression cassette and/or the third expression cassette further include a promoter and/or a terminator, respectively.
  • the promoters of the first expression cassette, the second expression cassette and the third expression cassette may be constitutive or inducible promoters.
  • the present invention also provides a vector containing the expression cassette of the present invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow transgenes to be stably integrated into the cell genome for a long time and replicate with the replication of the daughter cell genome.
  • Lentivirus vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells and have the advantage of low immunogenicity.
  • expression cassette or nucleic acid sequence of the present invention can be connected to the promoter downstream by routine operation, and incorporated into expression vector.
  • This vector can be integrated into the eukaryotic cell genome and replicated therewith.
  • Typical cloning vectors include transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression vectors of the present invention can also be used in standard gene delivery protocols for nucleic acid immunization and gene therapy. 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, which are incorporated herein by reference in their entirety.
  • 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, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, etc.
  • the expression vector can be provided to the cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described in, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001) and other virology and molecular biology manuals.
  • 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 at least one replication origin that works in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be used to Isolation and delivery to a subject cell in vivo or ex vivo.
  • retroviral systems are known in the art.
  • a lentiviral vector is used.
  • Many DNA viral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • promoter elements can regulate the frequency of transcription initiation.
  • these elements are located in the 30-110bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site.
  • the intervals between promoter elements are often flexible, so that when an element is inverted or moved relative to another element, the promoter function is maintained.
  • tk thymidine kinase
  • the intervals between promoter elements can be increased by 50bp, and activity begins to decline.
  • a single element can work cooperatively or independently to start transcription.
  • a suitable promoter is a 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 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus (Epstein-Barr virus, EBV) immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be part of the present invention.
  • an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence connected to the inducible promoter when desired, or turn off expression when not desired.
  • 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 any one or both of a selectable marker gene or a reporter gene, so that the expression cell can be identified and selected from a transfected or infected cell population by a viral vector.
  • selectable markers may be carried on a single section of DNA and used for cotransfection procedures. Both selectable marker genes and reporter genes may be flanked by suitable regulatory sequences so that they can be expressed in host cells.
  • Useful selectable marker genes include, for example, antibiotic resistance genes, such as neomycin, etc.
  • vectors can be easily introduced into host cells, for example, mammals (such as human T cells), bacteria, yeast or insect cells, by any method known in the art.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, cationic complex transfection, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for transfection of polynucleotides into host cells are well known in the art. See, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001). Preferred methods for introducing polynucleotides into host cells are liposome transfection and cationic complex polyethyleneimine 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 methods for inserting genes into mammalian cells such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. 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 liposomes.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to introduce a nucleic acid into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid.
  • Nucleic acids associated with lipids may be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids.
  • the lipids, lipid/DNA, or lipid/expression vectors associated with the composition are not limited to any specific structure in solution.
  • Lipids are lipid substances, which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • lentiviruses for transduction, direct transfection of mRNA or plasmids, or expression of artificial transcription factors, etc. can also be used to jointly express IL-7 and TGF ⁇ RII dominant inhibitory mutant molecules or shRNA targeting endogenous TGF ⁇ RII transcripts in immune cells such as T cells and TIL cells.
  • the present invention provides a preparation, which contains the engineered immune cells (such as CAR-T cells) described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the preparation is a liquid preparation.
  • the preparation is an injection.
  • the immune cells in the preparation are The concentration of immune cells (such as TIL cells or CAR-T cells) 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, etc.; 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 (e.g., aluminum hydroxide); and a preservative.
  • the formulation of the present invention is preferably formulated for intravenous administration.
  • the present invention provides therapeutic applications of cells (e.g., T cells and TIL cells, etc.) transduced with a vector (e.g., a lentiviral vector) containing the expression cassette of the present invention.
  • a vector e.g., a lentiviral vector
  • the transduced T cells or TIL cells can target surface markers of tumor cells and express IL-7 and TGF ⁇ RII dominant inhibitory mutant proteins, thereby synergistically and significantly improving their tumor cell killing efficiency.
  • the present invention also provides a method for stimulating an immune response mediated by T cells or TIL cells targeting a mammalian tumor cell population or tissue, comprising the following steps: administering the CAR-T cells of the present invention or administering the enhanced TIL cells of the present invention to a mammal.
  • the present invention provides a type of cell therapy in which the patient's autologous T cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR-T cells, which are then injected into the same patient.
  • This approach makes the probability of graft-versus-host reaction extremely low, and the antigen is recognized by the T cells in an MHC-free manner.
  • CAR-T can treat a variety of different cancers that express the antigen.
  • CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the present invention provides a type of cell therapy in which the patient's autologous TIL cells (or allogeneic donors) are isolated, activated and genetically modified to produce enhanced TIL cells, which are then injected into the same patient.
  • This approach makes the probability of occurrence of risks such as graft-versus-host reaction, cytokine storm, off-target, and tumor cell immune escape extremely low, and tumor cells are accurately recognized by TIL cells.
  • TIL cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells or enhanced TIL cells of the present invention can undergo stable in vivo expansion and can last for months to years.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells can induce a specific immune response to tumor cells that highly express the antigen recognized by the CAR antigen binding domain.
  • Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors.
  • the types of cancers treated with the CAR of the present invention include, but are not limited to, colorectal cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer, etc.
  • the present invention provides a method for treating cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of CAR-T cells or enhanced TIL cells of the present invention.
  • the CAR-T cells and enhanced TIL cells of the present invention can be administered alone, or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17, IL-7, IL-15, IL-21 or other cytokines or cell groups.
  • the pharmaceutical composition of the present invention may include a target cell population 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 manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.
  • the exact amount of the composition of the present invention to be administered can be determined by a physician, which takes into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject).
  • the pharmaceutical composition comprising the T cells or enhanced TIL cells described herein can be administered at a dose of 10 4 to 10 11 cells/kg body weight, preferably 10 5 to 10 8 cells/kg body weight (including all integer values within the range).
  • the T cell composition can also be administered multiple times at these doses.
  • the cells can be administered using injection techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • the optimal dose and treatment regimen for a specific patient can be easily determined by a technician in the medical field by monitoring the patient's disease signs and adjusting the treatment accordingly.
  • the administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation.
  • the composition described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranode, intraspinal, intramuscularly, by intravenous injection or intraperitoneal administration.
  • the T cell or enhanced TIL cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell or enhanced TIL cell composition of the present invention is preferably administered by intravenous injection.
  • the composition of T cell or enhanced TIL cell can be directly injected into a tumor, a lymph node or an infection site.
  • cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (ARA-C), or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (ARA-C), or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (ARA-C), or natalizumab treatment for MS patients or efavirenz treatment for p
  • the T cells or enhanced TIL cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents.
  • the invention The cell composition is administered to a patient in conjunction with (e.g., before, simultaneously with, or after) a bone marrow transplant, with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide.
  • a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant.
  • the subject receives an infusion of the expanded immune cells of the invention.
  • 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 treatment condition and the recipient of the treatment.
  • the dosage ratio for human administration can be implemented according to the practice accepted in the art.
  • 1 ⁇ 10 5 to 1 ⁇ 10 12 modified T cells or enhanced TIL cells of the present invention can be administered to the patient, for example, by intravenous reinfusion, per treatment or per course of treatment.
  • the combination of simultaneously activating the IL-7 pathway and inhibiting the TGF ⁇ pathway can be applied to a variety of different immune cells, such as TIL, CAR-T, TCR-T, NK and other immune cells.
  • the modification of the present invention by activating the IL-7 pathway and inhibiting the TGF ⁇ pathway unexpectedly improves the function of TIL cells to a greater extent than that of T cells.
  • the CAR-TIL cells (or enhanced TIL cells) of the present invention have a significantly more efficient ability to kill tumor cells.
  • the application of the CAR immune cells of the present invention in the treatment of solid tumors has great potential, especially in the treatment of colorectal cancer, ovarian cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, etc.
  • the present invention can synergistically further improve the proliferation ability of CAR-T cells, the release level of IFN- ⁇ and the killing effect on tumors.
  • the engineered immune cells of the present invention can improve the killing effect of CAR-T cells on tumor cells that highly express TGF ⁇ in vivo and in vitro, and unexpectedly reduce toxic side effects.
  • BW7TG is composed of the following structures in series: IL-7, self-cleaving peptide P2A (P2A for short), and TGF ⁇ RII dominant inhibitory mutant (dnTGF ⁇ RII for short).
  • BW197-2 is composed of the following structures in series: IL-7, self-cleaving peptide T2A (abbreviated as T2A), CCL19, P2A, and enhanced green fluorescent protein (abbreviated as eGFP).
  • BWTG is composed of the following structures in series: dnTGF ⁇ RII.
  • BC001 is composed of the following structures in series: human CD8 signal peptide [abbreviated as CD8(SP)], anti-human CEA single-chain antibody (abbreviated as CEA scFv), human CD8 hinge region [abbreviated as CD8(hinge)], human CD8 transmembrane domain [abbreviated as CD8(TM)], human 4-1BB intracellular co-stimulatory domain [abbreviated as 4-1BB(ID)], and human CD3 ⁇ intracellular signal transduction domain [abbreviated as CD3 ⁇ (ID)].
  • CD8(SP) human CD8 signal peptide
  • CEA scFv anti-human CEA single-chain antibody
  • CD8(hinge) human CD8 hinge region
  • CD8(TM) human CD8 transmembrane domain
  • 4-1BB(ID) human 4-1BB intracellular co-stimulatory domain
  • CD3 ⁇ intracellular signal transduction domain abbreviated as CD3 ⁇ (ID)
  • BC010 is composed of the following structures in series: CD8 (SP), CEA scFv, CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), self-cleaving peptide F2A (abbreviated as F2A), IL-7, T2A, and CCL19.
  • BC011 is composed of the following structures in series: CD8 (SP), CEA scFv, CD8 (hinge), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), F2A, IL-7, P2A, and dnTGF ⁇ RII.
  • the structural design of the CAR molecule targeting CD133 is shown in FIG3 .
  • BW133-2 is composed of the following structures in series: CD8 (SP), CD133 scFv, human IgG hinge region [abbreviated as IgG (Fc)], CD8 (TM), 4-1BB (ID), and CD3 ⁇ (ID).
  • SP CD8
  • Fc human IgG hinge region
  • TM CD8
  • ID 4-1BB
  • ID CD3 ⁇
  • BW133-12 is composed of the following structures in series: IL-7, T2A, CCL19, F2A, CD8 (SP), CD133 scFv, IgG (Fc), CD8 (TM), 4-1BB (ID), and CD3 ⁇ (ID).
  • BW133-13 is composed of the following structures in series: IL-7, P2A, dnTGF ⁇ RII, F2A, CD8 (SP), CD133 scFv, IgG (Fc), CD8 (TM), 4-1BB (ID), and CD3 ⁇ (ID).
  • BW133-11B is composed of the following structures in series: CD8 (SP), CD133 scFv, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), internal ribosome entry site (IRES), and dnTGF ⁇ RII.
  • BW133-6B is composed of the following structures in series: CD8 (SP), CD133 scFv, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), IRES, IL-7, T2A, and CCL19.
  • BW133-7B is composed of the following structures in series: CD8 (SP), CD133 scFv, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), IRES, IL-7, P2A, and dnTGF ⁇ RII.
  • BW133-7E is composed of the following structures in series: CD8 (SP), CD133 scFv, IgG (Fc), CD8 (TM), 4-1BB (ID), CD3 ⁇ (ID), IRES, and a fusion protein of IL-7 and dnTGF ⁇ RII (abbreviated as IL-7:dnTGF ⁇ RII).
  • amino acid sequence of the relevant structure is as follows:
  • the target gene fragments were constructed into the lentiviral expression vector pCDH-EF1 ⁇ -MCS, and then the vector plasmids were mixed with the lentiviral packaging plasmids pMDLg-pRRE, pRSV-Rev and pMD2.G, and transfected into 293T cells using Lipofectamine 3000 reagent according to the instructions.
  • the culture medium was replaced with complete medium 6 hours after transfection.
  • virus supernatants were collected 48h and 72h after transfection to obtain virus concentrates, which were named BW7TG, BW197-2, BWTG, BC001, BC010, BC011, BW133-2, BW133-12, BW133-13, BW133-11B, BW133-6B, BW133-7B, and BW133-7E.
  • the collected virus concentrates were stored at -80°C.
  • Jurkat cells were used as materials to detect the activity titer of the above lentivirus.
  • the single cell suspension obtained after tumor tissue digestion was cultured in a medium containing anti-human CD3 antibody and IL-2.
  • the cells were cultured in culture medium until the TIL cells entered the logarithmic growth phase.
  • the above lentiviral vectors were added at an MOI of 10 IU/ml, and the culture medium was replaced with X-VIVO 15 complete medium 72 hours after infection.
  • the enhanced TIL cells obtained were named BW7TG TIL, BW197-2 TIL, and BWTG TIL using the name of the lentivirus, and the T cells not transduced with the lentivirus were named Ctrl TIL.
  • the cells to be tested were washed twice with PBS and resuspended in FACS buffer. According to the antibody instructions, BV421-labeled anti-human CD3 antibody and APC-labeled anti-human TGF ⁇ RII antibody were added to the cell suspension to be tested, incubated at 4°C for 60 minutes, and TIL cells not transfected with lentivirus were used as negative controls. The expression rates of dnTGF ⁇ RII and eGFP in TIL cells were detected by flow cytometry to characterize the transduction efficiency. FlowJo software was used for analysis.
  • the expression rate of BW7TG in TIL cells was 67.0%
  • the expression rate of BWTG in TIL cells was 51.7%
  • the expression rate of BW197-2 in TIL cells was 30.8%, indicating that dnTGF ⁇ RII can be effectively expressed in TIL cells.
  • the results are shown in Figure 5.
  • the IL-7 secretion level of Ctrl TIL was 0.8 ⁇ 0.2pg/ml
  • that of BW7TG TIL was 8604.6 ⁇ 1003.8pg/ml
  • that of BWTG was 1.4 ⁇ 0.1pg/ml
  • that of BW197-2 was 1486.2 ⁇ 242.3pg/ml, indicating that IL-7 can be effectively expressed and secreted in TIL cells.
  • PBMC Peripheral blood mononuclear cells
  • CAR-T cells followed the name of the lentivirus and were named BC001, BC010, BC011, BW133-2, BW133-12, BW133-13, BW133-11B, BW133-6B, BW133-7B, BW133-7E, and T cells not transduced with lentivirus were named Ctrl T.
  • the Ctrl T, BC001, BC010 and BC011 cells to be tested were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, PE-labeled CEA protein and BV421-labeled anti-human CD3 antibody were added to the cell suspension to be tested and incubated at 4°C for 60 minutes. The expression rate of CEA CAR molecules in Ctrl T, BC001, BC010 and BC011 cells was detected by flow cytometry.
  • the Ctrl T and BC011 cells to be tested were washed twice with PBS and resuspended in FACS buffer. According to the antibody instructions, BV421-labeled anti-human CD3 antibody and APC-labeled anti-human TGF ⁇ RII antibody were added to the cell suspension to be tested, incubated at 4°C for 60 minutes, and Ctrl T cells not transfected with lentivirus were used as negative controls. The dnTGF ⁇ RII expression rate of Ctrl T and BC011 cells was detected by flow cytometry. FlowJo software was used for analysis.
  • AIM V medium to adjust the T cells to 1 ⁇ 10 5 /well. After 96 hours of culture, centrifuge and aspirate the supernatant of the co-cultured cells, and dilute the sample with PBS containing 1% BSA. At the same time, dissolve the IL-7 standard with ddH 2 O and dilute the standard according to the recommended gradient ratio. Add the standard and experimental samples to the reaction wells coated with IL-7 capture antibody, 100 ⁇ l per well. After incubation at room temperature for 1 to 3 hours, prepare 1 ⁇ washing solution, and wash each well with 300 ⁇ l washing solution 3 times. After patting the liquid in the well dry, add 100 ⁇ l enzyme-labeled IL-7 detection antibody to each well and incubate at room temperature for 1 to 3 hours.
  • the results are shown in Figure 8.
  • the IL-7 secretion levels of Ctrl T and BC001 cells were 0.0 ⁇ 0.0pg/ml and 0.4 ⁇ 0.0pg/ml.
  • the IL-7 secretion levels of BC010 and BC011 cells were 301.5 ⁇ 46.9pg/ml and 502.4 ⁇ 121.3pg/ml, indicating that the four-generation CAR structures can effectively secrete IL-7.
  • CD133 CAR molecules, dnTGF ⁇ RII and IL-7 in Ctrl T, BW133-2, BW133-12, BW133-13, BW133-11B, BW133-6B, BW133-7B and BW133-7E cells were detected according to the above method.
  • the expression rates of CD133 CAR molecules are shown in Figure 9 .
  • the expression rates of CAR molecules in BW133-2, BW133-12, BW133-13, BW133-11B, BW133-6B, BW133-7B, and BW133-7E cells were 78.7%, 57.2%, 42.4%, 74.9%, 67.7%, 61.8% and 70.0%, indicating that the CAR molecule was successfully expressed.
  • the expression rate of dnTGF ⁇ RII is shown in Figure 10.
  • the dnTGF ⁇ RII expression rates of BW133-13, BW133-11B, BW133-7B, and BW133-7E cells were 44.0%, 44.1%, 30.0% and 28.2%, indicating that the dnTGF ⁇ RII molecule was successfully expressed in these four generations of CAR.
  • the expression level of IL-7 is shown in Figure 11A.
  • the IL-7 secretion levels of Ctrl T, BW133-2, BW133-12, BW133-13, BW133-6B, and BW133-7B were 6.0 ⁇ 0.9pg/ml, 6.6 ⁇ 0.2pg/ml, 7338.6 ⁇ 166.3pg/ml, and 5244.9 ⁇ 271.8pg/ml, respectively.
  • the expression level of membrane-bound IL-7 is shown in Figure 11B.
  • the expression rate of membrane-bound IL-7 in BW133-7E was 17.3%, indicating that IL-7 can be successfully expressed in these four generations of CAR structures.
  • the tumor cells used in this example are all reporter cell lines expressing mCherry fluorescent protein: colorectal cancer cell HT55-Luc-mCherry (DMEM medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin), SW620-Luc-mCherry (Leibovitz L-15 medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin), HCT116-TGF ⁇ -Luc-mCherry and HT29-TGF ⁇ -Luc-mCherry.
  • SW620-Luc-mCherry Leibovitz L-15 medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin
  • HCT116-TGF ⁇ -Luc-mCherry and HT29-TGF ⁇ -Luc-m
  • rry (McCoy's 5A medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin); gastric cancer cell HGC-27-Luc-mCherry, pancreatic cancer Capan-2-Luc-mCherry (McCoy's 5A medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin) and liver cancer cell SK-Hep1-Luc-mCherry (EMEM medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin).
  • the cells to be tested were washed twice with PBS and resuspended with FACS buffer. According to the antibody instructions, the PE-Cy7 labeled anti-CD133 antibody was added to the cell suspension to be tested and incubated at 4°C for 60 minutes. The target cells incubated with the corresponding isotype antibody were used as negative controls, and the CD133 expression rate of the target cells was detected by flow cytometry. FlowJo software was used for analysis, and the results are shown in Table 1.
  • SK-Hep1-Luc-mCherry was used to detect the killing effect of enhanced TIL cells.
  • the target cells were digested and counted, and the target cells were inoculated in a 96-well plate at a density of 1 ⁇ 10 4 /well. According to the experimental requirements, 1 ⁇ 10 4 /well or 2 ⁇ 10 4 /well TIL cells were added for co-culture.
  • 10ng/ml TGF ⁇ was added to the co-culture system, and the changes in the target cell area over time were detected using the IncuCyte live cell analyzer. The smaller the target cell area, the higher the degree of target cell death and the better the T cell killing effect.
  • the concentrations of effector cells and target cells were adjusted with culture medium and inoculated into 96-well plates at a ratio of 1:1, so that the total number of cells was 1 ⁇ 10 5 /well (i.e., the number of effector cells and target cells was 0.5 ⁇ 10 5 /well, respectively). Subsequently, 10 ng/ml of TGF- ⁇ 1 was added, and three replicates were placed in a 37°C, 5% CO 2 incubator for co-culture. After 72 hours, the culture supernatant was collected and the concentration of IFN- ⁇ in the supernatant was detected by ELISA.
  • the results are shown in Figure 13.
  • the IFN- ⁇ release levels of Ctrl TIL, BW197-2 TIL, BWTG TIL, and BW7TG TIL were 8417.8 ⁇ 1004.7pg/ml, 10712.3 ⁇ 1521.3pg/ml, 37561.7 ⁇ 3846.0pg/ml, and 52859.2 ⁇ 4101.4pg/ml, respectively.
  • the IFN- ⁇ release level of BW7TG TIL was significantly higher than that of other groups (P ⁇ 0.05). This indicates that the combined expression of IL-7 and dnTGF ⁇ RII can synergistically increase the activation level of TIL cells in the tumor microenvironment with high TGF ⁇ concentration.
  • HT55-Luc-mCherry was selected to detect the killing effect of CAR-T cells.
  • the target cells were digested and counted, and the target cells were inoculated in a 96-well plate at a density of 1 ⁇ 10 4 /well. According to the experimental requirements, 1 ⁇ 10 4 /well or 2 ⁇ 10 4 /well T cells were added for co-culture.
  • 10ng/ml TGF ⁇ was added to the co-culture system, and the changes in the target cell area over time were detected using the IncuCyte live cell analyzer. The smaller the target cell area, the higher the degree of target cell death and the better the T cell killing effect.
  • HCT116-Luc-mCherry was selected to detect the killing effect of CAR-T cells.
  • the target cells were digested and counted, and the target cells were inoculated in a 96-well plate at a density of 1 ⁇ 10 4 /well. According to the experimental requirements, 1 ⁇ 10 4 /well or 2 ⁇ 10 4 /well T cells were added for co-culture.
  • 10ng/ml TGF ⁇ was added to the co-culture system, and the long-term killing effect was detected using the IncuCyte live cell analyzer.
  • the target cells SW620 in the logarithmic growth phase and in good growth state were collected by trypsin digestion, washed once with physiological saline, and the cell density was adjusted to 3 ⁇ 10 7 /ml.
  • 100 ⁇ l of cell suspension was subcutaneously injected into the right side of B-NDG mice near the armpit, that is, 3 ⁇ 10 6 target cells were inoculated per mouse.
  • the average tumor volume was 50-100 mm 3
  • CAR-T cells (1 ⁇ 10 7 /mouse) and Ctrl T cells (1 ⁇ 10 7 /mouse) were injected through the tail vein, and the day of injection of the test substance was recorded as the 0th day of treatment.
  • the tumor size was measured every week.
  • IL-7 was fused with dnTGF ⁇ RII to express IL-7, so that IL-7 was anchored on the membrane of T cells through dnTGF ⁇ RII to obtain BW133-7E cells.
  • CAR-T cells that jointly express IL-7 and dnTGF ⁇ RII are also suitable for the treatment of malignant solid tumors other than colorectal cancer.
  • HCT116-TGF ⁇ -Luc-mCherry, HT-29-Luc-mCherry, HGC-27-Luc-mCherry, and Capan-2-Luc-mCherry cells were used to detect the killing effect of CAR-T cells.
  • Target cells were digested and counted, and then inoculated into 96-well plates at a density of 1 ⁇ 10 4 /well.
  • CAR-T cells (BW133-2, BW133-6B, BW133-11B, BW133-7E) were added at 1 ⁇ 10 4 /well or 2 ⁇ 10 4 /well according to experimental requirements for co-culture. After adding TGF ⁇ to the co-culture system, the long-term killing effect was detected using the Incucyte live cell analyzer.
  • Ctrl T, BW133-2 and BW133-7E cells were cultured in 96-well plates at different starting cell densities. AO/PI staining was performed after 72 h and the number of live cells was counted.
  • TGF ⁇ receptors in immune cells can effectively block the endogenous TGF ⁇ signaling pathway in cells, reduce the inhibitory effect of TGF ⁇ on immune cells, and thus maintain the activity of immune cells in the tumor microenvironment.
  • dnTGF ⁇ RII dominant inhibitory mutants of TGF ⁇ receptors
  • the activation degree of the IL-7 signal of the engineered immune cells is increased and the resistance to the tumor microenvironment is significantly provided, thereby significantly and synergistically increasing the IFN- ⁇ release level of the immune cells and enhancing the killing effect on tumor cells, thereby synergistically improving the efficacy of the engineered immune cells.
  • the engineered immune cells of the present invention taking CAR-T cells or TIL as an example
  • the risk of toxic side effects caused by overexpression of IL-7 is unexpectedly reduced by optimizing the structure of IL-7 and dnTGF ⁇ RII.

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Abstract

提供了功能增强型工程化免疫细胞及其制备和应用。通过同时上调IL-7通路和抑制TGFβ通路，显著提高了工程化免疫细胞的IL-7信号的激活程度和对肿瘤微环境的抵抗能力，同时降低IL-7过量表达而造成的毒副作用。提供了工程化免疫细胞尤其是TIL，可用于治疗实体瘤。

Description

功能增强型工程化免疫细胞及其制备和应用 技术领域
本发明属于肿瘤免疫和细胞治疗领域，具体地，涉及功能增强型工程化免疫细胞及其制备和应用。
背景技术
T细胞是参与细胞免疫的一类重要的淋巴细胞，通过抗原呈递细胞的辅助，可以特异性地识别并杀伤肿瘤细胞。然而，肿瘤细胞也会通过减少或丢失抗原表位、构建免疫抑制微环境、进化出不同异质性的肿瘤(即同一种恶性肿瘤在不同患者个体间或者同一患者体内不同部位肿瘤细胞间，从基因型到表型上存在的差异)等方式阻碍T细胞的特异性识别，从而逃避机体的免疫应答。
嵌合抗原受体T细胞(chimeric antigen receptor T cell,CAR-T)疗法正是针对该问题应运而生的。具体来说，CAR分子是一种人为设计和构建的受体分子，由信号肽、胞外抗原结合域、铰链区、跨膜区、共刺激结构域、胞内信号传导结构域等部分组成，具有特异性识别肿瘤表面抗原、激活T细胞杀伤活性和刺激T细胞增殖等功能。通过采集肿瘤患者的T细胞并以人工方法转导CAR分子的基因，使患者自体的T细胞表达CAR分子，获得CAR-T细胞。回输至患者体内后，CAR-T细胞可以通过CAR分子特异性且高效的识别并杀伤肿瘤细胞，达到癌症治疗的效果。
CAR-T疗法的概念从1989年首次提出以来，经历了三十年的发展及多轮的技术更迭。第一代CAR-T细胞仅有作为胞外抗原结合域的单链抗体和作为胞内信号传导结构域的CD3ζ组成，第一代CAR-T细胞无法完全激活T细胞的活性，治疗效果不佳。第二代CAR-T细胞在第一代CAR-T细胞的基础上引入了一个共刺激结构域，提高了T细胞的体外增殖能力和细胞因子释放水平。第三代CAR-T细胞在第二代CAR-T细胞的基础上，增加了一个共刺激结构域，虽然可以提高T细胞的杀伤活性，但有可能诱发细胞因子的过量释放，导致严重的副反应。因此，新一代CAR-T细胞在第二代CAR-T细胞的基础上联合表达其他辅助因子，如联合表达IL-12或某些细胞因子受体的细胞内结构域等，有助于提高肿瘤杀伤活性和安全性。
然而，目前开发的CAR-T细胞存在增殖能力低下、衰竭增加、持续性低、毒副作用高等缺点。临床研究表明，在进行实体癌的治疗中由于抑制性的肿瘤免疫微环境、免疫细胞存续能力较差等诸多因素的限制导致抗肿瘤药物“失效”进而引起肿瘤免疫逃逸。因此，通过表达辅助因子，促进免疫细胞对肿瘤杀伤能力的同时促进其增殖、延长存活时间以及提高记忆能力，是提高免疫细胞治疗效果的关键。
综上所述，本领域迫切需要开发更高效、存续期更长的针对肿瘤细胞的工程化免疫细胞，从而既能在机体内长期存活并能抵抗肿瘤微环境抑制作用。
发明内容
本发明的目的是针对恶性肿瘤(尤其是实体瘤)提供一种能更高效、治疗效果更好的工程化免疫细胞(如CAR-T细胞、TIL细胞)。
本发明的又一目的是提供一种调控IL-7和TGFβ信号通路的工程化免疫细胞(如CAR-T细胞、TIL细胞)及其制法和应用。
在本发明的第一方面，提供了一种工程化免疫细胞，所述工程化免疫细胞为T细胞、TIL细胞、或NK细胞，并且所述的免疫细胞具有以下特征：
(a)所述的免疫细胞表达外源的IL-7蛋白；和
(b)所述的免疫细胞表达外源的TGFβRII显性抑制突变体蛋白或靶向内源TGFβRII转录本的shRNA。
在另一优选例中，所述的T细胞包括αβT细胞、γδT细胞、NKT细胞、MAIT细胞，或其组合。
在另一优选例中，所述的工程化免疫细胞选自下组：
(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)；
(iii)嵌合抗原受体TIL细胞(CAR-TIL细胞)。
在另一优选例中，所述的工程化免疫细胞为TIL细胞。
在另一优选例中，所述的工程化免疫细胞为来自经放疗和/或化疗的患者的T细胞。
在另一优选例中，所述的工程化免疫细胞为来自经放疗和/或化疗的患者的自体TIL细胞。
在另一优选例中，所述的工程化免疫细胞为自体的或异体的。
在另一优选例中，所述的IL-7蛋白和/或TGFβRII显性抑制突变体可以是组成型表达或诱导型表达。
在另一优选例中，所述的IL-7蛋白为膜结合型或分泌型。
在另一优选例中，所述的TGFβRII显性抑制突变体为膜结合型。
在另一优选例中，所述免疫细胞表达嵌合抗原受体(chimeric antigen receptor，CAR)，其中所述CAR靶向肿瘤细胞的表面标志物。
在另一优选例中，所述的CAR为组成型表达或诱导型表达。
在另一优选例中，所述的IL-7蛋白的结构如式Z如下：
A-H-TM-D    (Z)
式中，
A为IL-7蛋白，或其活性片段，或其突变体；
H为无或铰链区；
TM为无或跨膜结构域；
D为无或降解结构域(包括野生型、或其突变体/修饰体)；所述“-”为连接肽或肽键。
在另一优选例中，所述A为人或非人哺乳动物的野生型或突变型IL-7蛋白。
在另一优选例中，所述A的氨基酸序列如SEQ ID No:1所示。
在另一优选例中，所述H为选自下组的蛋白的铰链区：CD8、CD28、CD137、IgG、或其组合。
在另一优选例中，所述H为CD8蛋白的铰链区。
在另一优选例中，所述H为IgG蛋白Fc的铰链区。
在另一优选例中，所述H的氨基酸序列如SEQ ID No:2所示。
在另一优选例中，所述TM为选自下组的蛋白的跨膜区：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233、CD314、或其突变/修饰体、或其组合。
在另一优选例中，所述的TM为CD8的跨膜区。
在另一优选例中，所述的TM的氨基酸序列如SEQ ID No:3所示。
在另一优选例中，所述的D为氧依赖性降解结构域(ODD)或其部分序列；
在另一优选例中，ODD的氨基酸序列如SEQ ID No:35所示。
在另一优选例中，所述的IL-7蛋白和TGFβRII显性抑制突变体的结构如式I所示：
Z0-Z1-Z2-D   (I)
式中，
Z0和Z2中的一个为IL-7蛋白，另一个为TGFβRII显性抑制突变体；
Z1为无、或连接肽；
D为无或降解结构域(包括野生型、或其突变体/修饰体)；
所述“-”为无、连接肽或肽键。
在另一优选例中，Z0为IL-7蛋白，Z2为TGFβRII显性抑制突变体。
在另一优选例中，在所述的IL-7蛋白的编码序列和TGFβRII显性抑制突变体编码序列，通过内部核糖体进入位点(IRES)、2A连接肽的编码序列、或柔性连接肽的编码序列连接。
在另一优选例中，所述的连接肽选自下组：2A连接肽、或柔性连接肽。
在另一优选例中，所述2A连接肽为P2A(SEQ ID No:4)、T2A(SEQ ID No:5)。
在另一优选例中，所述的柔性连接肽为一个或多个G4S构成的连接肽，如4×GGGGS(SEQ ID No:6)。
在另一优选例中，IRES的核苷酸序列如SEQ ID No:7所示。
在另一优选例中，所述IL-7蛋白(Z0)的氨基酸序列如SEQ ID No:1或SEQ ID No:8所示。
在另一优选例中，所述TGFβRII显性抑制突变体(Z2)的氨基酸序列如SEQ ID No:9或SEQ ID No:34所示。
在另一优选例中，所述的靶向内源TGFβRII转录本的shRNA抑制内源TGFβRII转录本的表达。
在另一优选例中，所述的TGFβRII显性抑制突变体蛋白可以替换为靶向内源TGFβRII转录本(SEQ ID No:10)的shRNA表达框。
在另一优选例中，所述的shRNA表达框由启动子和shRNA编码序列组成。
在另一优选例中，所述的shRNA表达框的启动子为U6启动子(SEQ ID No:11)。
在另一优选例中，所述的shRNA的结合位点位于TGFβRII基因开放读码框的703-738nt内(SEQ ID No:12)。
在另一优选例中，所述的shRNA的结合的核苷酸序列如SEQ ID No:13、SEQ ID No:14、SEQ ID No:15、SEQ ID No:16或SEQ ID No:17所示。
在另一优选例中，所述的shRNA表达框的核苷酸序列如SEQ ID No:18、SEQ ID No:19、SEQ ID No:20、SEQ ID No:21或SEQ ID No:22所示。
在另一优选例中，所述细胞为一种嵌合抗原受体T细胞(CAR-T细胞)，所述CAR-T细胞具有以下一个或多个特征：
(a)所述CAR-T细胞表达外源的IL-7蛋白；
(b)所述CAR-T细胞表达外源的TGFβRII显性抑制突变体蛋白或靶向内源TGFβRII转录本的shRNA；和
(c)任选的所述CAR-T细胞表达嵌合抗原受体CAR。
在另一优选例中，当所述CAR-T细胞接触诱导剂或诱导条件时，所述CAR-T细胞诱导表达CAR分子和/或IL-7和/或TGFβRII显性抑制突变体蛋白。
在另一优选例中，在所述的CAR-T细胞中，CAR、IL-7和TGFβRII显性抑制突变体蛋白是串联表达的。
在另一优选例中，在所述的CAR-T细胞中，CAR、IL-7、TGFβRII显性抑制突变体蛋白和靶向内源TGFβRII的shRNA是各自独立地表达的。
在另一优选例中，所述“激活”指所述CAR与肿瘤细胞的表面标志物结合。
在另一优选例中，所述“肿瘤的表面标志物”指肿瘤表面的特异性抗原。
在另一优选例中，所述的嵌合抗原受体CAR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，所述的IL-7蛋白和/或TGFβRII显性抑制突变体蛋白定位于所述CAR-T细胞的细胞膜。
在另一优选例中，所述的CAR靶向肿瘤细胞的表面标志物，所述标志物选自下组：CD19、CD20、CD22、CD123、CD47、CD138、CD33、CD30、CD271、GUCY2C、CD24、CD133、CD44、CD166、CD276、ABCB5、ALDH1、间皮素(mesothelin,MSLN)、EGFR、GPC3、BCMA、ErbB2、NKG2D配体(ligands)、LMP1、EpCAM、VEGFR-1、Lewis-Y、ROR1、Claudin18.2、CEA、TAG-72、TROP2、CD147、CLDN6。
在另一优选例中，所述CAR的结构如式II所示：
L-scFv1-H-TM-C-CD3ζ-D    (II)
式中，
L为无或信号肽序列；
scFv1为靶向肿瘤细胞的表面标志物的抗原结合结构域(如抗体或其活性片段)；
H为无或铰链区；
TM为跨膜结构域；
C为共刺激信号结构域；
CD3ζ为源于CD3ζ的胞浆信号传导序列(包括野生型、或其突变体/修饰体)；
D为无或降解结构域(包括野生型、或其突变体/修饰体)；
所述“-”为连接肽或肽键。
在另一优选例中，所述的抗原结合结构域(scFv1)选自下组：单链抗体scFv、单域抗体VHH、受体蛋白的胞外域、或其组合。
在另一优选例中，所述的肿瘤细胞的表面标志物为CD133(SEQ ID No:23)或CEA(SEQ ID No:24)。
在另一优选例中，所述L分别选自下组的蛋白的信号肽：CD8、GM-CSF、CD4、CD28、CD137、或其突变/修饰体、或其组合。
在另一优选例中，所述的L的氨基酸序列如SEQ ID No:25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、IgG、或其组合。
在另一优选例中，所述H选自下组的IgG蛋白Fc或CD8的铰链区。
在另一优选例中，所述H的氨基酸序列如SEQ ID No:26所示或SEQ ID No:27。
在另一优选例中，所述TM选自下组的蛋白的跨膜区：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233、CD314、或其突变/修饰体、或其组合。
在另一优选例中，所述的TM为CD8的跨膜区。
在另一优选例中，所述的TM的氨基酸序列如SEQ ID No:3所示。
在另一优选例中，所述C选自下组的蛋白的共刺激结构域：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、OX40L、或其突变/修饰体、或其组合。
在另一优选例中，C为4-1BB来源的共刺激结构域。
在另一优选例中，所述的C的氨基酸序列如SEQ ID No:28所示。
在另一优选例中，所述的CD3ζ的氨基酸序列如SEQ ID No:29所示。
在另一优选例中，所述scFv1的氨基酸序列如SEQ ID NO:30或SEQ ID NO:31或SEQ ID NO:32所示。
在另一优选例中，除了式II所示的第一CAR之外，所述CAR-T细胞还含有用于针对第二抗原的第二CAR，所述的第二CAR的结构如式III所示：
L-scFv2-H-TM-C-CD3ζ-D   (III)
式中，
L为无或信号肽序列；
scFv2为靶向第二肿瘤细胞的表面标志物的抗原结合结构域(如抗体或其活性片段)；
H为无或铰链区；
TM为跨膜结构域；
C为共刺激结构域；
CD3ζ为源于CD3ζ的胞浆信号传导序列或其突变/修饰体；
D为无或降解结构域(包括野生型、或其突变体/修饰体)；
所述“-”为连接肽或肽键。
在另一优选例中，所述的抗原结合结构域(scFv2)选自下组：单链抗体scFv、单域抗体VHH、受体蛋白的胞外域、或其组合。
在另一优选例中，式III中的scFv2与式II中的scFv1靶向不同的抗原。
在另一优选例中，所述scFv2为靶向肿瘤抗原的抗体单链可变区或单域抗体序列及其活性片段。
在另一优选例中，所述scFv2为靶向选自下组抗原的抗体单链可变区或单域抗体序列及其活性片段：CD19、CD20、CD22、CD123、CD47、CD138、CD33、CD30、CD271、GUCY2C、CD24、CD133、CD44、CD166、CD276、ABCB5、ALDH1、间皮素(mesothelin,MSLN)、EGFR、GPC3、BCMA、ErbB2、NKG2D配体(ligands)、LMP1、EpCAM、VEGFR-1、Lewis-Y、ROR1、Claudin18.2、TROP2、CEA、TAG-72、CD147、CLDN6或其组合。
在另一优选例中，式II所示的第一CAR和式III所示的第二CAR可合而为一，从而构成如式IVa或IVb所示的CAR：
L-scFv1-scFv2-H-TM-C-CD3ζ-D   (IVa)
L-scFv2-scFv1-H-TM-C-CD3ζ-D   (IVb)
式中，
L为无或信号肽序列；
scFv1为靶向第一肿瘤细胞的表面标志物的抗体或其活性片段；
scFv2为靶向第二肿瘤细胞的表面标志物的抗体或其活性片段；
H为无或铰链区；
TM为跨膜结构域；
C为共刺激结构域；
CD3ζ为源于CD3ζ的胞浆信号传导序列或其突变/修饰体；
D为无或降解结构域(包括野生型、或其突变体/修饰体)；
所述“-”为连接肽或肽键。
在本发明的第二方面，提供了一种制备如本发明第一方面所述的工程化免疫细胞的方法，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外源的IL-7蛋白和外源的TGFβRII显性抑制突变体蛋白或靶向内源TGFβRII转录本的shRNA，从而获得如本发明第一方面所述的工程化免疫细胞。
在另一优选例中，在步骤(B)中，包括：
(B1)将表达IL-7的第一表达盒导入所述免疫细胞；
(B2)将表达TGFβRII显性抑制突变体或靶向内源TGFβRII转录本的shRNA的第二表达盒导入所述免疫细胞；
其中所述的步骤(B1)和(B2)可以按任意次序进行，或合并进行。
在另一优选例中，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，所述的步骤(B)还包括(B3)将表达嵌合抗原受体CAR的第三表达盒导入所述免疫细胞；其中所述的步骤(B1)、(B2)、和(B3)可以按任意次序进行，或合并进行。
在另一优选例中，所述的“合并进行”指将所述的第一表达盒、第二表达盒、和任选的第三表达盒中的两个或三个合并为一个表达盒，并导入免疫细胞。
在另一优选例中，提供了一种制备所述的CAR-免疫细胞的方法，包括以下步骤：
(A)提供一种待改造的免疫细胞；
(B)对所述的免疫细胞进行改造，使得所述的免疫细胞表达所述的CAR分子 以及外源的IL-7蛋白和外源的TGFβRII显性抑制突变体蛋白或靶向内源TGFβRII转录本的shRNA，从而获得如本发明第一方面所述的工程化免疫细胞。
在另一优选例中，所述的免疫细胞为T细胞、TIL细胞、或NK细胞。
在另一优选例中，在步骤(B)中，包括：将表达IL-7的第一表达盒、表达TGFβRII显性抑制突变体或靶向内源TGFβRII转录本的shRNA的第二表达盒、和任选的表达所述CAR的第三表达盒导入所述免疫细胞；其中所述的导入步骤可以按任意次序进行，或合并进行。
在另一优选例中，对于所述的第一、第二和第三表达盒中任何两个表达盒(以第一表达盒和第二表达盒为例)，其转录方向是同向的(→→)、相向的(→←)或相背的(←→)。
在另一优选例中，所述的第一表达盒、第二表达盒和第三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、第二表达盒和第三表达盒位于同一载体。
在另一优选例中，当所述的CAR分子、外源的IL-7蛋白和外源的TGFβRII显性抑制突变体蛋白中的两个或三个串联表达时，在两个蛋白之间，还包括连接肽或IRES元件。
在另一优选例中，所述连接肽为P2A或T2A或4×GGGGS。
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、第二和第三表达盒。
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pCDH系列慢病毒载体。
在本发明的第三方面，提供了一种制剂，所述制剂含有如本发明第一方面所述的工程化免疫细胞，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，所述制剂含有本发明所述的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，所述制剂中所述工程化免疫细胞(如CAR-T细胞)的浓度为1×103-1×108个细胞/ml，较佳地1×104-1×107个细胞/ml。
在本发明的第四方面，提供了一种如本发明第一方面所述的工程化免疫细胞的用途，用于制备预防和/或治疗癌症的药物或制剂。
在另一优选例中，提供了如本发明第一方面所述的CAR-T细胞和TIL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，所述制剂含有CAR-T细胞和TIL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，所述肿瘤包括实体瘤。
在另一优选例中，所述肿瘤选自下组：结直肠癌、胃癌、胰腺癌、食管癌、乳腺癌、卵巢癌或肝癌。
在本发明的第五方面，提供了一种用于制备如本发明第一方面所述的工程化免疫细胞的试剂盒，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IL-7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表达TGFβRII显性抑制突变体或靶向内源TGFβRII转录本的shRNA的第二表达盒。
在另一优选例中，提供了一种用于制备如本发明第一方面所述的工程化免疫细胞的试剂盒，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IL-7的第一表达盒；
(2)第二核酸序列，所述第二核酸序列含有用于表达TGFβRII显性抑制突变体或靶向内源TGFβRII转录本的shRNA的第二表达盒；和
(3)任选的第三核酸序列，所述第三核酸序列含有用于表达所述CAR的第三表达盒。
在另一优选例中，所述的第一、第二和第三核酸序列为独立的或相连的。
在另一优选例中，所述的第一、第二和第三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、第二和第三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、第二和第三核酸序列位于同一载体。
在另一优选例中，当所述的第一、第二和第三核酸序列中两个或三个位于同一载体时，在两个相邻的表达盒之间还包括用于表达连接肽的编码序列或IRES元件。
在另一优选例中，所述连接肽为P2A或T2A或4×GGGGS。
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、第二和第三核酸序列。
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显示了辅助因子的结构设计。
图2显示了靶向CEA的二代CAR和四代CAR的结构设计。
图3显示了靶向CD133的二代CAR和四代CAR的结构设计。
图4显示了流式检测增强型TIL细胞中各辅助因子的表达率。
图5显示了增强型TIL细胞中IL-7的分泌水平。
图6显示了流式检测CEA CAR-T细胞的CAR分子表达率。
图7显示了流式检测CEA CAR-T细胞的dnTGFβRII表达率。
图8显示了CEA CAR-T细胞的IL-7分泌水平检测。
图9显示了流式检测CD133 CAR-T细胞的CAR分子表达率。
图10显示了流式检测CD133 CAR-T细胞的dnTGFβRII的表达率。
图11显示了CD133 CAR-T细胞的IL-7分泌水平检测。
图12显示了增强型TIL细胞对肝癌细胞的杀伤效果检测。
图13显示了增强型TIL细胞的IFN-γ释放水平。
图14显示了联合表达IL-7和dnTGFβRII的CEA CAR-T细胞对HT55的杀伤效果检测。
图15显示了联合表达IL-7和dnTGFβRII的CD133 CAR-T细胞对SW620的杀伤效果检测。
图16显示了联合表达IL-7和dnTGFβRII的CD133 CAR-T细胞的体内抑瘤效果。
图17显示了联合表达IL-7和dnTGFβRII融合蛋白的CAR-T细胞对多种实体瘤细胞的杀伤效果。
图18显示了联合表达IL-7和dnTGFβRII融合蛋白的CAR-T细胞的体外增殖能力。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的筛选，首次通过同时调控IL-7和TGFβ通路，即上调IL-7通路和抑制TGFβ通路，显著提高工程化免疫细胞的IL-7信号的激活程度和显著提高对肿瘤微环境的抵抗能力。实验表明，本发明的工程化免疫细胞(以TIL和CAR-T细胞为例)不仅显著提高了免疫细胞的增殖能力、减少衰竭、增强对免疫抑制微环境的抵抗力，而且与表达IL-7×CCL19的免疫细胞相比，还意外地降低了副作用。在此基础上完成了本发明。
本发明以TIL和CAR-T细胞为例，代表性地对本发明的工程化免疫细胞进行详细说明。本发明的工程化免疫细胞不限于上下文所述的TIL和CAR-T细胞，本发明的工程化免疫细胞具有与上下文所述的TIL和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细胞或TIL细胞等同于T细胞(即，T细胞可替换为NK细胞或TIL细胞)。
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。
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”或“施用”(administer or administration)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引入受试者，包括静脉内、瘤内、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。
“抗体”也包括单域抗体(也称纳米抗体)。单域抗体是在羊驼、骆驼、鲨鱼等动物外周血液中存在一种天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区。单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位。
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，单一Fv片段，或单域抗体的VHH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。
此外，本发明的免疫细胞还可含有额外的特异性识别肿瘤高表达的抗原的抗体，较佳地为单链抗体、Fv抗体、或单域抗体。
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点特异性结合结构域(也称为抗原结合结构域)。细胞内结构域包括共刺激结构域和CD3ζ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CD3ζ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4-1BB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。
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。
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。
在本发明的免疫细胞中，激活IL-7通路的构建物(construct)、抑制TGFβ通路的构建物以及CAR分子构建物，可以通过各种不同方式进行组合，代表性的方式包括(但并不限于)：2A肽、柔性连接肽、IRES元件、多启动子、共转染、共转导等形式。
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。
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细胞还具有以下优点，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。
TIL细胞
除了CAR-T细胞疗法外，肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes,TIL)疗法也能高效识别并杀伤肿瘤细胞，达到癌症治疗的效果。
TIL是一种从肿瘤组织中分离出的对肿瘤细胞有反应的浸润性免疫细胞。这些免疫细胞具有识别和攻击肿瘤细胞的能力，目前TIL细胞也被用于治疗肿瘤患者。与其他免疫疗法相比，如CAR-T细胞疗法、TCR-T细胞疗法以及PD-1/PD-L1免疫检查点抑制剂，TIL疗法具有特异性更强，安全性更高、治疗后复发率低等优势。然而，肿瘤组织中的TIL细胞数量较少且活性通常被免疫微环境所抑制，在临床治疗时需制备极大量TIL细胞才有可能获得理想的抗肿瘤效果。因此，TIL疗法还需要继续优化。
在本发明中，通过同时激活TIL细胞中的IL-7通路并抑制TGFβ通路，出乎 意料地获得了性能显著改善的新型TIL细胞。
IL-7
人白细胞介素7(interleukin-7,IL-7)是一种多效细胞因子，具有广泛的免疫效应。长期以来，对于IL-7的认识一直集中于其对B细胞和T细胞的生长、存活及分化的影响。外源性注射的IL-7在体内可以增强机体抗肿瘤免疫。IL-7强大的免疫效应，尤其是其调节T细胞增殖、维持细胞内环境稳定、增强T免疫应答等功能，使IL-7作为一种免疫调节因子越来越受到关注。
在本发明中，通过激活IL-7通路来提高工程化免疫细胞的治疗效果。代表性的激活IL-7通路的方法，包括(但并不限于)：表达分泌型或膜结合型的IL-7。
在本发明中，可以将IL-7与任何能锚定于细胞膜上的结构融合表达，从而实现膜结合型IL-7的表达。
在本发明中，术语“IL-7”包括人及其他哺乳动物的野生型和突变型的IL-7。应理解，该术语还包括IL-7类似物或IL-7的衍生蛋白，只要其具有或保留了野生型IL-7的基本功能。
TGF-β
转化生长因子-β(transforming growth factor-β,TGF-β)是一个由结构相关的多功能细胞因子组成的蛋白超家族，包括TGF-β1/2/3亚型、活化素、骨形成蛋白和生长分化因子等。TGF-β超家族配体通过7种I型TGF-β超家族受体(激活素样激酶-1至7)和5种II型TGF-β超家族受体(TβRII、ActRII、ActRIIB、AMHRII和BMPRII)调控细胞功能，在胚胎发育、组织修复和骨骼肌、心血管、神经、内分泌和免疫系统的稳态中发挥着重要作用。
TGF-β是一种肿瘤促进因子，能够诱导肿瘤细胞的上皮-间质转化(epithelial mesenchymal transition,EMT)，促进肿瘤细胞侵袭和转移。此外，TGF-β是T细胞应答的关键调节因子，在调节免疫应答中扮演着重要的角色，可以调控几乎每一种固有和适应性免疫细胞的功能，包括树突状细胞、B细胞、NK细胞、先天淋巴细胞和粒细胞等。在肿瘤晚期，大多数肿瘤细胞均可分泌TGF-β。TGF-β的浓度一旦升高，便可以阻断未成熟的T细胞向Th1细胞方向分化，并促进其向Treg亚群方向转化，同时抑制树突状细胞的抗原递呈功能，从而导致肿瘤细胞的免疫逃逸。
在本发明中，代表性的抑制TGFβ通路的方式，包括但并不限于：过表达TGFβ 受体的显性抑制突变体(dnTGFβRII)或TGFβ的抗体，通过RNAi的方式敲降TGFβ受体，通过基因敲除的方式敲除TGFβ受体(TGFβ受体包括TGFβRI和TGFβRII)。一种代表性的通过RNAi的方式是利用靶向内源TGFβRII转录本的shRNA。
应理解，在本发明中，dnTGFβRII可以替换为TGFβR抑制剂(膜结合/分泌型)、TGFβR信号通路中的相关蛋白的抑制剂/干扰相关基因的表达手段、免疫细胞中敲除、敲降TGFβ的受体TGFβRII，或过表达其显性抑制突变体dnTGFβRII，或过表达膜结合型TGFβ抗体。
TGFβRII显性抑制突变体(dnTGFβRII)
在TGFβ的信号通路中，TGFβ通过与TGFβRI和TGFβRII的结合启动相关信号的传到。具体的，TGFβ与TGFβRI和TGFβRII的胞外域结合后，可以使TGFβRI和TGFβRII在空间位置上相互靠近，并使TGFβRII的胞内域激活TGFβRI的胞内域且磷酸化，使TGFβRI表现出激酶活性，进而使转录因子Smad磷酸化并进入细胞核调控下游基因的表达。dnTGFβRII是TGFβRII的胞内域缺失突变体，该突变体仍可通过胞外域与TGFβ和TGFβRI形成复合物，但不能磷酸化TGFβRI的胞内域。因此，过表达的dnTGFβRII可以通过竞争作用，使内源的TGFβRII无法与结合TGFβ和TGFβRI结合或激活下游的Smad，从而达到阻断TGFβ信号通路的效果。
在本发明中，通过免疫细胞中敲降或敲除TGFβ的受体TGFβRII，或过表达其显性抑制突变体dnTGFβRII，可以有效阻断细胞内源TGFβRII的信号通路，从而降低TGFβ对免疫细胞产生的抑制作用，保持免疫细胞在肿瘤微环境中的活性，有效提高肿瘤治疗的效果。
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、第二表达盒和第三表达盒。本发明表达盒如本发明第五方面所述，其中，所述第一表达盒表达外源的IL-7蛋白，所述第二表达盒表达外源的TGFβRII显性抑制突变体蛋白，而任选的第三表达盒表达所述CAR。
在本发明中，IL-7蛋白、TGFβRII显性抑制突变体蛋白和CAR分子可以各自独立地组成型表达或诱导型表达。
在诱导表达情况下，在所述CAR-T细胞被相应诱导剂或相应诱导条件激活时，第一表达盒表达IL-7蛋白，第二表达盒表达TGFβRII显性抑制突变体蛋白，第三 表达盒表达CAR分子。这样，在本发明CAR-T细胞在未接触相应诱导剂时，第一表达盒不表达IL-7蛋白，第二表达盒不表达TGFβRII显性抑制突变体蛋白，而第三表达盒不表达CAR分子。
在一个实施方式中，所述第一表达盒、第二表达盒和/或第三表达盒分别还包括启动子和/或终止子。第一表达盒、第二表达盒和第三表达盒的启动子可以为组成型或诱导型启动子。
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、稳定地整合于细胞基因组中并随子细胞基因组的复制而复制。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，且具有低免疫原性的优点。
通常，可通过常规操作将本发明的表达盒或核酸序列连接至启动子下游，并将其并入表达载体。该载体可整合至真核细胞基因组中并随之复制。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。
本发明的表达载体也可用于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、复制载体等。
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Molecular Cloning:A Laboratory Manual(Sambrook等,Cold Spring Harbor Laboratory,New York,2001)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、腺相关病毒、疱疹病毒和慢病毒。通常，合适的载体包含至少一种在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)。
已经有许多基于病毒的系统被开发出来，并用于哺乳动物细胞的基因转导。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。该重组病毒随后可被 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。在一个实施方式中，使用慢病毒载体。许多DNA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。
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，这些元件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元件被倒置或移动时，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-巴尔病毒(Epstein-Barr virus，EBV)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，能够在需要时，启动连接诱导型启动子的多核苷酸序列的表达，或在不需要时关闭表达。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告基因中的任一个或两者，以便于通过病毒载体从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。可选择的标记基因和报告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。有用的可选择标记基因包括例如抗生素抗性基因，诸如neomycin等等。
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、阳离子复合物转染法、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Molecular Cloning:A Laboratory Manual(Sambrook等,Cold Spring Harbor Laboratory,New York,2001)。将多核苷酸引入宿主细胞的优选方法为脂质体法转染法和阳离子复合物聚乙烯亚胺转染法。
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。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。脂质为脂类物质，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的实施方式中，所述载体为慢病毒载体。
应理解，在本发明中，除了采用多个慢病毒进行转导，还可采用直接转染mRNA或质粒，或者通过表达人工转录因子等方法，从而在T细胞、TIL细胞等免疫细胞中联合表达IL-7和TGFβRII显性抑制突变体分子或靶向内源TGFβRII转录本的shRNA。
制剂
本发明提供了一种制剂，其含有本发明第一方面所述的工程化免疫细胞(如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，所述制剂中所述免 疫细胞(如TIL细胞或CAR-T细胞)的浓度为1×103-1×108个细胞/ml，更优地1×104-1×107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
本发明提供了利用含本发明表达盒的载体(如慢病毒载体)转导的细胞(例如，T细胞和TIL细胞等)进行的治疗性应用。经转导的T细胞或TIL细胞可靶向肿瘤细胞的表面标志物并表达IL-7与TGFβRII显性抑制突变体蛋白，从而协同而显著地提高其对肿瘤细胞的杀伤效率。
因此，本发明也提供了刺激靶向哺乳动物肿瘤细胞群或组织的T细胞或TIL细胞所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细胞或施用本发明的增强型TIL细胞。
在一个实施方式中，本发明提供了一类细胞疗法，其中，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。这种方式使移植物抗宿主反应的发生概率极低，抗原被T细胞以无MHC限制方式识别。此外，CAR-T就可以治疗表达该抗原的多种不同癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续控制肿瘤的长期持久性。
在一个实施方式中，本发明提供了一类细胞疗法，其中，分离病人自体TIL细胞(或者异源供体)，激活并进行基因改造产生增强型TIL细胞，随后注入同一病人体内。这种方式使移植物抗宿主反应、细胞因子风暴、脱靶、肿瘤细胞免疫逃逸等风险的发生概率极低，肿瘤细胞被TIL细胞精确识别。不像抗体疗法，TIL细胞能够体内复制，产生可导致持续控制肿瘤的长期持久性。
在一个实施方式中，本发明的CAR-T细胞或增强型TIL细胞可经历稳定的体内扩增并可持续数月至数年的时间。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-T细胞可诱导对CAR抗原结合结构域所识别的抗原的高表达肿瘤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。用本发明的CAR治疗的癌症类型包括但不限于：结直肠癌、乳腺癌、肝癌、胰腺癌、胃癌、食管癌、卵巢癌等。
通常地，如本文所述活化和扩增的细胞可用于治疗和预防肿瘤等疾病。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-T细胞或增强型TIL细胞。
本发明的CAR-T细胞和增强型TIL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、IL-7、IL-15、IL-21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如患者的病症、和患者疾病的类型和严重度等因素确定，或可由临床试验确定。
当指出“治疗有效量”、“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。包括本文描述的T细胞或增强型TIL细胞的药物组合物可以以104至1011个细胞/kg体重的剂量，优选105至108个细胞/kg体重的剂量(包括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，New Eng.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由医学领域技术人员通过监测患者的疾病迹象容易地确定，并以此调整治疗。
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细胞或增强型TIL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细胞或增强型TIL细胞组合物优选通过静脉内注射施用。T细胞或增强型TIL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(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细胞或增强型TIL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1×105个至1×1012个本发明经修饰的T细胞或增强型TIL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)同时激活IL-7通路并抑制TGFβ通路的组合，可应用于多种不同免疫细胞，如应用于TIL、CAR-T、TCR-T、NK等免疫细胞。
(2)本发明中激活IL-7通路并抑制TGFβ通路的改造，对TIL细胞功能提升的幅度出乎意料地高于其对T细胞功能提升的幅度。本发明的CAR-TIL细胞(或增强型TIL细胞)具有显著更高效的杀伤肿瘤细胞的能力。
(3)出乎意料地，与健康供体的T细胞相比，当采用来自经放疗和/或化疗的患者的T细胞或TIL细胞(原本这些细胞常常因免疫能力下降或耗竭程度较高，而不适合用于制备工程化免疫细胞)来制备工程化免疫细胞时，本发明的激活IL-7通路并抑制TGFβ通路策略所带来的功能提升的幅度更高。
(4)本发明的CAR免疫细胞在实体瘤治疗中的应用具有巨大潜力，特别是在治疗结直肠癌、卵巢癌、乳腺癌、肝癌、胰腺癌、胃癌、食管癌等。
(5)与仅联合表达dnTGFβRII或TGFβRII-IL-7RA融合蛋白的CAR-T细胞相比，本发明可协同地进一步提高CAR-T细胞的增殖能力、IFN-γ的释放水平以及对肿瘤的杀伤效果。
(6)与联合表达IL-7和CCL19的CAR-T细胞相比，本发明的工程化免疫细胞可以在体内外提高CAR-T细胞对高表达TGFβ的肿瘤细胞杀伤效果，且意外地降低了毒副作用。
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《分子克隆：实验室手册》(Sambrook等人，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。
除非另外说明，实施例中采用的试剂和材料为市售获得。
材料与方法
辅助因子与CAR分子的结构设计
各辅助因子的结构设计如图1所示。
(1)BW7TG由以下结构依次串联组成：IL-7、自剪切肽P2A(简称P2A)、TGFβRII显性抑制突变体(简称dnTGFβRII)。
(2)BW197-2由以下结构依次串联组成：IL-7、自剪切肽T2A(简称T2A)、CCL19、P2A、增强型绿色荧光蛋白白(简称eGFP)。
(3)BWTG由以下结构依次串联组成：dnTGFβRII。
以CEA为靶点的CAR分子的结构设计如图2所示。
(1)BC001由以下结构依次串联组成：人CD8信号肽[简称CD8(SP)]、抗人CEA单链抗体(简称CEA scFv)、人CD8铰链区[简称CD8(hinge)]、人CD8跨膜结构域[简称CD8(TM)]、人4-1BB胞内共刺激结构域[简称4-1BB(ID)]、人CD3ζ胞内信号转导结构域[简称CD3ζ(ID)]。
(2)BC010由以下结构依次串联组成：CD8(SP)、CEA scFv、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、自剪切肽F2A(简称F2A)、IL-7、T2A、CCL19。
(3)BC011由以下结构依次串联组成：CD8(SP)、CEA scFv、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、F2A、IL-7、P2A、dnTGFβRII。
以CD133为靶点的CAR分子的结构设计如图3所示。
(1)BW133-2由以下结构依次串联组成：CD8(SP)、CD133 scFv、人IgG铰链区[简称IgG(Fc)]、CD8(TM)、4-1BB(ID)、CD3ζ(ID)。
(2)BW133-12由以下结构依次串联组成：IL-7、T2A、CCL19、F2A、CD8(SP)、CD133 scFv、IgG(Fc)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)。
(3)BW133-13由以下结构依次串联组成：IL-7、P2A、dnTGFβRII、F2A、CD8(SP)、CD133 scFv、IgG(Fc)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)。
(4)BW133-11B由以下结构依次串联组成：CD8(SP)、CD133 scFv、IgG(Fc)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、内部核糖体进入位点(简称IRES)、dnTGFβRII。
(5)BW133-6B由以下结构依次串联组成：CD8(SP)、CD133 scFv、IgG(Fc)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、IRES、IL-7、T2A、CCL19。
(6)BW133-7B由以下结构依次串联组成：CD8(SP)、CD133 scFv、IgG(Fc)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、IRES、IL-7、P2A、dnTGFβRII。
(7)BW133-7E由以下结构依次串联组成：CD8(SP)、CD133 scFv、IgG(Fc)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、IRES、IL-7与dnTGFβRII的融合蛋白(简称IL-7:dnTGFβRII)。
相关结构的氨基酸序列如下：



实施例1慢病毒载体的制备
分别将上述各目的基因片段构建于慢病毒表达载体pCDH-EF1α-MCS中，再将各载体质粒分别与慢病毒包装质粒pMDLg-pRRE、pRSV-Rev和pMD2.G混合，并按说明书用Lipofectamine 3000试剂转染至293T细胞中。转染后6h更换为完全培养基。
分别收集转染后48h和72h的病毒上清液，获得病毒浓缩液，分别命名为BW7TG、BW197-2、BWTG、BC001、BC010、BC011、BW133-2、BW133-12、BW133-13、BW133-11B、BW133-6B、BW133-7B、BW133-7E。将收集的病毒浓缩液置于-80℃保存。
最后，以Jurkat细胞为材料，对上述慢病毒进行活性滴度检测。
实施例2增强型TIL细胞的制备和检测
(1)增强型TIL细胞的制备
将肿瘤组织经消化处理后获得的单细胞悬液在含有抗人CD3抗体和IL-2的培 养基中培养，直至TIL细胞进入对数生长期。以MOI＝10IU/ml的用量，分别加入上述慢病毒载体，感染后72h更换为X-VIVO 15完全培养基。获得的增强型TIL细胞沿用慢病毒的名称，分别命名为BW7TG TIL、BW197-2 TIL、BWTG TIL，未转导慢病毒的T细胞命名为Ctrl TIL。
(2)增强型TIL细胞的转导效率和IL-7表达检测
用PBS清洗待检测的细胞两次，并用FACS缓冲液重悬。按照抗体说明书将BV421标记的抗人CD3抗体和APC标记的抗人TGFβRⅡ抗体加入待检测细胞悬液中，4℃孵育60min，以未用慢病毒转染的TIL细胞为阴性对照，用流式细胞仪检测TIL细胞的dnTGFβRII和eGFP表达率，以表征转导效率。采用FlowJo软件分析。
结果如图4所示，BW7TG在TIL细胞中的表达率为67.0％，BWTG在TIL细胞中的表达率为51.7％，BW197-2在TIL中的表达率为30.8％，说明dnTGFβRII在TIL细胞中能有效表达。
用X-VIVO 15培养基分别调整待测TIL细胞至1×105个/孔。培养96h后，离心并吸取上述共培养的细胞上清，用含1％BSA的PBS稀释样本。同时用ddH2O溶解IL-7标准品，将标准品按推荐梯度倍比稀释。分别将标准品和实验样品加入包被有IL-7捕获抗体反应孔中，每孔100μl。室温孵育1～3h后，配制1×清洗液，每孔用300μl清洗液清洗3次。将孔中液体拍干后，每孔加入100μl酶标IL-7检测抗体，室温孵育1～3h。每孔用300μl清洗液清洗3次。将孔中液体拍干后，加入100μl显色底物。室温避光孵育20min后，每孔加入50μl终止液，用酶标仪测定450nm的吸光值并计算IL-7的浓度。
结果如图5所示，Ctrl TIL的IL-7分泌水平为0.8±0.2pg/ml，BW7TG TIL为8604.6±1003.8pg/ml，BWTG为1.4±0.1pg/ml，BW197-2为1486.2±242.3pg/ml，说明IL-7在TIL细胞中能有效表达和分泌。
实施例3 CAR-T细胞的制备和检测
(1)CAR-T细胞的制备
将健康供者的外周血单个核细胞(peripheral blood mononuclear cell,PBMC)复苏于AIM V培养基中，并用抗人CD3/CD28抗体刺激培养T细胞。以MOI＝5IU/ml的用量，分别用慢病毒转导上述细胞。转导后第7天，检测相应CAR分子的表达，转导后第10天收取细胞，液氮保存备用。获得的CAR-T细胞沿用慢病毒的名称，分别命名为BC001、BC010、BC011、BW133-2、BW133-12、BW133-13、BW133-11B、 BW133-6B、BW133-7B、BW133-7E，未转导慢病毒的T细胞命名为Ctrl T。
(2)CEA CAR-T细胞中CAR分子、dnTGFβRII和IL-7的表达检测
用PBS清洗待检测的Ctrl T、BC001、BC010和BC011细胞两次，并用FACS缓冲液重悬。按照抗体说明书将PE标记的CEA蛋白、BV421标记的抗人CD3抗体加入待检测细胞悬液中，4℃孵育60min。用流式细胞仪检测Ctrl T、BC001、BC010和BC011细胞的CEA CAR分子的表达率。
结果如图6所示，BC001、BC010和BC011细胞CAR分子的表达率分别为80.8％、88.1％和86.5％。
用PBS清洗待检测的Ctrl T和BC011细胞两次，并用FACS缓冲液重悬。按照抗体说明书将BV421标记的抗人CD3抗体以及APC标记的抗人TGFβRⅡ抗体加入待检测细胞悬液中，4℃孵育60min,以未用慢病毒转染的Ctrl T细胞为阴性对照，用流式细胞仪检测Ctrl T和BC011细胞的dnTGFβRII表达率。采用FlowJo软件分析。
结果如图7所示，BC011细胞的dnTGFβRII表达率为34.2％，表明BC011细胞成功的表达dnTGFβRII分子。
用AIM V培养基分别调整待测T细胞至1×105个/孔。培养96h后，离心并吸取上述共培养的细胞上清，用含1％BSA的PBS稀释样本。同时用ddH2O溶解IL-7标准品，将标准品按推荐梯度倍比稀释。分别将标准品和实验样品加入包被有IL-7捕获抗体反应孔中，每孔100μl。室温孵育1～3h后，配制1×清洗液，每孔用300μl清洗液清洗3次。将孔中液体拍干后，每孔加入100μl酶标IL-7检测抗体，室温孵育1～3h。每孔用300μl清洗液清洗3次。将孔中液体拍干后，加入100μl显色底物。室温避光孵育20min后，每孔加入50μl终止液，用酶标仪测定450nm的吸光值并计算IL-7的浓度。
结果如图8所示，Ctrl T和BC001细胞的IL-7分泌水平为0.0±0.0pg/ml和0.4±0.0pg/ml。与之相比，BC010和BC011细胞的IL-7分泌水平为301.5±46.9pg/ml和502.4±121.3pg/ml，说明四代CAR结构都能有效分泌IL-7。
(3)CD133 CAR-T细胞中CAR分子、dnTGFβRII和IL-7的表达检测
按上述方法检测Ctrl T、BW133-2、BW133-12、BW133-13、BW133-11B、BW133-6B、BW133-7B、BW133-7E细胞中CD133 CAR分子、dnTGFβRII和IL-7的表达情况。
CD133 CAR分子的表达率如图9所示，BW133-2、BW133-12、BW133-13、BW133-11B、BW133-6B、BW133-7B、BW133-7E细胞的CAR分子表达率分别为 78.7％、57.2％、42.4％、74.9％、67.7％、61.8％和70.0％，表明CAR分子成功表达。dnTGFβRII的表达率如图10所示，BW133-13、BW133-11B、BW133-7B、BW133-7E细胞的dnTGFβRII表达率为44.0％、44.1％、30.0％和28.2％，表明dnTGFβRII分子在这些四代CAR中都表达成功。
IL-7的表达水平如图11A所示，Ctrl T、BW133-2、BW133-12、BW133-13、BW133-6B、BW133-7B的IL-7分泌水平分别为6.0±0.9pg/ml、6.6±0.2pg/ml、7338.6±166.3pg/ml和5244.9±271.8pg/ml。膜结合型IL-7的表达水平如图11B所示，BW133-7E中膜结合型的IL-7表达率为17.3％，说明IL-7在这些四代CAR结构中都能成功表达。
实施例4靶细胞检测
(1)靶细胞的培养条件
本实施例中使用的肿瘤细胞均为表达mCherry荧光蛋白的报告细胞系：结直肠癌细胞HT55-Luc-mCherry(DMEM培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)、SW620-Luc-mCherry(Leibovitz L-15培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)、HCT116-TGFβ-Luc-mCherry和HT29-TGFβ-Luc-mCherry(McCoy's 5A培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)；胃癌细胞HGC-27-Luc-mCherry、胰腺癌Capan-2-Luc-mCherry(McCoy's 5A培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)以及肝癌细胞SK-Hep1-Luc-mCherry(EMEM培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)。
(2)靶细胞的CEA表达检测
用PBS清洗待检测细胞两次，并用FACS缓冲液重悬。按照抗体说明书将AF700标记的抗CEA抗体加入待检测细胞悬液中，4℃孵育60min。以加入相应Isotype抗体孵育的靶细胞作为阴性对照，用流式细胞仪检测靶细胞的CEA表达率。
用PBS清洗待检测细胞两次，并用FACS缓冲液重悬。按照抗体说明书将PE-Cy7标记的抗CD133抗体加入待检测细胞悬液中，4℃孵育60min。以加入相应Isotype抗体孵育的靶细胞作为阴性对照，用流式细胞仪检测靶细胞的CD133表达率。采用FlowJo软件分析，结果如表1所示。
表1肿瘤细胞中CEA和CD133的表达率

实施例5增强型TIL细胞功能研究
(1)增强型TIL细胞杀伤效果检测
用SK-Hep1-Luc-mCherry进行增强型TIL细胞的杀伤效果检测。消化靶细胞并计数，以1×104/孔的密度将靶细胞接种于96孔板，根据实验需求加入1×104/孔或2×104/孔的TIL细胞进行共培养。另一方面为考察dnTGFβRⅡ功能，在共培养体系中加入10ng/ml的TGFβ，用IncuCyte活细胞分析仪检测靶细胞面积随时间的变化情况。靶细胞面积越小，说明靶细胞的死亡程度越高，T细胞杀伤效果越好。
结果如图12所示。BW197-2 TIL、BWTG TIL、BW7TG TIL组的杀伤效果都显著优于Ctrl TIL对照组。随着靶细胞刺激的次数增加，BW197-2的杀伤效果显著减弱。在与靶细胞共培养第11天时，BWTG TIL和BW7TG TIL细胞仍保持较强的杀伤效果，但BW7TG TIL的杀伤效果显著强于其他各组别(P<0.05)(图12)。这表明，同时激活IL-7通路并抑制TGFβ通路，可以意外地显著提升TIL细胞抵抗肿瘤微环境免疫抑制的能力，同时进一步增强其对肿瘤细胞的杀伤效果。
(2)增强型TIL细胞的IFN-γ释放水平
用培养基分别调整效应细胞和靶细胞的浓度并按1:1的比例接种于96孔板，使总细胞数为1×105/孔(即效应细胞和靶细胞细胞数分别为0.5×105/孔)。随后加入10ng/ml的TGF-β1，每组三个重复，放入37℃，5％CO2培养箱中共培养。72h后，收集培养上清液，用ELISA法检测上清中IFN-γ的浓度。
结果如图13所示。Ctrl TIL、BW197-2 TIL、BWTG TIL、BW7TG TIL的IFN-γ释放水平分别为8417.8±1004.7pg/ml、10712.3±1521.3pg/ml、37561.7±3846.0pg/ml和52859.2±4101.4pg/ml，BW7TG TIL的IFN-γ释放水平显著高于其他组别(P<0.05)。这表明，联合表达IL-7和dnTGFβRII可以协同地提高TIL细胞在TGFβ浓度较高的肿瘤微环境中的激活水平。
实施例6 CAR-T细胞的功能研究
(1)CEA CAR-T细胞杀伤效果检测
根据上述靶点抗原的流式检测结果，选用HT55-Luc-mCherry进行CAR-T细胞的杀伤效果检测。消化靶细胞并计数，以1×104/孔的密度将靶细胞接种于96孔板，根据实验需求加入1×104/孔或2×104/孔的T细胞进行共培养。另一方面，为考察dnTGFβRⅡ功能，在共培养体系中加入10ng/ml的TGFβ，用IncuCyte活细胞分析仪检测靶细胞面积随时间的变化情况。靶细胞面积越小，说明靶细胞的死亡程度越高，T细胞杀伤效果越好。
结果如图14所示。虽然BC001和BC010对靶细胞有一定的杀伤能力，但在共培养第5天靶细胞的面积仍有明显增长，说明BC001和BC010的杀伤效果受到TGFβ的抑制。相反，表达IL-7和dnTGFβRⅡ的CAR-T细胞BC011能有效且持续的抑制靶细胞的生长，杀伤效果显著强于其他各组别(P<0.05)。这说明联合表达IL-7和dnTGFβRⅡ能有效抵抗TGFβ对T细胞的抑制作用，增强CAR-T细胞对肿瘤的杀伤能力。
(2)CD133 CAR-T细胞杀伤效果检测
根据上述靶点抗原的流式检测结果，选用HCT116-Luc-mCherry进行CAR-T细胞的杀伤效果检测。消化靶细胞并计数，以1×104/孔的密度将靶细胞接种于96孔板，根据实验需求加入1×104/孔或2×104/孔的T细胞进行共培养。另一方面，为考察dnTGFβRⅡ功能，在共培养体系中加入10ng/ml的TGFβ，用IncuCyte活细胞分析仪进行长时间杀伤效果检测。
结果如图15所示。与CEA CAR-T细胞的结果相似，BW133-13组对肿瘤细胞的杀伤效果显著强于其他各组别(P<0.05)。这一结果说明，联合表达IL-7和dnTGFβRⅡ能有效抵抗TGFβ对T细胞的抑制作用，增强CAR-T对肿瘤的杀伤能力。
(3)体内抑瘤实验。
用胰酶消化法收集处于对数生长期且生长状态良好的靶细胞SW620，用生理盐水洗涤1次后，调整细胞密度为3×107/ml。在B-NDG小鼠右侧靠近腋下部位皮下注射100μl细胞悬液，即每只小鼠接种3×106的靶细胞，当肿瘤平均体积为50～100mm3时)，通过尾静脉分别注射CAR-T细胞(1×107/只)和Ctrl T细胞(1×107/只)，注射受试物当天记为治疗的第0天，每周测量肿瘤大小。
结果如图16所示。在SW620皮下瘤模型中，CAR-T细胞回输第25天后，联合表达IL-7和dnTGFβRII的BW133-13组的肿瘤体积为654.3±255.8mm3，显著小于BW133-2对照组(肿瘤体积为1503.2±365.4mm3)(P<0.05)。虽然在注射 CAR-T细胞后的第14天，联合表达IL-7和CCL19的BW133-12组的肿瘤体积已有缩小的趋势，但之后小鼠出现明显的副反应并死亡，说明该CAR-T细胞存在一定的安全风险。
上述结果表明，在激活IL-7通路的同时抑制TGFβ的信号通路，不仅可以在保持IL-7对T细胞增强作用的同时，还意外地显著降低在体内的毒副作用，比现有的联合表达IL-7和CCL19技术的安全性更好。
实施例7 CAR-T细胞适应症研究
(1)联合表达IL-7和dnTGFβRII的CAR-T细胞对多种实体瘤细胞的杀伤效果检测
为了进一步提升IL-7和dnTGFβRII的CAR-T细胞的安全性，将IL-7与dnTGFβRII融合表达，使IL-7通过dnTGFβRII锚定在T细胞的膜上，获得BW133-7E细胞。同时，为了研究联合表达IL-7和dnTGFβRII的CAR-T细胞是否也适用于治疗结直肠癌以外的恶性实体瘤。用HCT116-TGFβ-Luc-mCherry、HT-29-Luc-mCherry、HGC-27-Luc-mCherry、Capan-2-Luc-mCherry细胞进行CAR-T细胞的杀伤效果检测。消化靶细胞并计数，以1×104/孔的密度将靶细胞接种于96孔板，根据实验需求加入1×104/孔或2×104/孔的CAR-T细胞(BW133-2、BW133-6B、BW133-11B、BW133-7E)进行共培养。在共培养体系中加入TGFβ后，用Incucyte活细胞分析仪进行长时间杀伤效果检测。
结果如图17所示。各实验组的杀伤效果都显著优于Ctrl T对照组。随着靶细胞刺激的次数增加，表达IL-7和dnTGFβRII融合蛋白的CAR-T细胞，对HCT116-Luc-mCherry结直肠癌细胞(图17A)、HT-29-Luc-mCherry结直肠癌细胞(图17B)、Capan-2-Luc-mCherry胰腺癌细胞(图17C)和HGC-27-Luc-mCherry胃癌细胞(图17D)的杀伤效果显著强于其他组别(P<0.05)。
以不同的起始细胞密度将Ctrl T、BW133-2和BW133-7E细胞至于96孔板中培养，72h后进行AO/PI染色并统计活细胞数目。
当起始细胞密度为1×105/ml至2×105/ml时，BW133-7E组的细胞扩增倍数与BW133-2组没有显著差异(图18A和图18B)。当起始细胞密度为5×105/ml时，BW133-2组的扩增倍数为1.48±0.13倍，BW133-7E组的扩增倍数为2.08±0.19倍，显著高于BW133-2组(P<0.05,图18C)。以上结果说明，表达IL-7和dnTGFβRII融合蛋白的CAR-T细胞的细胞增殖能力可以根据细胞密度进行自我调控。当聚集于肿瘤病灶时，细胞增殖能力可以显著提升，而在非肿瘤部位则不会不受控地增 殖。
讨论
对于实体瘤的治疗，目前的免疫细胞疗法存在多种缺点。例如，传统的二代CAR-T细胞在体内的存活时间较短，扩增能力较弱。在二代CAR-T细胞的基础上联合表达IL-7，可以有效促进T细胞的增殖、存活、免疫记忆能力。研究表明，进一步联合表达IL-7和趋化因子CCL19或CCL21，虽然可以促进CAR-T细胞向肿瘤部位浸润和存活，但仍未能有效解决实体瘤中免疫抑制性的微环境对免疫细胞的抑制作用。
一方面，在免疫细胞中敲降或敲除TGFβ的受体，或过表达TGFβ受体的显性抑制突变体(如dnTGFβRII)，都可以有效阻断细胞内源的TGFβ信号通路，降低TGFβ对免疫细胞产生的抑制作用，从而保持免疫细胞在肿瘤微环境中的活性。然而，虽然这些技术能一定程度上提高T细胞的在肿瘤微环境中的杀伤效果，但并不能保持CAR-T细胞在体内的记忆和存续能力。
另一方面，为了解决肿瘤微环境的抑制作用和工程化免疫细胞在体内存续时间短、记忆能力差等问题，有研究在CD19 CAR-T的基础上联合表达由TGFβRII胞外域和IL-7RA胞内域组成的融合蛋白，以将TGFβ的抑制信号逆转为IL-7的激活信号。然而，完整的IL-7激活信号需要IL-2RG和IL-7RA形成复合体并共同传导。因此，所述融合蛋白仅能通过IL-7RA胞内域进行信号转导，激活程度是不完整的。此外，由于该技术需要TGFβ的存在才能激活免疫细胞的IL-7通路。因此，在当免疫细胞所处的环境中TGFβ浓度较低，便难以通过IL-7通路维持自身的存续。
在本发明中，通过上调IL-7并同时下调TGFβ通路，提高工程化免疫细胞的IL-7信号的激活程度和显著提供对肿瘤微环境的抵抗能力，从而显著而协同地提高免疫细胞地IFN-γ释放水平并增强对肿瘤细胞的杀伤效果，进而协同提高工程化免疫细胞的疗效。实验表明，本发明的工程化免疫细胞(以CAR-T细胞或TIL为例)具有显著提高的免疫细胞的增殖能力、显著减少的衰竭、显著增强对免疫抑制环境的抵抗力。在本发明中，还通过优化的IL-7和dnTGFβRII的结构，意外地降低了IL-7过量表达而造成的毒副作用的风险。
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 (15)

  1. 一种工程化免疫细胞，其特征在于，所述工程化免疫细胞为T细胞、TIL细胞、或NK细胞，并且所述的免疫细胞具有以下特征：
    (a)所述的免疫细胞表达外源的IL-7蛋白；和
    (b)所述的免疫细胞表达外源的TGFβRII显性抑制突变体蛋白或靶向内源TGFβRII转录本的shRNA。
  2. 如权利要求1所述的工程化免疫细胞，其特征在于，所述免疫细胞表达嵌合抗原受体(chimeric antigen receptor，CAR)，其中所述CAR靶向肿瘤细胞的表面标志物。
  3. 如权利要求1所述的工程化免疫细胞，其特征在于，所述的IL-7蛋白的结构如式Z如下：
    A-H-TM-D  (Z)
    式中，
    A为IL-7蛋白，或其活性片段，或其突变体；
    H为无或铰链区；
    TM为无或跨膜结构域；
    D为无或降解结构域(包括野生型、或其突变体/修饰体)；所述“-”为连接肽或肽键。
  4. 如权利要求3所述的工程化免疫细胞，其特征在于，所述H为选自下组的蛋白的铰链区：CD8、CD28、CD137、IgG、或其组合。
  5. 如权利要求3所述的工程化免疫细胞，其特征在于，所述TM为选自下组的蛋白的跨膜区：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233、CD314、或其突变/修饰体、或其组合。
  6. 如权利要求1所述的工程化免疫细胞，其特征在于，所述的IL-7蛋白和TGFβRII显性抑制突变体的结构如式I所示：
    Z0-Z1-Z2-D  (I)
    式中，
    Z0和Z2中的一个为IL-7蛋白，另一个为TGFβRII显性抑制突变体；
    Z1为无、或连接肽；
    D为无或降解结构域(包括野生型、或其突变体/修饰体)；
    所述“-”为无、连接肽或肽键。
  7. 如权利要求6所述的工程化免疫细胞，其特征在于，所述IL-7蛋白(Z0)的氨基酸序列如SEQ ID No:1或SEQ ID No:8所示。
  8. 如权利要求6所述的工程化免疫细胞，其特征在于，所述TGFβRII显性抑制突变体(Z2)的氨基酸序列如SEQ ID No:9或SEQ ID No:34所示。
  9. 如权利要求6所述的工程化免疫细胞，其特征在于，所述的TGFβRII显性抑制突变体蛋白可以替换为靶向内源TGFβRII转录本(SEQ ID No:10)的shRNA表达框。
  10. 如权利要求2所述的工程化免疫细胞，其特征在于，所述CAR的结构如式II所示：
    L-scFv1-H-TM-C-CD3ζ-D  (II)
    式中，
    L为无或信号肽序列；
    scFv1为靶向肿瘤细胞的表面标志物的抗原结合结构域(如抗体或其活性片段)；
    H为无或铰链区；
    TM为跨膜结构域；
    C为共刺激信号结构域；
    CD3ζ为源于CD3ζ的胞浆信号传导序列(包括野生型、或其突变体/修饰体)；
    D为无或降解结构域(包括野生型、或其突变体/修饰体)；
    所述“-”为连接肽或肽键。
  11. 如权利要求10所述的工程化免疫细胞，其特征在于，除了式II所示的第一CAR之外，所述CAR-T细胞还含有用于针对第二抗原的第二CAR，所述的第二CAR的结构如式III所示：
    L-scFv2-H-TM-C-CD3ζ-D  (III)
    式中，
    L为无或信号肽序列；
    scFv2为靶向第二肿瘤细胞的表面标志物的抗原结合结构域(如抗体或其活性片段)；
    H为无或铰链区；
    TM为跨膜结构域；
    C为共刺激结构域；
    CD3ζ为源于CD3ζ的胞浆信号传导序列或其突变/修饰体；
    D为无或降解结构域(包括野生型、或其突变体/修饰体)；
    所述“-”为连接肽或肽键。
  12. 一种制备如权利要求1所述的工程化免疫细胞的方法，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外源的IL-7蛋白和外源的TGFβRII显性抑制突变体蛋白或靶向内源TGFβRII转录本的shRNA，从而获得权利要求1所述的工程化免疫细胞。
  13. 一种制剂，其特征在于，所述制剂含有如权利要求1所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  14. 一种如权利要求1所述的工程化免疫细胞的用途，其特征在于，用于制备预防和/或治疗癌症的药物或制剂。
  15. 一种用于制备如权利要求1所述的工程化免疫细胞的试剂盒，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IL-7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表达TGFβRII显性抑制突变体或靶向内源TGFβRII转录本的shRNA的第二表达盒。
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