WO2024041618A1 - 联合表达cd40l的工程化免疫细胞及其制备和应用 - Google Patents

联合表达cd40l的工程化免疫细胞及其制备和应用 Download PDF

Info

Publication number
WO2024041618A1
WO2024041618A1 PCT/CN2023/114797 CN2023114797W WO2024041618A1 WO 2024041618 A1 WO2024041618 A1 WO 2024041618A1 CN 2023114797 W CN2023114797 W CN 2023114797W WO 2024041618 A1 WO2024041618 A1 WO 2024041618A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
car
cell
tumor
cea
Prior art date
Application number
PCT/CN2023/114797
Other languages
English (en)
French (fr)
Inventor
黄智宏
张曦
燕妮
朱晓娜
刘秋燕
韩德平
Original Assignee
广州百吉生物制药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州百吉生物制药有限公司 filed Critical 广州百吉生物制药有限公司
Priority to CN202380012661.3A priority Critical patent/CN117940559A/zh
Publication of WO2024041618A1 publication Critical patent/WO2024041618A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of tumor immunity and cell therapy, and specifically relates to an engineered immune cell that jointly expresses CD40L.
  • Cellular immunotherapy is an emerging tumor treatment model with significant efficacy and a new autoimmune anti-cancer treatment method. It is a method that uses biotechnology and biological agents to culture, modify and amplify immune cells collected from patients in vitro and then inject them back into the patient's body to stimulate and enhance the body's own immune function, thereby achieving the purpose of treating tumors.
  • T cells are an important type of lymphocytes that participate in cellular immunity. They can specifically recognize and kill tumor cells through signal transmission from antigen-presenting cells. However, tumor cells can also be affected by the reduction or loss of antigenic epitopes, immunosuppression, and tumor heterogeneity (i.e., the same malignant tumor exists from genotype to phenotype among different patients or between tumor cells in different parts of the same patient's body). differences) and other ways to hinder the specific recognition of T cells, thereby evading the body's immune response.
  • CAR-T Chimeric antigen receptor T cell
  • a CAR molecule is an artificially designed and constructed receptor molecule, which consists of a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane region, a costimulatory domain, and an intracellular signaling domain. Therefore, CAR molecules have the functions of specifically recognizing tumor surface antigens, activating T cell killing activity, and stimulating T cell proliferation.
  • the patient's own T cells can express CAR molecules. After being reinfused back into the patient's body, T cells can efficiently and specifically recognize and kill tumor cells through CAR molecules, achieving the effect of cancer treatment.
  • the first-generation CAR-T only has a single-chain antibody as the extracellular antigen-binding domain and CD3 ⁇ as the intracellular signaling domain. It cannot fully activate the activity of T cells and has poor therapeutic effect.
  • the second-generation CAR-T introduced a costimulatory domain based on the first-generation CAR-T, which improved the in vitro proliferation ability and cytokine release level of T cells.
  • the third-generation CAR-T adds a costimulatory domain based on the second-generation CAR-T. Although it can improve the killing activity of T cells, it may induce excessive release of cytokines.
  • the new generation of CAR-T combines the expression of other cofactors on the basis of the second generation of CAR-T, such as the combined expression of IL-12 or the intracellular STAT3/5 binding domain of IL-2R ⁇ , which helps to improve tumor killing. activity and safety.
  • CAR-T therapy has achieved satisfactory results in hematological tumors, there is still much room for improvement in the therapeutic effect of CAR-T on solid tumors.
  • the main reasons include: (1) Many solid tumors are difficult to detect at an early stage. It was found that it has the characteristics of high malignancy, high recurrence rate, and poor prognosis. (2) During the treatment of solid tumors, tumor tissues often have an immunosuppressive microenvironment, which can hinder the migration and infiltration of CAR-T cells. (3) Many malignant solid tumors are also characterized by high heterogeneity. Therefore, CAR-T cell therapy for patients with malignant solid tumors such as colorectal cancer, ovarian cancer, pancreatic cancer, breast cancer, liver cancer, etc. needs to further improve its efficiency and effectiveness.
  • the purpose of the present invention is to provide an engineered immune cell (such as CAR-T cell) that is more efficient and has better therapeutic effect for malignant tumors (especially solid tumors).
  • an engineered immune cell such as CAR-T cell
  • Another object of the present invention is to provide an engineered immune cell (such as CAR-T cell) that jointly expresses CD40L and its preparation method and application.
  • an engineered immune cell such as CAR-T cell
  • a first aspect of the invention provides an engineered immune cell, the engineered immune cell is a T cell or an NK cell, and the immune cell has the following characteristics:
  • the immune cell expresses a chimeric antigen receptor (CAR), wherein the CAR targets a surface marker of a tumor cell, and wherein the antigen-binding domain of the CAR includes one targeting CEA or CD133 Antigen binding domain; and
  • CAR chimeric antigen receptor
  • the immune cells express exogenous CD40L protein.
  • the T cells include ⁇ T, ⁇ T cells, NKT cells, MAIT cells, or combinations thereof.
  • the engineered immune cells are selected from the following group:
  • CAR-NK cells Chimeric antigen receptor NK cells
  • the CD40L protein can be expressed constitutively or inducibly.
  • a chimeric antigen receptor T cell (CAR-T cell) is provided, and the CAR-T cell has one or more of the following characteristics:
  • the cells express a chimeric antigen receptor CAR that targets a surface marker of a tumor cell
  • CAR and CD40L protein are expressed in tandem.
  • CAR and CD40L proteins are expressed independently.
  • the "activation" refers to the binding of the CAR to surface markers of tumor cells.
  • the "tumor surface marker” refers to a specific antigen on the tumor surface.
  • 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 CD40L protein is secreted and expressed.
  • the structure of the CAR is shown in Formula I: L-ABD-H-TM-C-CD3 ⁇ (I)
  • L is none or signal peptide sequence
  • ABD antigen-binding domain
  • CEA or CD133 such as CEA scFv or CD133 scFv
  • H is no or hinge area
  • TM is the transmembrane domain
  • C is the costimulatory signal domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ (including wild type, or mutants/modified forms thereof);
  • the "-" refers to the connecting peptide or peptide bond.
  • the ABD can be derived from a single-chain variable fragment (scFv) of a traditional antibody containing a light chain and a heavy chain, or can be derived from a single domain containing only a heavy chain.
  • the heavy chain variable region fragment of the antibody (variable domain of heavy chain antibody, V HH ).
  • the L is selected from the signal peptide of the following group of proteins: CD8, GM-CSF, CD4, CD28, CD137, or mutations/modifications thereof, or combinations thereof.
  • the H is selected from the hinge region of the following group of proteins: CD8, CD28, CD137, IgG, or a combination thereof.
  • the TM is selected from the transmembrane region of the following group of proteins: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 , CD137, CD154, CD278, CD152, CD279, CD233, CD314, or mutations/modifications thereof, or combinations thereof.
  • the C is selected from the costimulatory domain of the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dapl0 , CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, or mutations/modifications thereof, or combinations thereof.
  • proteins OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dapl0 , CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, or mutations/modifications thereof, or combinations thereof.
  • C is a costimulatory domain derived from 4-1BB.
  • the amino acid sequence of the CEA scFv is shown in SEQ ID NO:4; the amino acid sequence of the CD133 scFv is shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15 .
  • the CAR also has a human IgG Fc domain, and the amino acid sequence of the human IgG Fc domain is shown in SEQ ID NO: 12.
  • the CD40L protein includes full-length CD40L protein or an active fragment thereof (that is, an active fragment or a mutant thereof that retains the function of binding to CD40).
  • amino acid sequence of the CD40L protein is shown in SEQ ID NO: 2.
  • the CAR cell in addition to the first CAR shown in Formula I, also contains a second CAR for targeting the second antigen, and the structure of the second CAR is shown in Formula II: L-ABD2-H-TM-C-CD3 ⁇ (II)
  • L is none or signal peptide sequence
  • ABD2 is the antigen-binding domain or active fragment thereof targeting the second target.
  • ABD2 can be derived from the single-chain variable region fragment scFv of a traditional antibody containing light and heavy chains, or from a single-domain antibody containing only heavy chains.
  • H is no or hinge area
  • TM is the transmembrane domain
  • C is the costimulatory domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ or a mutation/modification thereof;
  • the "-" refers to the connecting peptide or peptide bond.
  • the scFv is a single-chain variable region sequence of an antibody targeting a tumor antigen.
  • the VHH is the heavy chain variable region sequence of a single domain antibody targeting a tumor antigen.
  • the scFv and VHH are antibody single chain variable region sequences targeting antigens selected from the following group: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, CD276, GUCY2C, CD24, CD133, CD44, CD166, CEACAM5, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, ErbB2, NKG2D ligands (ligands), LMP1, EpCAM, EphA2, VEGFR-1, Lewis-Y, ROR1, Claudin18.2, TAG-72 or combinations thereof.
  • first CAR represented by Formula I and the second CAR represented by Formula II can be combined into one, thereby forming a CAR represented by Formula IIIa or IIIb: L-ABD1-ABD2-H-TM-C-CD3 ⁇ (IIIa) L-ABD2-ABD1-H-TM-C-CD3 ⁇ (IIIb)
  • L is none or signal peptide sequence
  • ABD1 is an antigen-binding domain or active fragment targeting CEA or CD133. It can be derived from a single-chain variable region fragment scFv of a traditional antibody containing light and heavy chains, or from a single-domain antibody containing only a heavy chain. Chain variable region fragment V HH ;
  • ABD2 is the antigen-binding domain of the second target or its active fragment; ABD2 can be derived from the single-chain variable region fragment scFv of a traditional antibody containing light and heavy chains, or it can be derived from the heavy chain of a single-domain antibody containing only heavy chains. Chain variable region fragment V HH ;
  • H is no or hinge area
  • TM is the transmembrane domain
  • C is the costimulatory domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ or a mutation/modification thereof;
  • the "-" refers to the connecting peptide or peptide bond.
  • a second aspect of the present invention provides a method for preparing the engineered immune cells described in the first aspect of the present invention, comprising the following steps:
  • (B) Transform the immune cells so that the immune cells express CAR molecules and exogenous CD40L protein, thereby obtaining the engineered immune cells according to the first aspect of the present invention, wherein the CAR targets A surface marker of tumor cells, wherein the antigen-binding domain of the CAR includes an antigen-binding domain targeting CEA or CD133.
  • step (B) includes:
  • Step (B1) described therein can be performed before, after, simultaneously, or alternately with step (B2).
  • a method for preparing the CAR-T cells of the present invention comprising the following steps:
  • step (B) includes (B1) introducing a first expression cassette expressing the CAR into the T cell; and (B2) introducing a second expression cassette expressing CD40L into the T cell. cells; step (B1) may be performed before, after, simultaneously, or alternately with step (B2).
  • step (B) when the T cells to be modified in step (A) already express CAR, then in step (B), the second expression cassette including (B2) is introduced into the T cells.
  • the transcription directions of the first expression cassette and the second expression cassette are in the same direction ( ⁇ ), opposite ( ⁇ ) or opposite ( ⁇ ).
  • first expression cassette and the second expression cassette are located on the same or different vectors.
  • first expression cassette and the second expression cassette are located in the same vector.
  • a third expression cassette for expressing the connecting peptide is further included between the first and second expression cassettes.
  • the connecting peptide is P2A or T2A or F2A.
  • the upstream of the connecting peptide contains a Furin cleavage site and or an SGSG connecting sequence.
  • the vector is a viral vector, preferably the viral vector contains the first and second expression cassettes in tandem form.
  • the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or combinations thereof.
  • the vector is a pCDH series lentiviral vector.
  • a third aspect of the present invention provides a preparation, which contains the engineered immune cells described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the preparation contains the CAR-T cells of 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 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.
  • the preparation also contains PBMC.
  • a fourth aspect of the present invention provides the use of engineered immune cells as described in the first aspect of the present invention for preparing drugs or preparations for preventing and/or treating cancer or tumors.
  • CAR-T cells as described in the first aspect of the present invention is provided for preparing drugs or preparations for preventing and/or treating cancer or tumors.
  • the preparation contains CAR-T cells and pharmaceutically acceptable carriers, diluents or excipients.
  • the medicine or preparation also contains PBMC.
  • the tumor is selected from the group consisting of solid tumors, or a combination thereof.
  • the tumor is selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, and liver cancer.
  • the tumor is a tumor with high expression (positive) of CEA or CD133.
  • the tumor is a tumor with low CEA expression or CEA negative.
  • the tumor is a tumor with low expression of CD133 or negative CD133.
  • the tumor is a tumor with high CD40 expression (positive).
  • the tumor is a tumor with low CD40 expression or CD40 negative.
  • the tumor is a tumor with high expression (positive) of NKG2D ligand.
  • the NKG2D ligand is selected from the following group: MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP-4, ULBP-5, ULBP-6, or a combination thereof.
  • the tumor is (a) a tumor with high expression of CEA or CD133 and (b) high expression of CD40.
  • the tumor is (a) a tumor with high expression of CEA or CD133 and (b) low expression of CD40.
  • the tumor is (a) a tumor with low expression of CEA or CD133 and (b) high expression of CD40.
  • the tumor is (a) a tumor with low expression of CEA or CD133 and (b) low expression of CD40.
  • the tumor is (a) a tumor with high expression of CEA or CD133 and (b) high expression of NKG2D ligand.
  • the fifth aspect of the present invention provides a kit for preparing the engineered immune cells according to the first aspect of the present invention.
  • the kit contains a container, and located in the container:
  • kits for preparing the engineered immune cells according to the first aspect of the present invention contains a container, and located in the container:
  • first and second nucleic acid sequences are independent or connected.
  • first and second nucleic acid sequences are located in the same or different containers.
  • first and second nucleic acid sequences are located on the same or different vectors.
  • first and second nucleic acid sequences are located in the same vector.
  • a third nucleic acid sequence is also included between the first and second nucleic acid sequences, and the third nucleic acid sequence contains A third expression cassette for expressing the linker peptide.
  • the connecting peptide is P2A or T2A or F2A.
  • the upstream of the connecting peptide contains a Furin cleavage site and or an SGSG connecting sequence.
  • the vector is a viral vector, preferably the viral vector contains the first and second nucleic acid sequences in tandem form.
  • Figure 1 shows the structure of CAR molecules through the ages.
  • Figure 2 shows the structure of the CAR molecule.
  • Figure 3 shows the expression rates of CEA and CD133 CAR-T cells detected by flow cytometry.
  • Figure 4 shows the expression rate of each NKG2D CAR-T cell detected by flow cytometry.
  • Figure 5 shows the expression rates of CEA and CD133 in target cells detected by flow cytometry.
  • Figure 6 shows the expression rate of NKG2D ligand (MICA/MICB) in target cells detected by flow cytometry.
  • Figure 7 shows the expression rate of NKG2D ligand (ULBP-1) in target cells detected by flow cytometry.
  • Figure 8 shows the expression rate of NKG2D ligand (ULBP-2/5/6) in target cells detected by flow cytometry.
  • Figure 9 shows the expression rate of NKG2D ligand (ULBP-3) in target cells detected by flow cytometry.
  • Figure 10 shows the expression rate of NKG2D ligand (ULBP-4) in target cells detected by flow cytometry.
  • Figure 11 shows the expression rate of CD40 in target cells detected by flow cytometry.
  • Figure 12 shows the killing effect of each CAR-T cell on target-positive tumor cells detected by EuTDA.
  • Figure 13 shows the IFN- ⁇ release levels of CEA and NKG2D CAR-T cells detected by ELISA.
  • Figure 14 shows the luciferase method to detect the killing effect of CEA CAR-T on HCT116 cells.
  • Figure 15 shows the luciferase method to detect the killing effect of PBMC in assisting CEA CAR-T in killing SW480 cells.
  • Figure 16 shows the level of activation of CEA CAR-T cells by B cells.
  • Figure 17 shows the tumor suppressive function of CEA CAR-T cells in animal efficacy experiments.
  • the inventors jointly expressed specific CAR and CD40L proteins, that is, CAR and CD40L targeting CEA or CD133, in CAR-T cells for the first time.
  • the immune cells of the present invention can not only target malignant solid tumors such as colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, liver cancer, etc. through CEA or CD133 CAR, but can also effectively activate CD40L in the body.
  • Source of natural and adaptive immune responses thus helping T cells overcome the immunosuppressive tumor microenvironment, collaboratively kill tumor cells, improve tumor treatment, and reduce the risk of tumor recurrence. On this basis, the present invention was completed.
  • the present invention takes CAR-T cells as an example to representatively describe in detail the engineered immune cells of the present invention.
  • the engineered immune cells of the present invention are not limited to the CAR-T cells described in the context.
  • the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described in the context. Specifically, when immune cells express chimeric antigen receptor CARs, NK cells are equivalent to T cells (or T cells can be replaced by NK cells).
  • administering refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenously, intratumorally, intramuscularly, subcutaneously, intraperitoneally , spinal or other parenteral route of administration, such as by injection or infusion.
  • antibody shall include, but is not limited to, immunoglobulins, including traditional antibodies and single domain antibodies.
  • traditional antibodies have the activity of specifically binding antigens and include at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or the antigen-binding portion thereof.
  • H chain contains a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (herein abbreviated as VL) and a light chain constant region.
  • the light chain constant region contains a constant domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Each VH and VL contains 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 binding domains that interact with the antigen.
  • single domain antibodies only contain one heavy chain variable region (V HH ) and CH2 and CH3 regions.
  • V HH heavy chain variable region
  • CH2 and CH3 regions CH2 and CH3 regions.
  • the light chain and CH1 region of single domain antibodies are naturally missing. They are the smallest naturally occurring fragments that can bind to antigens and are also called nanobodies.
  • antigen binding domain refers to a Fab fragment, a Fab' fragment, an F(ab') fragment, a single Fv fragment, or a V HH fragment from a single domain antibody that has antigen-binding activity .
  • Fv antibodies are the smallest antibody fragments that contain an antibody heavy chain variable region, a light chain variable region, but no constant region, and have all antigen-binding sites. Typically, 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 a scFv or VHH .
  • Single chain antibodies are preferably one amino acid chain sequence encoded by one nucleotide chain.
  • the scFv includes CEA scFv and CD133 scFv that specifically recognize antigens highly expressed by tumors.
  • the immune cells of the present invention may also contain additional antibodies that specifically recognize antigens highly expressed by tumors, preferably single-chain antibodies, Fv antibodies or VHH .
  • 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 called an antigen-binding domain).
  • the intracellular domain includes the costimulatory domain and the CD3 ⁇ chain portion.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the CD3 ⁇ chain.
  • the antigen binding domain is fused to an intracellular domain that is a combination of a 4-1BB signaling domain and a CD3 ⁇ signaling domain.
  • CAR-T cell As used herein, the terms “CAR-T cell”, “CAR-T” and “CAR-T cell of the present invention” all refer to the CAR-T cell described in the first aspect of the present invention.
  • the CAR-T cells of the present invention can be used to treat tumors with high expression of CEA or CD133, such as colorectal cancer, ovarian cancer, pancreatic cancer, breast cancer, liver cancer, etc.
  • CAR-T cells have the following advantages over other T cell-based treatments: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigen, targeting a certain tumor Once the CAR gene construction of the antigen is completed, it can be widely used; (3) CAR can use both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) Use patients' autologous The cells reduce the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • CAR-NK cells As used herein, the terms “CAR-NK cells”, “CAR-NK” and “CAR-NK cells of the present invention” all refer to the CAR-NK cells described in the first aspect of the present invention.
  • the CAR-NK cells of the present invention can be used to treat tumors with high CEA expression, such as colorectal cancer, ovarian cancer, pancreatic cancer, etc.
  • 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 enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) directly killing tumor cells by releasing perforin and granzyme, without killing normal cells of the body; (2) they release A very small amount of cytokines thus reduces the risk of cytokine storm; (3) It is easy to amplify in vitro and develop into "off-the-shelf" products. Otherwise, it is similar to CAR-T cell therapy.
  • CEA stands for carcinoembryonic antigen and belongs to the immunoglobulin superfamily.
  • CEA usually refers to the CEACAM-5 protein (also known as CD66e) in this protein family.
  • CEA is a glycoprotein with a molecular weight of approximately 180kDa and is involved in cell adhesion.
  • CEA is usually produced in the fetal digestive system, but because it is suppressed after birth, CEA levels in the colon and blood of normal adults are usually very low.
  • various types of cancer such as colorectal cancer, lung cancer, and breast cancer, can cause elevated serum levels of CEA. Therefore, CEA is widely used as a broad-spectrum tumor marker.
  • CEA is not specific for any type of cancer, but the concentration of CEA in the blood can predict the treatment effect, progression and prognosis of cancer.
  • accession number of the amino acid sequence of human CEA protein is NP_004354.3, and the accession number of the nucleotide sequence is NM_004363.6.
  • the full-length amino acid sequence of human CEA is as follows:
  • CD133 (Prominin-1) is a member of the Prominin family of five-times-transmembrane glycoproteins. Studies have shown that CD133 is a marker for the prognosis of various tumor treatments and can interact with substances such as vascular endothelial growth factor. CD133 is also an important membrane and cytoplasmic protein involved in cell functions and can regulate tumor cell survival by affecting glucose uptake, autophagy response and ATP synthesis. CD133 is also a signature molecule that is abnormally highly expressed in colorectal cancer. CD133 is also a cancer stem cell marker. Studies have found that cancer stem cells that highly express CD133 can be isolated and identified from a variety of solid tumors, such as colon cancer, brain tumors, lung cancer, ovarian cancer, gastric cancer, melanoma, etc.
  • accession number of the amino acid sequence of human CD133 protein is NP_001139319.1, and the accession number of the nucleotide sequence is NM_001145847.2.
  • the full-length amino acid sequence of human CD133 is as follows:
  • CD40L Cluster of differentiation 40 ligand
  • CD154 tumor necrosis factor-associated activation protein
  • CD40L and its receptor CD40 are a pair of costimulatory molecules in the inflammatory and immune response systems in the body.
  • CD40L/CD40 costimulatory pathway is an important trigger in the monocyte maturation process, mainly driving the differentiation of monocytes into macrophages and DC cells of the M1 lineage.
  • this pathway can also promote the release of cytokines and chemokines from DC cells, induce the expression of other costimulatory molecules, and promote the cross-presentation of antigens.
  • this pathway is also involved in the T cell-dependent B lymphocyte response process, the formation of germinal centers, the production of long-term memory B cells, antibody production and antibody class switching.
  • this pathway can promote T cell activation and amplify T cell-mediated immune responses. It plays an important role in the differentiation process of CD4+ T cells and can also promote the expansion and pluripotency of CD8+ T cells. memory-producing CD8+ The basis of T cells.
  • the CD40L/CD40 costimulatory pathway also plays a variety of roles, such as activating T cell proliferation and cytokine release, inducing M2 lineage macrophages to migrate towards M1 lineage macrophages with anti-tumor activity. transformation etc. In tumors where certain target antigens are lost but CD40 is highly expressed, this pathway can also mediate the killing effect of T cells on tumor cells.
  • suitable CD40L includes wild-type and mutant CD40L, as long as the mutant CD40L has the basic function of wild-type CD40L.
  • preferred CD40L is from mammals, such as humans and non-human mammals.
  • accession number of the amino acid sequence of human CD40L protein is NP_000065.1, and the accession number of the nucleotide amino acid sequence is NM_000074.3.
  • the full-length amino acid sequence of human CD40L is as follows:
  • NKG2D protein can recognize a variety of target antigens (including MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6) on the surface of malignant tumor cells such as colorectal cancer, ovarian cancer, and pancreatic cancer. Its extracellular domain can be used as The antigen-binding domain of the CAR molecule.
  • NKG2D includes wild type or mutant forms thereof or derivative forms thereof or active fragments thereof.
  • Preferred NKG2Ds are NKG2Ds derived from mammals (eg, humans and non-human primates).
  • accession number of the amino acid sequence of human NKG2D protein is NP_031386, and the accession number of the nucleotide amino acid sequence is NM_007360.
  • the amino acid sequence of the extracellular domain of human NKG2D is as follows:
  • expression cassette or "expression cassette of the invention” includes a first expression cassette and a second expression cassette.
  • the expression cassette of the present invention is as described in the fifth aspect of the present invention, and the first expression cassette contains the nucleic acid sequence encoding the CAR.
  • the second expression cassette expresses exogenous CD40L protein.
  • the CD40L protein can be expressed constitutively or inducibly.
  • the second expression cassette expresses the CD40L protein; in this way, when the CAR-T cells of the present invention do not contact tumor cells or are not exposed to corresponding inducers, the second expression cassette expresses CD40L protein. When used as an inducer, the second expression cassette does not express CD40L protein.
  • the first expression cassette and the second expression cassette respectively further include a promoter and/or a terminator.
  • the promoter of the second expression cassette can be a constitutive or inducible promoter.
  • the invention also provides vectors containing the expression cassette of the invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for long-term gene transfer because they allow long-term, stable integration of transgenes into the cellular matrix. Replicated within the genome and as the genome of the daughter cell replicates.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus in that they can transduce non-proliferating cells and have the advantage of low immunogenicity.
  • the expression cassette or nucleic acid sequence of the invention can be ligated downstream of the promoter and incorporated into an expression vector by routine operations.
  • the vector can be integrated into the eukaryotic cell genome and subsequently replicated.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate 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 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, for example, in 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, herpesviruses, and lentiviruses.
  • a suitable vector will contain at least one origin of replication functional in the organism, a promoter sequence, convenient restriction enzyme sites and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected genes can be inserted into the vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • lentiviral vectors are used.
  • DNA virus systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • promoter elements can modulate the frequency with which transcription is initiated.
  • these elements are located in a region of 30-110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when one element is inverted or moved relative to another element.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased by 50 bp before activity begins to decrease.
  • individual elements appear to act cooperatively or independently to initiate transcription.
  • a suitable promoter is the 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 (EBV) immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters such as but not limited to actin protein promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • EBV Epstein-Barr virus
  • Ruth's sarcoma virus promoter and human gene promoters such as but not limited to actin protein promoter, myosin promoter,
  • an inducible promoter provides a molecular switch that enables expression of the polynucleotide sequence linked to the inducible promoter to be turned on when desired, or turned off when not desired.
  • inducible promoters include, but are not limited to, metallothionein promoters, Glucocorticoid promoter, progesterone promoter, and tetracycline promoter.
  • the expression vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a transfected or infected cell population by the viral vector.
  • the selectable marker can be carried on a separate stretch of DNA and used in co-transfection procedures.
  • Both the selectable marker gene and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neomycin and the like.
  • the vector can be readily introduced into a host cell, eg, a mammalian (eg, human T cell), bacterial, yeast or insect cell, by any method known in the art.
  • a host cell eg, a mammalian (eg, human T cell), bacterial, yeast or insect cell, 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 of producing cells including vectors and/or exogenous nucleic acids 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 polyethylenimine transfection.
  • Biological methods of introducing polynucleotides into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenovirus and adeno-associated virus, among others. See, for example, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Exemplary colloidal systems useful as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).
  • an exemplary delivery vehicle is liposomes.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids can be encapsulated into the aqueous interior of the liposomes, dispersed within the lipid bilayer of the liposomes, attached via linker molecules associated with both the liposomes and the oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexed with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in solution.
  • Lipids are lipid substances, which may be naturally occurring or synthetic lipids.
  • lipids include lipid droplets that occur naturally in the cytoplasm as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the invention provides a device containing the engineered immune cells (such as CAR-T cells) described in the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation For liquid preparations.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • the present invention includes therapeutic applications of cells (eg, T cells) transduced with vectors (eg, lentiviral vectors) containing expression cassettes of the invention.
  • the transduced T cells can target surface markers of tumor cells and express CD40L protein, synergistically and significantly improving their killing efficiency against tumor cells.
  • the present invention also provides a method for stimulating an immune response mediated by T cells targeting a mammalian tumor cell population or tissue, which includes the following steps: administering the CAR-T cells of the present invention to the mammal.
  • the present invention includes a type of cell therapy in which a patient's autologous T cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • This method makes the probability of graft-versus-host reaction extremely low, and the antigen is recognized by T cells in an MHC-free manner.
  • one CAR-T can treat all cancers that express this antigen.
  • CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the CAR-T 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 the adoptive immunotherapy step, in which CAR-T cells can induce a specific immune response on tumor cells with high expression of the antigen recognized by the CAR antigen-binding domain.
  • the CAR-T cells of the present invention elicit specific immune responses against tumor cells with high expression of CEA or CD133.
  • Treatable cancers include tumors that are not vascularized or substantially unvascularized, as well as tumors that are vascularized.
  • Cancer types treated with the CAR of the present invention include, but are not limited to: colorectal cancer, gastric cancer, ovarian cancer, breast cancer, liver cancer and pancreatic cancer.
  • cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors. Accordingly, the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell of the invention.
  • the CAR-T cells of the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • a pharmaceutical composition of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions of the present invention may 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.
  • compositions of the invention to be administered can be determined by the physician, who takes into account the patient (subject) ) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease.
  • Pharmaceutical compositions including T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 7 cells/kg body weight (including all integer values within the range). T cell compositions can also be administered multiple times at these dosages.
  • Cells can be injected using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally.
  • the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by intravenous injection.
  • the composition of T cells can be injected directly into the tumor, lymph node or site of infection.
  • cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., before , simultaneously or subsequently) administered to a patient, such forms of treatment include, but are not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as for ARA-C) or natalizumab treatment in patients with MS or elfalizumab treatment in patients with psoriasis or other treatments in patients with PML.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as for ARA-C) or natalizumab treatment in patients with MS or elfalizumab treatment in patients with psoriasis or other treatments in patients with PML.
  • the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cellular compositions of the invention are administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, use of a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatments administered to a patient will vary depending on the precise nature of the condition being treated and the recipient of the treatment. Dosage proportions for human administration may be implemented in accordance with art-accepted practice. Generally, 1 ⁇ 10 5 to 1 ⁇ 10 10 modified T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, by intravenous infusion.
  • the engineered immune cells of the present invention can not only specifically target target antigens on the surface of malignant tumor cells through CEA or CD133 CAR molecules, but also activate the body's endogenous immune system through CD40L, improving the therapeutic effect while also Prevent tumor recurrence.
  • CD40L also unexpectedly promoted a significant increase in the expression of CAR molecules targeting CEA or CD133.
  • the in vitro killing effect on tumor cells can be significantly improved synergistically, and the tumor suppressive effect can be significantly improved and tumor recurrence can be prevented in mouse models.
  • NKG2D CAR-T cells were used as controls for CEA and CD133 CAR-T cells.
  • Each CAR molecule contains the following partial structures: human CD8 signal peptide [CD8(SP)], anti-human CEA single chain antibody [CEA scFv], anti-human CD133 single chain antibody [CD133 scFv], human NKG2D extracellular domain [NKG2D(ED)], optimized human CD8 hinge region [CD8(hinge)], human IgG Fc domain [IgG(Fc)], human CD8 transmembrane domain [CD8(TM)], Human 4-1BB intracellular domain [referred to as 4-1BB(ID)], human CD3 ⁇ intracellular signal transduction domain [referred to as CD3 ⁇ (ID)], self-cleaving peptide P2A or F2A, and human CD40L.
  • CD8(SP) human CD8 signal peptide
  • CEA scFv anti-human CEA single chain antibody
  • CD133 scFv anti-human
  • the new generation CEA CAR molecule that jointly expresses CD40L is named BC004, and the second generation CEA CAR molecule used as a control is named BC001;
  • the new generation NKG2D CAR molecule that jointly expresses CD40L is named BN004, and the second generation NKG2D CAR molecule used as a control is named BN001;
  • the second generation CD133 CAR molecule is named BW133-2.
  • the specific structure is shown in Figure 2:
  • BC001 is composed of CD8(SP), CEA scFv, CD8(hinge), CD8(TM), 4-1BB(ID), and CD3 ⁇ (ID) in series from its amino terminus to its carboxyl terminus;
  • BC004 is composed of CD8(SP), CEA scFv, CD8(hinge), CD8(TM), 4-1BB(ID), CD3 ⁇ (ID), P2A, and CD40L in series from its amino terminus to its carboxyl terminus;
  • BW133-2 is composed of CD8 (SP), CD133 scFv, IgG (Fc), CD8 (TM), 4-1BB (ID), and CD3 ⁇ (ID) in series from its amino terminus to its carboxyl terminus;
  • BN001 is composed of CD8(SP), NKG2D(ED), CD8(hinge), CD8(TM), 4-1BB(ID), and CD3 ⁇ (ID) in series from its amino terminus to its carboxyl terminus;
  • BN004 is composed of CD8(SP), NKG2D(ED), CD8(hinge), CD8(TM), 4-1BB(ID), CD3 ⁇ (ID), P2A, and CD40L in series from its amino terminus to its carboxyl terminus.
  • nucleotide sequences of BC001, BC004, BW133-2, BN001 and BN004 were completely synthesized, codon optimized, and then connected to the lentiviral vector pCDH-EF1-MCS-T2A-copGFP plasmid through molecular cloning. It is expressed under the control of human EF1 ⁇ promoter and Kozak sequence.
  • the cells in each well were gently mixed and transferred to a 1.5 ml centrifuge tube, washed twice with staining buffer (100 ml PBS+1% BSA), and centrifuged at 800 g for 3 min each time.
  • staining buffer 100 ml PBS+1% BSA
  • the above cells were stained with corresponding antibodies, and then flow cytometry was used to detect the proportion of Jurkat cells successfully transduced by lentivirus.
  • BC001 titer was 2.58 ⁇ 10 9 TU/ml
  • BC004 titer was 3.26 ⁇ 10 8 TU/ml
  • BN001 titer was 5.46 ⁇ 10 8 TU/ml
  • BN004 titer was 6.31 ⁇ 10 8 TU/ml
  • the titer of BW133-2 is 4.28 ⁇ 10 8 TU/ml.
  • the obtained T cells were washed, and the cell density was adjusted to 4 ⁇ 10 6 /ml.
  • Add lentivirus at MOI 1 ⁇ 10TU/cell for transduction, and supplement 50ng/ml anti-CD3 antibody, 50ng/ml anti-CD28 antibody, and 200IU/ml recombinant IL-2, and culture in a cell culture incubator ( The culture temperature is 37°C and the carbon dioxide concentration is 5%). After 24 hours, the cell density was adjusted to 1.5-2 ⁇ 10 6 /ml, and 300IU/ml IL-2 was supplemented.
  • CAR-T cells follow the naming of the corresponding CAR molecules, namely BC001, BC004, BW133-2, BN001 and BN004.
  • the CAR-T cells obtained by co-transduction of BW133-2 and lentivirus overexpressing CD40L are named BW133- 2+CD40L, T cells that were not transduced with lentivirus were named Ctrl T.
  • the Ctrl T, BC001 and BC004 cells to be detected were washed twice with PBS, resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA) and subjected to flow cytometric detection.
  • the results are shown in Figure 3A.
  • the CD40L expression rate of BC001 cells is approximately 17.67%, and the CD40L expression rate of BC004 cells is approximately 70.93%, indicating that exogenous CD40L protein has been successfully expressed in BC004 cells.
  • the CD40L expression rate of BW133-2 cells was approximately 55.7%, and the CD40L expression rate of BW133-2+CD40L cells was approximately 92.4%, indicating that exogenous CD40L protein has been successfully expressed in BW133-2+CD40L cells.
  • the CD40L expression rate of BN001 is approximately 27.91%, and the CD40L expression rate of BN004 is approximately 84.88%, indicating that exogenous CD40L protein has been successfully expressed in BN004 cells.
  • LS174T (EMEM medium + 10% fetal calf serum + 100 U/ml penicillin + 100 ⁇ g/ml streptomycin), LoVo (F-12K medium + 10% fetal calf serum + 100 U/ml penicillin + 100 ⁇ g/ml streptomycin) ), SW480 (Leibovitz's L-15 medium + 10% fetal bovine serum + 100U/ml penicillin + 100 ⁇ g/ml streptomycin), HCT116 (McCoy's 5A medium + 10% fetal calf serum + 100U/ml penicillin + 100 ⁇ g/ ml streptomycin), Caco-2 (EMEM medium + 20% fetal calf serum + 100 U/ml penicillin + 100 ⁇ g/ml streptomycin).
  • the cells to be detected were washed twice with PBS and resuspended in FACS buffer. Add FITC-labeled anti-CEA antibody to the cell suspension to be detected according to the antibody instructions, and incubate at 4°C for 60 minutes. Target cells incubated without antibody were used as negative controls, and flow cytometry was used to detect the CEA expression rate of target cells. Analyzed using CytExpert software.
  • the results are shown in Figure 5A.
  • the CEA expression rate of LS174T was approximately 90.7%
  • the CEA expression rate of LoVo cells was approximately 60%
  • the CEA expression rate of SW480 was approximately 8.2%
  • the CEA expression rate of HCT116 was approximately 2.5%.
  • the cells to be detected were washed twice with PBS and resuspended in FACS buffer. Add APC-labeled anti-CD133 antibody to the cell suspension to be detected according to the antibody instructions, and incubate at 4°C for 60 minutes. Target cells incubated without antibody were used as negative controls, and flow cytometry was used to detect the CD133 expression rate of target cells. Analyzed using CytExpert software. The results are shown in Figure 5B, the CD133 expression rate of Caco-2 exceeded 99%.
  • the above target cells were washed twice with PBS and resuspended in FACS buffer. According to the antibody instructions, APC was Labeled anti-human MICA/MICB antibodies were added to each target cell suspension and incubated at 4°C for 30 minutes. Target cells incubated without adding antibodies were used as negative controls, and flow cytometry was used to detect the MICA/MICB expression rate of target cells. Analyzed using CytExpert software.
  • the results are shown in Figure 6.
  • the MICA/MICB expression rate of LS174T is approximately 96.6%
  • the MICA/MICB expression rate of LoVo is approximately 7.8%
  • the MICA/MICB expression rate of Caco-2 is approximately 5.7%.
  • the above target cells were washed twice with PBS and resuspended in FACS buffer. Add PE-labeled anti-human ULBP-1 antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 minutes. Target cells incubated with IgG were used as negative controls, and flow cytometry was used to detect the ULBP-1 expression rate of target cells. Analyzed using CytExpert software.
  • the results are shown in Figure 7.
  • the ULBP-1 expression rate of LS174T was approximately 1.8%
  • the ULBP-1 expression rate of LoVo was approximately 9.1%
  • the ULBP-1 expression rate of Caco-2 was approximately 13.1%.
  • the above target cells were washed twice with PBS and resuspended in FACS buffer. Add PE-labeled anti-human ULBP-2/5/6 antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 minutes. Target cells incubated with IgG were used as negative controls, and flow cytometry was used to detect the expression rate of ULBP-2/5/6 in target cells. Analyzed using CytExpert software.
  • the results are shown in Figure 8.
  • the ULBP-2/5/6 expression rate of LS174T is approximately 33.4%
  • the ULBP-2/5/6 expression rate of LoVo is approximately 86.8%
  • the ULBP-2/5/6 expression rate of Caco-2 The expression rate is approximately 78.2%.
  • the above target cells were washed twice with PBS and resuspended in FACS buffer. Add PE-labeled anti-human ULBP-3 antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 minutes. Target cells incubated with IgG were used as negative controls, and flow cytometry was used to detect the ULBP-3 expression rate of target cells. Analyzed using CytExpert software.
  • the results are shown in Figure 9.
  • the ULBP-3 expression rate of LS174T was approximately 46.6%
  • the ULBP-3 expression rate of LoVo was approximately 21.1%
  • the ULBP-3 expression rate of Caco-2 was approximately 3.9%.
  • the above target cells were washed twice with PBS and resuspended in FACS buffer. Add PE-labeled anti-human ULBP-4 antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 minutes. Target cells incubated with IgG were used as negative controls, and flow cytometry was used to detect the ULBP-4 expression rate of target cells. Analyzed using CytExpert software.
  • the results are shown in Figure 10.
  • the ULBP-4 expression rate of LS174T was approximately 82.0%
  • the ULBP-4 expression rate of LoVo was approximately 82.2%
  • the ULBP-4 expression rate of Caco-2 was approximately 5.3%.
  • the above target cells were washed twice with PBS and resuspended in FACS buffer. Add APC-labeled anti-human CD40 antibody to each target cell suspension according to the antibody instructions, and incubate at 4°C for 60 minutes. Target cells incubated without antibody were used as negative controls, and flow cytometry was used to detect the CD40 expression rate of target cells. Analyzed using CytExpert software.
  • Target cells once with AIM-V medium. Adjust the target cell density to 1 ⁇ 10 6 /ml, add DELFIA BATDA Reagent at a dosage of 2 ⁇ l/ml, mix well, and incubate at 37°C for 30 minutes. After washing the target cells three times with AIM-V medium, the target cells were seeded in a 96-well plate at a density of 1 ⁇ 10 4 /well. Add 100 ⁇ l of T cells (effect-to-target ratios are 2.5:1, 5:1 and 10:1) and place them in a carbon dioxide incubator for 2 hours (culture temperature is 37°C and carbon dioxide concentration is 5%).
  • CEA CAR-T cells All were higher than the expression rate of CEA (60%). However, under the same effect-to-target ratio, the killing rate of CEA CAR-T cells on target cells is significantly higher than that of NKG2D CAR. The reason may be that the binding ability of CEA scFv to the corresponding target is stronger than that of NKG2D, indicating that for CEA CAR-T The cells are more suitable for killing tumor cell lines with high CEA expression (although they also highly express NKG2D ligand).
  • the expression rate of CD40 was lower than 3%, which may be the reason why the increase in the specific killing rate of BN004 compared to BN001 was not detected (P>0.05).
  • the specific killing rate of BC004 in each effective target ratio was significantly higher than that of BC001 (P ⁇ 0.05), with an increase of 10%-16%, indicating that the combined expression of CD40L in CEA CAR-T cells can be effective Improve the killing effect of T cells on tumor cells.
  • the EuTDA method was used to detect the killing level of Caco-2 by BW133-2 and BW133-2+CD40L. The results are shown in Table 3 and Figure 12B. Compared with Ctrl T, BW133-2, BW133-2+CD40L, BN001 and BN004 have significant killing effects on Caco-2 cells. Taking the killing rate of Ctrl T as the background value, the specific killing rate of each CAR-T cell on target cells can be obtained after deducting the corresponding background value (Table 4).
  • the CD40 expression rate of Caco-2 cells was lower than 3%, which may be the reason why no increase in the specific killing rate of BN004 compared to BN001 was detected (P>0.05).
  • the specific killing rate of BW133-2+CD40L was significantly higher than that of BW133-2 (P ⁇ 0.05), with an increase of 8.3%-18.1%, indicating that the combined expression of CD40L in CD133 CAR-T cells can effectively improve its effect on tumors. Cell killing effect.
  • Ctrl T, BC001, BC004, BN001 and BN004 were co-cultured with the corresponding target cells in AIM-V medium without IL-2 (the effect-to-target ratio was 2.5:1). After 24 hours, dissolve the INF- ⁇ standard with ddH 2 O, leave it at room temperature for 15 to 20 minutes to ensure full dissolution, and dilute the standard according to the recommended gradient ratio. Aspirate the above-mentioned co-cultured cell supernatant and dilute it 2-fold and 20-fold with ddH 2 O. Add the standard and experimental samples to the corresponding reaction wells respectively, 100 ⁇ l per well.
  • the luciferase method was used to detect the killing levels of BC001 and BC004 on CEA-negative CD40-positive HCT116 cells (firefly luciferase was overexpressed in these cells).
  • the target cells were seeded in a 96-well plate at a density of 1 ⁇ 10 4 /well. Add 100 ⁇ l of T cells (effect-to-target ratios are 0.5:1, 1:1 and 2:1) and place them in a carbon dioxide incubator for 24 hours (culture temperature is 37°C and carbon dioxide concentration is 5%).
  • the cell pellet was collected by centrifugation at 500 ⁇ g for 5 min, resuspended in 50 ⁇ l of RPMI-1640 medium, and 50 ⁇ l of Steady-Glo luciferase detection reaction reagent was added to each well. After mixing with a gun, take out 80 ⁇ l of the mixed solution and add it to a black 96-well plate. Place it in a microplate reader to detect the luciferase content of the remaining HCT116 viable cells in each sample. After HCT116 cells are killed by CAR-T cells, they rupture and release intracellular luciferase into the culture supernatant. Therefore, the lower the luciferase signal measured from the collected cell pellets, it means that there are fewer remaining HCT116 viable cells and the better the killing effect of T cells.
  • the luciferase method was used to detect the killing levels of BC001 and BC004 on CEA and CD40 double-negative SW480 cells (the cells overexpressed firefly luciferase). The results are shown in Table 8 and Figure 15A. Under each effect-to-target ratio, the killing effects of BC001 and BC004 were weak, and there was no significant difference (P>0.05).
  • Table 9 ELISA detects the degree of activation of PBMC-assisted CEA CAR-T cells by SW480 cells
  • CD40L can promote the killing effect of CEA CAR-T cells on target-negative tumor cells. Therefore, its safety also needs to be evaluated.
  • BC001 and BC004 were co-cultured with CD40-positive and CEA-negative B cells respectively, and the release level of IFN- ⁇ in the supernatant was detected. The results are shown in Figure 16A. Compared with Ctrl T cells, the IFN- ⁇ release levels of BC001 and BC004 after co-culture with B cells did not increase significantly (P>0.05), indicating that normal cells that highly express CD40 do not BC004 will be activated.
  • B cells were stained with live cell fluorescent dyes, and then the IncuCyte live cell real-time imaging analysis system was used to observe whether BC001 and BC004 would affect the long-term growth of B cells.
  • the results are shown in Figure 16B, Within 48 hours of co-culture, the growth of B cells co-cultured with BC004 was not significantly affected, indicating that although BC004 can kill target-negative tumor cells, it has no significant killing effect on normal cells that highly express CD40, and has great Good security.
  • NSG-B2M mice aged 6 to 8 weeks were selected for subcutaneous tumor drug efficacy experiments, and were randomly divided into 3 groups of 8 mice each, namely the Ctrl T control group, the BC001 control group, and the BC004 experimental group.
  • CAR-T cells (1 ⁇ 10 7 /mouse) and Ctrl T cells (1 ⁇ 10 7 /mouse) were injected through the tail vein.
  • mice injected with BC001 had reached 430 ⁇ 378 mm 3 , while no tumor recurrence was seen in mice injected with BC004.
  • the results show that the combination of CEA CAR-T cells expressing CD40L can not only eliminate tumors quickly and effectively, but also effectively inhibit the recurrence of tumors.
  • CEA is mainly expressed in the epithelial cells of the digestive tract during the embryonic stage and stops expression after birth. Therefore, the expression level of CEA in healthy tissue cells is extremely low.
  • studies have found that CEA is expressed at a high level in various tumor tissues such as colorectal cancer cells and is related to the adhesion and invasion of tumor cells.
  • CD133 is a marker for the prognosis of various tumor treatments and a cancer stem cell marker.
  • CEA and CD133 as tumor treatment targets, can enable CAR-T cells to effectively target solid tumor cells with extremely high safety and have important application value in tumor cell therapy.
  • the present invention combines expression of CD40L with CEA CAR and CD133 CAR targeting broad-spectrum solid tumor antigens. Therefore, the CAR-T cells in the present invention can efficiently target malignant solid tumor cells through CEA CAR and CD133 CAR molecules. At the same time, they can also combine CD40 on tumor cells or immune cells with jointly expressed CD40L and costimulate through CD40/CD40L. pathway, activating the proliferation of T cells and the release of cytokines, and at the same time improving the response of T cells to tumor cells that have lost certain antigens but have high CD40 expression. cell killing effect.
  • CD40L does not depend on the improvement of CAR-T cell function.
  • the scheme of the present invention that combines expression of CD40L with CEA CAR and CD133 CAR has certain particularity and has important application value in the field of solid tumor treatment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

本发明涉及联合表达CD40L的工程化免疫细胞。具体地，本发明的工程化免疫细胞为T细胞或NK细胞。本发明将含有靶向CEA或CD133的CAR分子和CD40L联合表达于免疫细胞中，既能靶向高表达CEA或CD133的恶性肿瘤细胞，也能通过CD40L激活机体内源的免疫系统，提高治疗效果的同时，防止肿瘤复发。

Description

联合表达CD40L的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，涉及一种联合表达CD40L的工程化免疫细胞。
背景技术
细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式，是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养、修饰改造和扩增后回输到病人体内的方法，以激发、增强机体自身免疫功能，从而达到治疗肿瘤的目的。
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信号传递，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、免疫抑制作用、肿瘤异质性(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。因此，CAR分子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。通过采集培养肿瘤患者的T细胞并以人工方法转导CAR分子的编码基因，使患者自体的T细胞表达CAR分子。回输至患者体内后，T细胞可以通过CAR分子高效且特异性地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年首次被首次提出以来，经历了三十年的发展及多轮的技术更迭(图1)。第一代CAR-T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，无法完全激活T细胞的活性，治疗效果不佳。第二代CAR-T在第一代CAR-T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在第二代CAR-T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。因此，新一代CAR-T在第二代CAR-T的基础上联合表达其他辅助因子，例如联合表达IL-12或IL-2Rβ胞内的STAT3/5结合结构域等，有助于提高肿瘤杀伤活性和安全性等效果。
虽然在血液瘤中CAR-T治疗获得令人满意的效果，但CAR-T对于实体瘤的治疗效果仍有很大的提升空间，主要的原因包括：(1)很多实体肿瘤都难以在早期被发现，具有恶性程度高、复发率高、预后差等特点。(2)在实体瘤治疗过程中，肿瘤组织往往具有免疫抑制性的微环境，可以阻碍CAR-T细胞的迁移和浸润。(3)很多恶性实体肿瘤还具有异质性高的特点。因此，用于结直肠癌、卵巢癌、胰腺癌、乳腺癌、肝癌等恶性实体肿瘤患者的CAR-T细胞疗法，需要更进一步提高其作用效率和有效性。
综上所述，本领域仍然需要进一步的研究，针对恶性肿瘤(尤其是实体瘤)开发一种能更高效、治疗效果更好的工程化免疫细胞。
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)。
本发明的又一目的是提供一种联合表达CD40L的工程化免疫细胞(如CAR-T细胞)及其制法和应用。
本发明的第一方面，提供了一种工程化免疫细胞，所述工程化免疫细胞为T细胞或NK细胞，并且所述的免疫细胞具有以下特征：
(a)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，其中所述CAR的抗原结合结构域包括靶向CEA或CD133的抗原结合域；和
(b)所述的免疫细胞表达外源的CD40L蛋白。
在另一优选例中，所述的T细胞包括αβT、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)。
在另一优选例中，所述的CD40L蛋白可以是组成型表达或诱导型表达。
在另一优选例中，提供了一种嵌合抗原受体T细胞(CAR-T细胞)，所述CAR-T细胞具有以下一个或多个特征：
(a)所述细胞表达嵌合抗原受体CAR，所述CAR靶向肿瘤细胞的表面标志物；和
(b)当所述CAR-T细胞接触肿瘤细胞或诱导剂时，所述CAR-T细胞诱导表达CD40L蛋白。
在另一优选例中，在所述的CAR细胞中，CAR和CD40L蛋白是串联表达的。
在另一优选例中，在所述的CAR细胞中，CAR和CD40L蛋白各自独立地表达的。
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CD40L蛋白为分泌表达。
在另一优选例中，所述CAR的结构如式I所示：
L-ABD-H-TM-C-CD3ζ   (I)
式中，
L为无或信号肽序列；
ABD(antigen-binding domain)为靶向CEA或CD133的抗原结合域或其活性片段(如CEA scFv或CD133 scFv)；
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)；
所述“-”为连接肽或肽键。
在另一优选例中，所述的ABD可以来自含有轻链和重链的传统抗体的单链可变区片段(single-chain fragment variable,scFv)，也可以是来自只含有重链的单域抗体的重链可变区片段(variable domain of heavy chain antibody,VHH)。
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TM选自下组的蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、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CEA scFv的氨基酸序列如SEQ ID NO:4所示；所述CD133 scFv的氨基酸序列如SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15所示。
在另一优选例中，所述CAR还具有人IgG Fc结构域，所述人IgG Fc结构域的氨基酸序列如SEQ ID NO:12所示。
在另一优选例中，所述的CD40L蛋白包括全长CD40L蛋白或其活性片段(即保留可与CD40发生结合的功能的活性片段或其突变体)。
在另一优选例中，所述CD40L蛋白的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，除了式I所示的第一CAR之外，所述CAR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II所示：
L-ABD2-H-TM-C-CD3ζ   (II)
式中，
L为无或信号肽序列；
ABD2为靶向第二靶点的抗原结合域或其活性片段，ABD2可以来自含有轻链和重链的传统抗体的单链可变区片段scFv，也可以是来自只含有重链的单域抗体的重链可变区片段VHH；
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；
所述“-”为连接肽或肽键。
在另一优选例中，所述scFv为靶向肿瘤抗原的抗体的单链可变区序列。
在另一优选例中，所述VHH为靶向肿瘤抗原的单域抗体的重链可变区序列。
在另一优选例中，所述scFv和VHH为靶向选自下组抗原的抗体单链可变区序列：CD19、CD20、CD22、CD123、CD47、CD138、CD33、CD30、CD271、CD276、GUCY2C、CD24、CD133、CD44、CD166、CEACAM5、ABCB5、ALDH1、间皮素(mesothelin,MSLN)、EGFR、GPC3、BCMA、ErbB2、NKG2D配体(ligands)、LMP1、EpCAM、EphA2、VEGFR-1、Lewis-Y、ROR1、Claudin18.2、TAG-72或其组合。
在另一优选例中，式I所示的第一CAR和式II所示的第二CAR可合而为一，从而构成如式IIIa或IIIb所示的CAR：
L-ABD1-ABD2-H-TM-C-CD3ζ   (IIIa)
L-ABD2-ABD1-H-TM-C-CD3ζ   (IIIb)
式中，
L为无或信号肽序列；
ABD1为靶向CEA或CD133的抗原结合域或其活性片段，可以来自含有轻链和重链的传统抗体的单链可变区片段scFv，也可以是来自只含有重链的单域抗体的重链可变区片段VHH；
ABD2为第二靶点的抗原结合域或其活性片段；ABD2可以来自含有轻链和重链的传统抗体的单链可变区片段scFv，也可以是来自只含有重链的单域抗体的重链可变区片段VHH；
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；
所述“-”为连接肽或肽键。
本发明的第二方面，提供了一种制备本发明第一方面所述的工程化免疫细胞的方法，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CAR分子和外源的CD40L蛋白，从而获得本发明第一方面所述的工程化免疫细胞，其中所述CAR靶向肿瘤细胞的表面标志物，其中所述CAR的抗原结合结构域包括靶向CEA或CD133的抗原结合域。
在另一优选例中，在步骤(B)中，包括：
(B1)将表达所述CAR的第一表达盒导入所述免疫细胞；和(B2)将表达CD40L的第二表达盒导入所述免疫细胞；
其中所述的步骤(B1)可在步骤(B2)之前、之后、同时、或交替进行。
在另一优选例中，提供了一种制备本发明所述的CAR-T细胞的方法，包括以下步骤：
(A)提供一种待改造的T细胞；
(B)对所述的T细胞进行改造，使得所述的T细胞表达所述的CAR分子和外源的CD40L蛋白，从而获得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)将表达所述CAR的第一表达盒导入所述T细胞；和(B2)将表达CD40L的第二表达盒导入所述T细胞；其中所述的步骤(B1)可在步骤(B2)之前、之后、同时、或交替进行。
在另一优选例中，当步骤(A)中的待改造的T细胞已经表达CAR时，则在步骤(B)中，包括(B2)第二表达盒导入所述T细胞。
在另一优选例中，所述的第一表达盒和第二表达盒的转录方向是同向的(→→)、相向的(→←)或相背的(←→)。
在另一优选例中，所述的第一表达盒、第二表达盒位于相同或不同的载体上。
在另一优选例中，所述的第一表达盒、第二表达盒位于同一载体。
在另一优选例中，当所述的第一、第二表达盒位于同一载体时，在所述第一、第二表达盒之间，还包括用于表达连接肽的第三表达盒。
在另一优选例中，所述连接肽为P2A或T2A或F2A。
在另一优选例中，所述连接肽上游含有Furin酶切位点和或SGSG连接序列。
在另一优选例中，所述的载体为病毒载体，较佳地所述病毒载体含有串联形式的第一和第二表达盒。
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。
本发明的第三方面，提供了一种制剂，所述制剂含有本发明第一方面所述的工程化免疫细胞，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。
在另一优选例中，所述制剂还含有PBMC。
本发明的第四方面，提供了如本发明第一方面所述的工程化免疫细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，提供了如本发明第一方面所述的CAR-T细胞的用途，用于制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述制剂含有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，所述药物或制剂还含有PBMC。
在另一优选例中，所述肿瘤选自下组：实体瘤、或其组合。
在另一优选例中，所述肿瘤选自下组：结直肠癌、结肠癌、直肠癌、胃癌、卵巢癌、胰腺癌、乳腺癌、肝癌。
在另一优选例中，所述肿瘤为CEA或CD133高表达(阳性)的肿瘤。
在另一优选例中，所述肿瘤为CEA低表达或CEA阴性的肿瘤。
在另一优选例中，所述肿瘤为CD133低表达或CD133阴性的肿瘤。
在另一优选例中，所述肿瘤为CD40高表达(阳性)的肿瘤。
在另一优选例中，所述肿瘤为CD40低表达或CD40阴性的肿瘤。
在另一优选例中，所述肿瘤为NKG2D配体高表达(阳性)的肿瘤。
在另一优选例中，所述NKG2D配体选自下组：MICA、MICB、ULBP-1、ULBP-2、ULBP-3、ULBP-4、ULBP-5、ULBP-6，或其组合。
在另一优选例中，所述肿瘤为(a)CEA或CD133高表达和(b)CD40高表达的肿瘤。
在另一优选例中，所述肿瘤为(a)CEA或CD133高表达和(b)CD40低表达的肿瘤。
在另一优选例中，所述肿瘤为(a)CEA或CD133低表达和(b)CD40高表达的肿瘤。
在另一优选例中，所述肿瘤为(a)CEA或CD133低表达和(b)CD40低表达的肿瘤。
在另一优选例中，所述肿瘤为(a)CEA或CD133高表达和(b)NKG2D配体高表达的肿瘤。
本发明的第五方面，提供了一种用于制备本发明第一方面所述的工程化免疫细胞的试剂盒，所述试剂盒含有容器，以及位于容器内的：
(1)第一核酸序列，所述第一核酸序列含有用于表达所述CAR的第一表达盒，其中所述CAR的抗原结合结构域为靶向CEA或CD133的抗原结合域；和
(2)第二核酸序列，所述第二核酸序列含有用于联合表达CD40L的第二表达盒。
在另一优选例中，提供了一种用于制备本发明第一方面所述的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所述CAR的第一表达盒；和
(2)第二核酸序列，所述第二核酸序列含有用于联合表达CD40L的第二表达盒。
在另一优选例中，所述的第一、第二核酸序列为独立的或相连的。
在另一优选例中，所述的第一、第二核酸序列位于相同或不同的容器内。
在另一优选例中，所述的第一、第二核酸序列位于相同或不同的载体上。
在另一优选例中，所述的第一、第二核酸序列位于同一载体。
在另一优选例中，当所述的第一、第二核酸序列位于同一载体时，在所述第一、第二核酸序列之间，还包括第三核酸序列，所述第三核酸序列含有用于表达连接肽的第三表达盒。
在另一优选例中，所述连接肽为P2A或T2A或F2A。
在另一优选例中，所述连接肽上游含有Furin酶切位点和或SGSG连接序列。
在另一优选例中，所述的载体为病毒载体，较佳地所述病毒载体含有串联形式的第一和第二核酸序列。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了历代CAR分子的结构。
图2显示了CAR分子的结构。
图3显示了流式检测CEA和CD133 CAR-T细胞的表达率。
图4显示了流式检测各NKG2D CAR-T细胞的表达率。
图5显示了流式检测靶细胞中CEA和CD133的表达率。
图6显示了流式检测靶细胞中NKG2D配体(MICA/MICB)的表达率。
图7显示了流式检测靶细胞中NKG2D配体(ULBP-1)的表达率。
图8显示了流式检测靶细胞中NKG2D配体(ULBP-2/5/6)的表达率。
图9显示了流式检测靶细胞中NKG2D配体(ULBP-3)的表达率。
图10显示了流式检测靶细胞中NKG2D配体(ULBP-4)的表达率。
图11显示了流式检测靶细胞中CD40的表达率。
图12显示了EuTDA检测各CAR-T细胞对靶点阳性的肿瘤细胞的杀伤效果。
图13显示了ELISA检测CEA和NKG2D CAR-T细胞的IFN-γ释放水平。
图14显示了荧光素酶法检测CEA CAR-T对HCT116细胞的杀伤效果。
图15显示了荧光素酶法检测PBMC辅助CEA CAR-T杀伤SW480细胞的杀伤效果。
图16显示了B细胞对CEA CAR-T细胞的激活水平。
图17显示了CEA CAR-T细胞在动物药效实验中的抑瘤功能。
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，首次将特定的CAR和CD40L蛋白，即将靶向CEA或CD133的CAR和CD40L联合表达于CAR-T细胞中。与现有技术相比，本发明的免疫细胞既能通过CEA或CD133 CAR靶向结直肠癌、胃癌、卵巢癌、胰腺癌、乳腺癌、肝癌等恶性实体肿瘤，同时可通过CD40L有效激活机体内源的天然和适应性免疫应答，从而有助于T细胞克服免疫抑制的肿瘤微环境，协同地杀灭肿瘤细胞，提高肿瘤治疗，降低肿瘤复发风险。在此基础上完成了本发明。
本发明以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CAR-T细胞，本发明的工程化免疫细胞具有与上下文所述的CAR-T细胞相同或类似的技术特征和有益效果。具体地，当免疫细胞表达嵌合抗原受体CAR时，NK细胞等同于T细胞(或T细胞可替换为NK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”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内、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，包括传统抗体和单域抗体。其中，传统抗体具有特异性结合抗原的活性，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。
另外，单域抗体则是只包含一个重链可变区(VHH)和CH2，CH3区。相比于其他抗体，单域抗体的轻链和CH1区天然缺失，是自然存在的可以和抗原结合的最小片段，也被成为纳米抗体。
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”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，或来自单域抗体的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或VHH。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。
在本发明中，所述scFv包含特异性识别肿瘤高表达的抗原的CEA scFv、CD133 scFv。
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VHH。
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。本发明CAR-T细胞可用于治疗CEA或CD133高表达的肿瘤，如结直肠癌、卵巢癌、胰腺癌、乳腺癌、肝癌等。
CAR-T细胞较其它基于T细胞的治疗方式存在以下优势：(1)CAR-T细胞的作用过程不受MHC的限制；(2)鉴于很多肿瘤细胞表达相同的肿瘤抗原，针对某一种肿瘤抗原的CAR基因构建一旦完成，便可以被广泛利用；(3)CAR既可以利用肿瘤蛋白质抗原，又可利用糖脂类非蛋白质抗原，扩大了肿瘤抗原的靶点范围；(4)使用患者自体细胞降低了排异反应的风险；(5)CAR-T细胞具有免疫记忆功能，可以长期在体内存活。
嵌合抗原受体NK细胞(CAR-NK细胞)
如本文所用，术语“CAR-NK细胞”、“CAR-NK”、“本发明CAR-NK细胞”均指本发明第一方面所述的CAR-NK细胞。本发明CAR-NK细胞可用于治疗CEA高表达的肿瘤，如结直肠癌、卵巢癌、胰腺癌等。
自然杀伤(NK)细胞是一类主要的免疫效应细胞，通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能，包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与自体CAR-T细胞相比，CAR-NK细胞还具有以下优点，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。
CEA
CEA全称为癌胚抗原(carcinoembryonic antigen)，属于免疫球蛋白超级家族。CEA通常指该蛋白家族中的CEACAM-5蛋白(又称为CD66e)。CEA是一种分子量约为180kDa的糖蛋白，参与细胞黏附。CEA通常在胎儿消化系统产生,但由于出生后被抑制,正常成人结肠和血液中CEA含量通常很低。然而,结直肠癌、肺癌和乳腺癌等多种类型的癌症的发生可以引起CEA血清水平升高。因此，CEA被广泛用作广谱肿瘤标志物。CEA并不特异性的针对任何种类的癌症,但是血液中CEA的浓度可以预测癌症的治疗效果、进展和预后。
人CEA蛋白的氨基酸序列的登录号为NP_004354.3，核苷酸序列的登录号为NM_004363.6。人CEA的全长氨基酸序列如下所示：

CD133
CD133(Prominin-1)是五次跨膜糖蛋白Prominin家族的成员之一。研究表明,CD133是多种肿瘤治疗预后的标志物,能与血管内皮生长因子等物质相互作用。CD133也是一个重要的、参与细胞功能的膜和胞浆蛋白，可通过影响葡萄糖摄取，自噬反应和ATP合成而调节肿瘤细胞生存。CD133也是结直肠癌中异常高表达的标志性分子。CD133也是肿瘤干细胞标志物。已有研究发现，可以从多种实体肿瘤中分离鉴定出高表达CD133的肿瘤干细胞，例如结肠癌、脑瘤、肺癌、卵巢癌、胃癌、黑色素瘤等。
人CD133蛋白的氨基酸序列的登录号为NP_001139319.1，核苷酸序列的登录号为NM_001145847.2。人CD133的全长氨基酸序列如下所示：
CD40L
白细胞分化抗原40配体(cluster of differentiation 40 ligand,CD40L)，也称CD154或肿瘤坏死因子相关激活蛋白(tumor necrosis factor-associated activation protein,TRAP)。CD40L主要表达于活化的CD4+T淋巴细胞、活化的CD8+T细胞、嗜碱性粒细胞、肥大细胞、和NK细胞。CD40L与其受体CD40是体内炎症和免疫反应系统中的一对共同刺激分子。在天然免疫中，CD40L/CD40共刺激途径是单核细胞成熟过程的重要触发因素，主要驱动单核细胞分化为M1谱系的巨噬细胞和DC细胞。同时，该途径也可促进DC细胞释放细胞因子和趋化因子，诱导其他共刺激分子的表达，并促进抗原的交叉呈递。在体液免疫中，该途径也参与T细胞依赖性的B淋巴细胞应答过程、生发中心的形成、长期记忆性B细胞的产生、抗体的产生及抗体类别转换。在细胞免疫中，该途径能促进T细胞活化并放大T细胞介导的免疫应答，在CD4+T细胞分化的过程中起重要作用，也能够促进CD8+T细胞的扩增和多能性，是产生记忆性CD8+ T细胞的基础。
在抗肿瘤免疫反应中，CD40L/CD40共刺激途径也发挥多种作用，如激活T细胞的增殖和细胞因子的释放，诱导M2谱系的巨噬细胞向具有抗肿瘤活性的M1谱系的巨噬细胞转变等。在某些靶点抗原丢失但CD40高表达的肿瘤中，该途径还可以介导T细胞对肿瘤细胞的杀伤作用。
在本发明中，合适的CD40L包括野生型和突变型的CD40L，只要该突变型CD40L具有野生型CD40L的基本功能。此外，在本发明中，优选的CD40L来自哺乳动物，如人和非人哺乳动物。
人CD40L蛋白的氨基酸序列的登录号为NP_000065.1，核苷酸氨基酸序列的登录号为NM_000074.3。人CD40L的全长氨基酸序列如下所示：
NKG2D
NKG2D蛋白可识别结直肠癌、卵巢癌、胰腺癌等恶性肿瘤细胞表面的多种靶点抗原(包括MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6)，其胞外结构域可作为CAR分子的抗原结合结构域。在本发明实施例中，NKG2D包括野生型或其突变型或其衍生形式或其活性片段。优选的NKG2D来自于哺乳动物(如人和非人灵长动物)的NKG2D。
人NKG2D蛋白的氨基酸序列的登录号为NP_031386，核苷酸氨基酸序列的登录号为NM_007360。人NKG2D的胞外结构域氨基酸序列如下所示：
表达盒
如本文所用，“表达盒”或“本发明表达盒”包括第一表达盒和第二表达盒。本发明表达盒如本发明第五方面所述，第一表达盒包含编码所述CAR的核酸序列。所述第二表达盒表达外源的CD40L蛋白。
在本发明中，CD40L蛋白可以是组成型表达或诱导型表达。
在诱导表达情况下，在所述CAR-T细胞接触肿瘤细胞或被相应诱导剂激活时，第二表达盒表达CD40L蛋白；这样，在本发明CAR-T细胞在未接触肿瘤细胞或未接触相应诱导剂时，第二表达盒不表达CD40L蛋白。
在一个实施方式中，所述第一表达盒和第二表达盒分别还包括启动子和/或终止子。第二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，还可以用直接转染mRNA或质粒，或者通过表达人工转录因子等方法，从而在T细胞等免疫细胞中联合表达CD40L和CEA或CD133 CAR分子。
制剂
本发明提供了一种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
本发明包括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)进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的表面标志物并表达CD40L蛋白，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。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式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，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续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。例如，本发明的CAR-T细胞引起针对CEA或CD133高表达的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、胃癌、卵巢癌、乳腺癌、肝癌和胰腺癌。
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。
本发明的CAR-T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由如患者的病症、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“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。包括本文描述的T细胞的药物组合物可以以104至109个细胞/kg体重的剂量，优选105至107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，NewEng.J.of  Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可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细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过静脉内注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×105个至1×1010个本发明经修饰的T细胞，通过例如静脉回输的方式，施用于患者。
本发明的主要优点
(1)本发明的工程化免疫细胞，既能通过CEA或CD133 CAR分子特异性靶向恶性肿瘤细胞表面的靶点抗原，也能通过CD40L激活机体内源的免疫系统，提高治疗效果的同时，防止肿瘤复发。
(2)CD40L的表达也意外地促进了靶向CEA或CD133的CAR分子的表达显著上升。
(3)当本发明CEA或CD133 CAR分子和外源CD40L蛋白的联合表达时，可协同地显著提高针对肿瘤细胞的体外杀伤作用，以及在小鼠模型中显著提高抑瘤效果并防止肿瘤复发。
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。
材料与方法
CAR分子及其结构
在实施例中，以NKG2D CAR-T细胞作为CEA和CD133 CAR-T细胞的对照。各CAR分子包含以下部分结构：人CD8信号肽[简称CD8(SP)]、抗人CEA单链抗体[简称CEA scFv]、抗人CD133单链抗体[简称CD133 scFv]、人NKG2D胞外结构域[简称NKG2D(ED)]、优化了的人CD8铰链区[简称CD8(hinge)]、人IgG Fc结构域[简称IgG(Fc)]、人CD8跨膜结构域[简称CD8(TM)]、人4-1BB胞内结构域[简称4-1BB(ID)]、人CD3ζ胞内信号转导结构域[简称CD3ζ(ID)]、自剪切肽P2A或F2A、人CD40L。
联合表达CD40L的新一代CEA CAR分子命名为BC004，作为对照的第二代CEA CAR分子命名为BC001；联合表达CD40L的新一代NKG2D CAR分子命名为BN004，作为对照的第二代NKG2D CAR分子命名为BN001；第二代CD133 CAR分子命名为BW133-2。具体结构如图2所示：
BC001从其氨基端至羧基端由CD8(SP)、CEA scFv、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)依次串联组成；
BC004从其氨基端至羧基端由CD8(SP)、CEA scFv、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、P2A、CD40L依次串联组成；
BW133-2从其氨基端至羧基端由CD8(SP)、CD133 scFv、IgG(Fc)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)依次串联组成；
BN001从其氨基端至羧基端由CD8(SP)、NKG2D(ED)、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)依次串联组成；
BN004从其氨基端至羧基端由CD8(SP)、NKG2D(ED)、CD8(hinge)、CD8(TM)、4-1BB(ID)、CD3ζ(ID)、P2A、CD40L依次串联组成。
序列信息


实施例1慢病毒制备
1.1慢病毒载体质粒的获得
全基因合成BC001、BC004、BW133-2、BN001和BN004的核苷酸序列，并进行密码子优化，再通过分子克隆的方式连接到慢病毒载体pCDH-EF1-MCS-T2A-copGFP质粒中，使之在人EF1α启动子和Kozak序列的调控下表达。
1.2慢病毒载体质粒转染293T细胞
将上述各慢病毒载体质粒与慢病毒包装质粒pMD2.G、pRSV-Rev和pMDLg/pRRE用聚乙烯亚胺转染试剂混合，共转染293T细胞。培养48h后，分 别收集病毒上清液，于4℃下4500rpm离心10～15min，经0.5μm孔径的滤膜过滤后用中空纤维柱超滤系统进行慢病毒浓缩，再用层析法进行慢病毒纯化，最后用0.22μm孔径的滤膜过滤除菌后分装置于-80℃保存。
1.3慢病毒滴度测定
将Jurkat细胞的浓度调整至1×105个/300μl，充分混匀后取300μl重悬的细胞至24孔板的每个孔中。取70μl慢病毒浓缩液用Opti-MEM培养基进行5倍梯度稀释。将各稀释梯度的慢病毒以200μl/孔的用量加入上述24孔板中，使慢病毒感染Jurkat细胞(阴性对照组的Jurkat细胞只加入Opti-MEM培养基)，并置于细胞培养箱中培养(培养温度为37℃，二氧化碳浓度为5％)。培养3天后，将各孔内的细胞轻柔混匀并转移至1.5ml离心管中，用染色缓冲液(100ml PBS+1％BSA)清洗两次，每次800g离心3min。用相应抗体对上述细胞进行染色，再用流式细胞仪进行检测成功被慢病毒转导的Jurkat细胞的比例。将Jurkat细胞的慢病毒感染率记为P，通过以下公式计算慢病毒滴度：
慢病毒滴度(TU/ml)＝P/V×103×105
结果：BC001滴度为2.58×109TU/ml，BC004滴度为3.26×108TU/ml，BN001滴度为5.46×108TU/ml，BN004滴度为6.31×108TU/ml，BW133-2滴度为4.28×108TU/ml。
实施例2 CAR-T细胞的制备和检测
2.1 T细胞制备
将健康供者的外周血单个核细胞密度调整至2×106/ml，加入50ng/ml抗CD3抗体、50ng/ml抗CD28抗体，以及200IU/ml重组IL-2，置于细胞培养箱中培养24h(培养温度为37℃，二氧化碳浓度为5％)。
2.2慢病毒转导T细胞
清洗获得的T细胞，并将细胞密度调整至4×106/ml。以MOI＝1～10TU/cell的用量加入慢病毒进行转导，同时补充50ng/ml抗CD3抗体、50ng/ml抗CD28抗体，以及200IU/ml重组IL-2，置于细胞培养箱中培养(培养温度为37℃，二氧化碳浓度为5％)。24h后，将细胞密度调整至1.5～2×106/ml，并补充300IU/ml的IL-2。转导后第4天，清洗细胞以去除上清中残留的慢病毒粒子，并继续置于细胞培养箱中培养5天(培养温度为37℃，二氧化碳浓度为5％)，期间保持细胞密度为1～2×106/ml。转导后第10天收取细胞，并用冻存液(含有5％人血清白蛋白的冻存培养基:生理盐水＝1:1)冻存于液氮中备用。获得的CAR-T细胞沿用相应CAR分子的命名，分别为BC001、BC004、BW133-2、BN001和BN004，BW133-2与过表达CD40L的慢病毒共转导获得的CAR-T细胞命名为BW133-2+CD40L，未用慢病毒转导的T细胞命名为Ctrl T。
2.3 CEA CAR-T细胞的表达检测
用PBS清洗待检测的Ctrl T、BC001和BC004细胞两次，用FACS缓冲液(含0.1％叠氮化钠和0.4％BSA的PBS)重悬并进行流式检测。结果如图3A所示，BC001细胞的CAR分子表达率约为71.82％，BC004细胞的CAR分子表达率约为81.41％。这表明，联合表达CD40L使CEA CAR分子的表达率意外地显著上升，上升幅度为13.4％[(81.41％-71.82％)/71.82％＝13.4％]。
BC001细胞的CD40L表达率约为17.67％，BC004细胞的CD40L表达率约为70.93％，表明外源CD40L蛋白已经在BC004细胞中成功表达。
2.4 CD133 CAR-T细胞的表达检测
用PBS清洗待检测的Ctrl T、BW133-2和BW133-2+CD40L细胞两次，用FACS缓冲液重悬并进行流式检测。
结果如图3B所示，BW133-2细胞和BW133-2+CD40L细胞的CAR分子表达率均超过70％。
BW133-2细胞的CD40L表达率约为55.7％，BW133-2+CD40L细胞的CD40L表达率约为92.4％，表明外源CD40L蛋白已经在BW133-2+CD40L细胞中成功表达。
2.5 NKG2D CAR-T细胞的表达检测
用PBS清洗待检测的Ctrl T、BN001和BN004细胞两次，用FACS缓冲液重悬并进行流式检测。结果如图4所示，BN001细胞的CAR分子表达率约为79.05％，BN004细胞的CAR分子表达率约为81.80％。这表明，联合表达CD40L只能使NKG2D CAR分子的表达率有小幅上升，上升幅度仅为3.5％[(81.8％-78.4％)/79.05％＝3.5％]。
BN001的CD40L表达率约为27.91％，BN004的CD40L表达率约为84.88％，表明外源CD40L蛋白已经在BN004细胞中成功表达。
以上结果表明，虽然外源CD40L蛋白已经在BN004细胞中成功表达，但外源CD40L蛋白对提升NKG2D CAR分子的表达率的作用比较有限。
实施例3靶细胞检测
3.1靶细胞培养条件
LS174T(EMEM培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)，LoVo(F-12K培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)，SW480(Leibovitz's L-15培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)，HCT116(McCoy's 5A培养基+10％胎牛血清+100U/ml青霉素+100μg/ml链霉素)，Caco-2(EMEM培养基+20％胎牛血清+100U/ml青霉素+100μg/ml链霉素)。
3.2 CEA和CD133的表达检测
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FITC标记的抗CEA抗体加入待检测细胞悬液中，4℃孵育60min。以不加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。采用CytExpert软件分析。
结果如图5A所示，LS174T的CEA表达率约为90.7％，LoVo细胞的CEA表达率约为60％，SW480的CEA表达率约为8.2％，HCT116的CEA表达约为2.5％。
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APC标记的抗CD133抗体加入待检测细胞悬液中，4℃孵育60min。以不加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CytExpert软件分析。结果如图5B所示，Caco-2的CD133表达率超过99％。
3.3 NKG2D配体(MICA/MICB)的表达检测
用PBS清洗上述靶细胞两次，并用FACS缓冲液重悬。按照抗体说明书将APC 标记的抗人MICA/MICB抗体加入各靶细胞悬液中，4℃孵育30min。以不加入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MICA/MICB表达率。采用CytExpert软件分析。
结果如图6所示，LS174T的MICA/MICB表达率约为96.6％，LoVo的MICA/MICB表达率约为7.8％，Caco-2的MICA/MICB表达率约为5.7％。
3.4 NKG2D配体(ULBP-1)的表达率
用PBS清洗上述靶细胞两次，并用FACS缓冲液重悬。按照抗体说明书将PE标记的抗人ULBP-1抗体加入各靶细胞悬液中，4℃孵育60min。以加入IgG孵育的靶细胞作为阴性对照，用流式细胞仪检测靶细胞的ULBP-1表达率。采用CytExpert软件分析。
结果如图7所示，LS174T的ULBP-1表达率约为1.8％，LoVo的ULBP-1表达率约为9.1％，Caco-2的ULBP-1表达率约为13.1％。
3.5 NKG2D配体(ULBP-2/5/6)的表达率
用PBS清洗上述靶细胞两次，并用FACS缓冲液重悬。按照抗体说明书将PE标记的抗人ULBP-2/5/6抗体加入各靶细胞悬液中，4℃孵育60min。以加入IgG孵育的靶细胞作为阴性对照，用流式细胞仪检测靶细胞的ULBP-2/5/6表达率。采用CytExpert软件分析。
结果如图8所示，LS174T的ULBP-2/5/6表达率约为33.4％，LoVo的ULBP-2/5/6表达率约为86.8％，Caco-2的ULBP-2/5/6表达率约为78.2％。
3.6 NKG2D配体(ULBP-3)的表达率
用PBS清洗上述靶细胞两次，并用FACS缓冲液重悬。按照抗体说明书将PE标记的抗人ULBP-3抗体加入各靶细胞悬液中，4℃孵育60min。以加入IgG孵育的靶细胞作为阴性对照，用流式细胞仪检测靶细胞的ULBP-3表达率。采用CytExpert软件分析。
结果如图9所示，LS174T的ULBP-3表达率约为46.6％，LoVo的ULBP-3表达率约为21.1％，Caco-2的ULBP-3表达率约为3.9％。
3.7 NKG2D配体(ULBP-4)的表达率
用PBS清洗上述靶细胞两次，并用FACS缓冲液重悬。按照抗体说明书将PE标记的抗人ULBP-4抗体加入各靶细胞悬液中，4℃孵育60min。以加入IgG孵育的靶细胞作为阴性对照，用流式细胞仪检测靶细胞的ULBP-4表达率。采用CytExpert软件分析。
结果如图10所示，LS174T的ULBP-4表达率约为82.0％，LoVo的ULBP-4表达率约为82.2％，Caco-2的ULBP-4表达率约为5.3％。
3.8 CD40的表达率
用PBS清洗上述靶细胞两次，并用FACS缓冲液重悬。按照抗体说明书将APC标记的抗人CD40抗体加入各靶细胞悬液中，4℃孵育60min。以不加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CD40表达率。采用CytExpert软件分析。
结果如图11所示，LS174T、LoVo、SW480、Caco-2的表达率均低于3％，HCT116的表达率约为99.9％。
实施例4 CAR-T细胞的体外功能
4.1对靶点阳性的肿瘤细胞的杀伤效果检测
用AIM-V培养基清洗靶细胞一次。将靶细胞密度调节至1×106/ml，并以2μl/ml的用量加入DELFIA BATDA Reagent混匀，37℃下孵育30min。用AIM-V培养基清洗靶细胞三次后，以1×104/孔的密度将靶细胞接种于96孔板中。加入100μl T细胞(效靶比为2.5:1、5:1和10:1)，并置于二氧化碳培养箱培养2h(培养温度为37℃，二氧化碳浓度为5％)。最后，以500×g离心5min，取20μl上清液转移至加有Europium solution(200μl/孔)的96孔板中。室温孵育15min后，在酶标仪中检测。
结果如表1和图12A所示，与Ctrl T相比，BC001、BC004、BN001和BN004对各靶细胞都有显著的杀伤效果。以Ctrl T的杀伤率为本底值，扣除相应本底值后可获得各CAR-T细胞对靶细胞的特异性杀伤率(表2)。值得注意的是，LS174T和LoVo中能检测到NKG2D配体的显著表达。例如，LS174T的MICA/MICB表达率为96.6％，高于其CEA的表达率(90.7％)；LoVo的ULBP-2/5/6表达率为86.8％，ULBP-4表达率约为82.2％，均高于其CEA的表达率(60％)。然而，在相同的效靶比下，CEA CAR-T细胞对靶细胞的杀伤率都显著高于NKG2D CAR，原因可能在于CEA scFv与相应靶点的结合能力强于NKG2D，说明对于CEA CAR-T细胞更适合用于杀伤CEA高表达的肿瘤细胞系(尽管其同时高表达NKG2D配体)。
另外，在LS174T和LoVo中，CD40的表达率都低于3％，可能是未检测到BN004相对于BN001的特异性杀伤率的提升(P＞0.05)的原因。然而，意外发现，BC004在各效靶比中的特异性杀伤率都显著高于BC001(P＜0.05)，提升幅度为10％-16％，说明在CEA CAR-T细胞中联合表达CD40L可以有效提升T细胞对肿瘤细胞的杀伤效果。
表1 EuTDA检测CEA和NKG2D CAR-T细胞对LS174T和LoVo细胞细胞的杀伤率
表2 EuTDA检测CEA和NKG2D CAR-T细胞对LS174T和LoVo细胞的特异性杀伤率

用EuTDA法检测BW133-2和BW133-2+CD40L对Caco-2的杀伤水平。结果如表3和图12B所示，与Ctrl T相比，BW133-2、BW133-2+CD40L、BN001和BN004对Caco-2细胞具有显著的杀伤效果。以Ctrl T的杀伤率为本底值，扣除相应本底值后可获得各CAR-T细胞对靶细胞的特异性杀伤率(表4)。
Caco-2细胞的CD40的表达率低于3％，可能是未检测到BN004相对于BN001的特异性杀伤率的提升(P＞0.05)的原因。然而，BW133-2+CD40L的特异性杀伤率显著高于BW133-2(P＜0.05)，提升幅度为8.3％-18.1％，说明在CD133 CAR-T细胞中联合表达CD40L可以有效提升其对肿瘤细胞的杀伤效果。
表3 EuTDA检测CD133和NKG2D CAR-T细胞对Caco-2细胞的杀伤率
表4 EuTDA检测CD133和NKG2D CAR-T细胞对Caco-2细胞的特异性杀伤率
将Ctrl T、BC001、BC004、BN001和BN004分别与相应的靶细胞共培养于不含IL-2的AIM-V培养基中(效靶比为2.5:1)。24h后，用ddH2O溶解INF-γ标准品，室温放置15～20min保证充分溶解，将标准品按推荐梯度倍比稀释。吸取上述共培养的细胞上清，用ddH2O进行2倍和20倍稀释。分别将标准品和实验样品加入相应反应孔中，每孔100μl。室温孵育1～3h后，配制1×清洗液，每孔用360μl清洗液清洗4次，并将孔中液体拍干，每孔加入200μl酶标检测抗体，室温孵育1～3h。每孔用360μl清洗液清洗4次，并将孔中液体拍干后，加入200μl显色底物。室温避光孵育30～60min后，每孔加入50μl终止液，用酶标仪测定450nm的吸光值。
结果如表5和图13所示，与Ctrl T相比，各CAR-T细胞分别与各靶细胞共培养后都有显著的IFN-γ释放。BC004与LS174T或LoVo共培养时，IFN-γ 的释放水平显著高于BC001细胞。BN004只有与LS174T共培养时，IFN-γ的释放水平才高于BN001细胞。联合表达CD40L可以显著提升CAR-T细胞的IFN-γ的释放水平。其中，当CD40L与CEA CAR联合表达时能对更多的肿瘤细胞表现出该提升效果。
表5 ELISA检测LS174T和LoVo细胞对CEA和NKG2D CAR-T细胞激活程度

*信号低于检测阈值。
4.2对靶点阴性的肿瘤细胞的杀伤效果检测
用荧光素酶法检测BC001和BC004对CEA阴性CD40阳性的HCT116细胞(该细胞中过表达了萤火虫荧光素酶)的杀伤水平。用AIM-V培养基清洗靶细胞后，以1×104/孔的密度将靶细胞接种于96孔板中。加入100μl T细胞(效靶比为0.5:1、1:1和2:1)，并置于二氧化碳培养箱培养24h(培养温度为37℃，二氧化碳浓度为5％)。最后，以500×g离心5min收集细胞沉淀，用50μl的RPMI-1640培养基重悬并在每孔加入50μl的Steady-Glo荧光素酶检测反应试剂。排枪混匀后，取出80μl混合液加入黑色的96孔板中，置于酶标仪检测各样品中剩余的HCT116活细胞中荧光素酶的含量。HCT116细胞被CAR-T细胞杀伤后会破裂并将胞内的荧光素酶释放至培养上清中。因此，从收集的细胞沉淀中测得的荧光素酶信号越低，说明剩余的HCT116活细胞越少，T细胞的杀伤效果越好。
结果如表6和图14所示，与Ctrl T相比，BC001对HCT116细胞没有显著的杀伤效果(P>0.05)，而BC004的杀伤效果显著高于Ctrl T与BC001(P<0.05)。以Ctrl T的杀伤率为本底值，扣除相应本底值后可获得各CAR-T细胞对靶细胞的特异性杀伤率(表7)。BC004在各效靶比中的特异性杀伤率都显著高于BC001(P＜0.05)，提升幅度为29％-50％，说明在CEA CAR-T细胞中联合表达CD40L可以有效提升T细胞对靶点阴性的肿瘤细胞的杀伤效果。
表6荧光素酶法检测CEA CAR-T细胞对HCT116细胞的杀伤率
表7荧光素酶法检测CEA CAR-T细胞对HCT116细胞的特异性杀伤率

用荧光素酶法检测BC001和BC004对CEA和CD40双阴性的SW480细胞(该细胞中过表达了萤火虫荧光素酶)的杀伤水平。结果如表8和图15A所示，在各效靶比下，BC001和BC004的杀伤效果较弱，且没有显著差异(P>0.05)。然而，与现有技术相比(现有技术未发现联合表达CD40L可以提升CD19CAR-T细胞对CD19-CD40-的双阴性细胞的杀伤效果)，出乎意料的是，在共培养体系中加入与T细胞等量的PBMC后，BC004对SW480的杀伤率显著提升26.2％～52.8％(P<0.05)，且显著强于PBMC辅助的BC001(P<0.05)。收取共培养体系中的上清检测T细胞的IFN-γ释放水平，也可以发现PBMC辅助的BC004的激活程度显著上升(P<0.05)，IFN-γ释放水平提升5倍以上(表9和图15B)。相反，即使加入PBMC辅助，BC001的激活水平也没有显著提升(P>0.05)。这个结果说明，CD40L可以介导CEA CAR-T细胞与其他免疫细胞的相互作用，提升其对靶点阴性的肿瘤细胞的杀伤作用，有助于防止治疗后的肿瘤免疫逃逸。
表8荧光素酶法检测CEA CAR-T细胞对SW480细胞的杀伤率
表9 ELISA检测SW480细胞对PBMC辅助的CEA CAR-T细胞激活程度
4.3对正常细胞的杀伤效果检测
联合表达CD40L可以促进CEA CAR-T细胞对靶点阴性的肿瘤细胞的杀伤效果。因此，还需要对其安全性进行评价。将BC001和BC004分别与CD40阳性CEA阴性的B细胞共培养，检测上清中IFN-γ的释放水平。结果如图16A所示，与Ctrl T细胞相比，与B细胞共培养后的BC001和BC004的IFN-γ的释放水平都没有显著上升(P>0.05)，说明高表达CD40的正常细胞并不会激活BC004。
用活细胞荧光染料对B细胞进行染色，再用IncuCyte活细胞实时成像分析系统观察BC001和BC004是否会对B细胞的长期生长有影响。结果如图16B所示， 在共培养的48h内，与BC004共培养的B细胞的生长未受显著影响，说明虽然BC004可以杀伤靶点阴性的肿瘤细胞，但其对高表达CD40的正常细胞没有显著的杀伤效果，具有很好的安全性。
实施例5 CAR-T细胞的体内抑瘤功能
以BC001和BC004为效应细胞，以LoVo为靶细胞，进行小鼠皮下移植瘤抑制效果测试。用免疫缺陷的NSG-B2M小鼠进行实验，以观察联合表达CD40L的CEA CAR-T细胞对肿瘤浸润和抑制效果。方法如下：
取24只6～8周龄的NSG-B2M小鼠进行皮下瘤药效实验，随机分为3组，每组8只，分别为Ctrl T对照组、BC001对照组、BC004实验组。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，用生理盐水洗涤1次后，调整细胞密度为2×107/ml。在NSG-B2M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2×106的靶细胞，接种日记为第0天。接种靶细胞后第7天(或肿瘤平均体积为50～100mm3时)，通过尾静脉分别注射CAR-T细胞(1×107/只)、Ctrl T细胞(1×107/只)，注射受试物当天记为治疗的第0天。每周测量肿瘤大小和小鼠体重2～3次。
结果如图17所示，与注射Ctrl T细胞组别的小鼠相比，BC001和BC004可以有效控制小鼠的皮下瘤生长。从注射后第12天开始，注射BC001和BC004的组别中肿瘤体积都显著小于注射Ctrl T细胞的组别(P<0.05)，其中部分小鼠的肿瘤已被完全消除。注射后第16天，在所有注射BC001或BC004的小鼠中，肿瘤都被完全消除。然而，在注射后第19天开始，注射BC001的小鼠的肿瘤开始复发，随后肿瘤体积逐渐增大。至第54天，注射BC001的小鼠的肿瘤平均瘤径已达430±378mm3，而注射BC004的小鼠中未见肿瘤复发。结果说明，联合表达CD40L的CEA CAR-T细胞不仅可以快速有效地消除肿瘤，且可以有效抑制肿瘤的复发。
讨论
在针对结直肠癌、卵巢癌、胰腺癌、乳腺癌等恶性实体肿瘤的CAR-T治疗中，目前主要识别的靶点有CD133、CEA、EGFR、HER-2、MSLN、MUC-16和NKG2D配体等。CEA主要在胚胎阶段的消化道上皮细胞表达，并在出生后停止表达。因此，CEA在健康组织细胞中的表达水平极低。但研究发现，在结直肠癌细胞等多种肿瘤组织中，CEA都有较高水平的表达，与肿瘤细胞的粘附、侵袭相关。CD133是多种肿瘤治疗预后的标志物，也是肿瘤干细胞标志物。已有研究发现，可以从多种实体肿瘤中分离鉴定出高表达CD133的肿瘤干细胞。因此，CEA和CD133作为肿瘤治疗靶点，可以使CAR-T细胞有效靶向实体瘤细胞，同时具有极高的安全性，对肿瘤细胞治疗具有重要应用价值。
虽然CD40L具有激活机体免疫应答、提高肿瘤治疗效果并防止复发的功能，但现有技术只将CD40L应用于靶向CD19的血液瘤的免疫治疗中，未能满足实体瘤治疗的需求。为解决上述问题，本发明将CD40L与靶向广谱实体瘤抗原的CEA CAR、CD133 CAR联合表达。因此，本发明中的CAR-T细胞能通过CEA CAR、CD133 CAR分子高效靶向恶性实体瘤细胞，同时也能以联合表达的CD40L结合肿瘤细胞或免疫细胞上的CD40，通过CD40/CD40L共刺激通路，激活T细胞的增殖和细胞因子的释放，同时还能提高T细胞对某些抗原丢失但CD40高表达的肿瘤细 胞的杀伤作用。
然而，在未结合CD40时，CD40L对CAR-T细胞的功能则少有报道。本发明经过大量实验筛选和比较，意外发现在未与其他细胞共培养的情况下，联合表达CD40L即可显著提升CEA CAR分子在T细胞中的表达。在已有报道的联合表达CD40L的CD19CAR-T细胞中，未发现CD40L提升CD19CAR分子表达水平的作用。另外，与几乎不表达CD40的LS174T和LoVo共培养后，也发现CD40L提高了CEA CAR-T细胞对相应肿瘤细胞的杀伤效果，说明联合表达的CD40L对CAR-T细胞功能的提升作用并不依赖于CD40/CD40L共刺激通路。然而，联合表达的CD40L未能有效提升NKG2D CAR分子的表达和杀伤效果，说明上述功能还与CAR分子靶点选择和的设计有关。另外，现有文献未发现联合表达CD40L能提升CD19CAR-T细胞对CD19-CD40-双阴性细胞的杀伤效果。但本研究发现，联合表达CD40L可以显著提升CAR-T细胞对靶点和CD40双阴性的肿瘤细胞的杀伤效果。因此，本发明中联合表达CD40L与CEA CAR、CD133 CAR的方案具有一定的特殊性，在实体瘤治疗领域中有重要的应用价值。
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 (10)

  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细胞或NK细胞，并且所述的免疫细胞具有以下特征：
    (a)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，其中所述CAR的抗原结合结构域包括靶向CEA或CD133的抗原结合域；和
    (b)所述的免疫细胞表达外源的CD40L蛋白。
  2. 如权利要求1所述的工程化免疫细胞，其特征在于，所述CAR的结构如式I所示：
    L-ABD-H-TM-C-CD3ζ   (I)
    式中，
    L为无或信号肽序列；
    ABD(antigen-binding domain)为靶向CEA或CD133的抗原结合域或其活性片段(如CEA scFv或CD133 scFv)；
    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；
    所述“-”为连接肽或肽键。
  3. 如权利要求2所述的工程化免疫细胞，其特征在于，所述CEA scFv的氨基酸序列如SEQ ID NO:4所示；所述CD133 scFv的氨基酸序列如SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15所示。
  4. 如权利要求1所述的工程化免疫细胞，其特征在于，所述CD40L蛋白的氨基酸序列如SEQ ID NO:2所示。
  5. 一种制备权利要求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所述的CAR分子和外源的CD40L蛋白，从而获得权利要求1所述的工程化免疫细胞。
  6. 一种制剂，其特征在于，所述制剂含有权利要求1所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  7. 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，用于制备可预防和/或治疗癌症或肿瘤的药物或制剂。
  8. 如权利要求7所述的用途，其特征在于，所述肿瘤选自下组：结直肠癌、结肠癌、直肠癌、胃癌、卵巢癌、胰腺癌、乳腺癌、肝癌。
  9. 如权利要求7所述的用途，其特征在于，所述肿瘤为CEA或CD133高表达的肿瘤。
  10. 一种用于制备权利要求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所述CAR的第一表达盒，其中所述CAR的抗原结合结构域为靶向CEA或CD133的抗原结合域；和
    (2)第二核酸序列，所述第二核酸序列含有用于联合表达CD40L的第二表达盒。
PCT/CN2023/114797 2022-08-25 2023-08-24 联合表达cd40l的工程化免疫细胞及其制备和应用 WO2024041618A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202380012661.3A CN117940559A (zh) 2022-08-25 2023-08-24 联合表达cd40l的工程化免疫细胞及其制备和应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211028558 2022-08-25
CN202211028558.1 2022-08-25

Publications (1)

Publication Number Publication Date
WO2024041618A1 true WO2024041618A1 (zh) 2024-02-29

Family

ID=90012597

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/114797 WO2024041618A1 (zh) 2022-08-25 2023-08-24 联合表达cd40l的工程化免疫细胞及其制备和应用

Country Status (2)

Country Link
CN (1) CN117940559A (zh)
WO (1) WO2024041618A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111166876A (zh) * 2018-11-13 2020-05-19 北京启辰生生物科技有限公司 一种免疫增强剂组合和编码核酸及其应用
CN113234162A (zh) * 2020-12-24 2021-08-10 四川大学华西医院 一种靶向cd133的嵌合抗原受体t细胞
CN113621582A (zh) * 2021-09-23 2021-11-09 广州百吉生物制药有限公司 联合表达CCR2b的工程化免疫细胞及其制备和应用
CN113755448A (zh) * 2021-11-10 2021-12-07 广州百吉生物制药有限公司 联合表达CCR2b和CD40L的工程化免疫细胞及其制备和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111166876A (zh) * 2018-11-13 2020-05-19 北京启辰生生物科技有限公司 一种免疫增强剂组合和编码核酸及其应用
CN113234162A (zh) * 2020-12-24 2021-08-10 四川大学华西医院 一种靶向cd133的嵌合抗原受体t细胞
CN113621582A (zh) * 2021-09-23 2021-11-09 广州百吉生物制药有限公司 联合表达CCR2b的工程化免疫细胞及其制备和应用
CN113755448A (zh) * 2021-11-10 2021-12-07 广州百吉生物制药有限公司 联合表达CCR2b和CD40L的工程化免疫细胞及其制备和应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CURRAN K.J. ET AL.: "Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression", MOL. THER., vol. 23, no. 4, 10 February 2015 (2015-02-10), pages 769 - 778, XP002789068, DOI: 10.1038/mt.2015.4 *
DATABASE Protein 26 December 2021 (2021-12-26), ANONYMOUS : "prominin-1 isoform 2 precursor [Homo sapiens]", XP093142728, retrieved from NCBI Database accession no. NP_001139319.1 *
DATABASE Protein 31 October 2021 (2021-10-31), ANONYMOUS : "carcinoembryonic antigen-related cell adhesion molecule 5 isoform 1 preproprotein [Homo sapiens]", XP093142720, retrieved from NCBI Database accession no. NP_004354.3 *
KUHN NICHOLAS F.; PURDON TERENCE J.; VAN LEEUWEN DAYENNE G.; LOPEZ ANDREA V.; CURRAN KEVIN J.; DANIYAN ANTHONY F.; BRENTJENS RENIE: "CD40 Ligand-Modified Chimeric Antigen Receptor T Cells Enhance Antitumor Function by Eliciting an Endogenous Antitumor Response", CANCER CELL, CELL PRESS, US, vol. 35, no. 3, 1 January 1900 (1900-01-01), US , pages 473, XP085638453, ISSN: 1535-6108, DOI: 10.1016/j.ccell.2019.02.006 *
邹依地 等 (ZOU, YIDI ET AL.): "胰腺导管腺癌面临的挑战与最新研究进展 (Challenges and Recent Research Progress in Pancreatic Ductal Adenocarcinoma)", 生命科学 ("CHINESE BULLETIN OF LIFE SCIENCES"), vol. 31, no. 9, 30 September 2019 (2019-09-30) *

Also Published As

Publication number Publication date
CN117940559A (zh) 2024-04-26

Similar Documents

Publication Publication Date Title
US20210277114A1 (en) Car based immunotherapy
US10780120B2 (en) Prostate-specific membrane antigen cars and methods of use thereof
AU2014259675B2 (en) CS1-specific chimeric antigen receptor engineered immune effector cells
US20210395369A1 (en) Anti-b7-h3 monoclonal antibody and use thereof in cell therapy
WO2023046110A1 (zh) 联合表达CCR2b的工程化免疫细胞及其制备和应用
WO2019196713A1 (zh) 靶向bcma的嵌合抗原受体及其制法和应用
EP3964238A1 (en) Bcma-targeting engineered immune cell and use thereof
CN109575143B (zh) 双特异性cd20-cd19-car及其应用
WO2023083192A1 (zh) 联合表达CCR2b和CD40L的工程化免疫细胞及其制备和应用
US20240082401A1 (en) Bispecific cs1-bcma car-t cell and application thereof
CN107936120B (zh) Cd19靶向性的嵌合抗原受体及其制法和应用
WO2023051735A1 (zh) 嵌合抗原受体免疫细胞及其制法和应用
WO2021208750A1 (zh) 靶向cd22的嵌合抗原受体及其制法和应用
US20230103327A1 (en) Quantitative control of activity of engineered cells expressing spycatcher and spytag universal immune receptors
WO2024041618A1 (zh) 联合表达cd40l的工程化免疫细胞及其制备和应用
CN116496397B (zh) 靶向cd19 car-t细胞人源化抗体
US11992503B2 (en) Prostate-specific membrane antigen cars and methods of use thereof
WO2022143928A1 (zh) 膜融合蛋白及其在免疫细胞中的应用
CN118109416A (zh) 功能增强型工程化免疫细胞及其制备和应用
CN116814553A (zh) 杀伤和存续能力增强型car-t细胞及其制备和应用
CN114763388A (zh) 靶向b7-h3的car-t细胞及其在急性髓系白血病治疗中的应用

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 202380012661.3

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23856705

Country of ref document: EP

Kind code of ref document: A1